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emesis is important in terms of ridding the upper gastrointestinal tract of toxins and pathogens . the act of vomiting by an individual infected with an infectious disease such as norovirus disseminates the agent into the local environment . electron microscopic studies have suggested that as many as 310 norovirus particles could be distributed into the immediate surroundings following an episode of vomiting , based on a bolus of 30 ml i.e. 110 virus particles / l ( caul , 1995 ) . the robust nature of many infectious agents allows them to survive in the environment for protracted periods of time , particularly when associated with organic matter such as skin squames , faeces and vomit . this increases the likelihood of infection transmission if they are not removed from the environment . cleaning and decontamination of surfaces contaminated with an infectious agent post - emesis is therefore an important aspect of infection control . research relating to the physiology of vomiting has been carried out , although most published studies relating to the mechanics of vomiting date back to the early 1900s . such studies have demonstrated that the mechanism of emesis in humans and other mammals is complex ( lumsden and holden , 1969 ) and is still controversial ( pickering and jones , 2002 ) . it is accepted that the process comprises three key phases , which occur in the following order : nausea , retching and vomiting . projectile vomiting ( sometimes referred to as forced vomiting ) often occurs suddenly and without warning , i.e. without nausea and retching . consequently an individual may have an episode of projectile vomiting at any time , e.g. while standing in a queue , on public transport , or in a restaurant or hospital bed . based on the widely accepted text by guyton and hall ( 2011 ) , vomiting is initiated via a deep inspiratory breath , which allows the upper oesophageal sphincter to open , closing of the glottis and posterior nares , as well as a strong downward contraction of the diaphragm . contraction of all abdominal muscles occurs simultaneously , consequently squeezing the stomach between the two sets of muscles , i.e. the diaphragm and the abdominals , creating a high level of intragastric pressure . the lower oesophageal sphincter at the top of the stomach then relaxes , as does the perioesophageal ( crural ) portion of the diaphragm required to release the fluid into the oesophagus and out of the mouth ( guyton and hall , 2011 ) . many early studies have shown reasonably conclusively that the role of the stomach wall during emesis is minimal ( haskell , 1924 ) . projectile vomiting is likely to have the biggest impact in terms of splash distribution and provides a worst - case scenario for cleaning and disinfection following vomiting . this study describes the development of a novel simulated vomiting system ( vomiting larry ) to simulate projectile vomiting . this system would then enable research into environmental contamination and aid development and implementation of infection control practices . in order to develop a simulated vomiting system , key pieces of information regarding vomiting were required . these were typical distances travelled by ejected vomitus fluid , the flow rate or intragastric pressure exerted during an episode of projectile vomiting , and the typical volumes of fluid produced . one study by brimacombe and keller ( 2006 ) established that a patient who had an episode of projectile vomiting during the testing of a laryngeal mask airway achieved a distance of 1.2 metres . evidence relating to the velocity or flow rate of emitted fluid during vomiting was not found . one study by iqbal et al ( 2008 ) showed that intragastric pressure changes during a range of activities including vomiting . this study used a transnasally placed manometry catheter to measure intragastric pressure and found that the maximum intragastric pressure exerted during vomiting was 38.66 kpa , with a mean of 10.93 kpa after 40 episodes of vomiting from 10 volunteers . this research was used as a starting point in the development of the model with regards to pressure for the system , in an attempt to expel the main bulk of the fluid a distance of 1.2 metres . the volume of fluid produced through projectile vomiting is likely to vary from person to person and the volume of digester , and there are limited data on volumes of vomit emitted ; none have been identified for symptomatic norovirus patients . a study by saetta and quinton ( 1991 ) on the residual gastric content after gastric lavage and ipecacuanha - induced emesis in self - poisoned patients showed that the volumes of vomitus produced from 13 ipecacuanha - treated patients varied between 0.4 and 1.35 l. based on this information , it was decided that for this study , a volume of 1 l would be used . the simulated vomiting system was therefore required to hold 1 l of fluid , which was to be ejected in a similar fashion to that of a human during an episode of projectile vomiting . the bulk of the expelled fluid was to land 1.2 m from the outlet source possibly using a pressure of 10.93 kpa . projectile vomiting can occur without warning , and thus the simulated vomiting system was designed to represent a person projectile vomiting whilst in a standing position . the stomach of the system encompassed a plastic cylinder 3 mm thick , 95 mm in diameter to the outer edge and 300 mm in length to comfortably hold 1 l of fluid . the base of the cylinder was attached to a double acting actuator ( pneumatic ram , pra/182063/m/250 , norgren ltd . ) , which would operate in a piston pump fashion to force the fluid out of the cylinder by forcing compressed air in at the base of the actuator to push the piston up the shaft of the cylinder to force the fluid out . a stainless steel disc ( 91.5 mm diameter 15 mm thick ) was attached to the top of the piston head , i.e. at the top of the actuator . a rubber seal ( 6 mm thick ) was placed around the edge of the disc to ensure a snug fit into the cylinder and prevent fluid ( i.e. simulated vomit ) leaking from the cylinder into the actuator . two stainless steel cuffs ( 10 mm thick ) were located 100 mm from the top and bottom of the cylinder for support during simulated vomiting . the base and top of the cylinder were fitted with stainless steel plates ( 120 mm 120 mm 20 mm ) . the base plate comprised a centrally located hole ( 65 mm diameter ) to allow the piston of the actuator to move freely allowing the piston head to move up and down the cylinder . the top plate contained a 120 mm long stainless steel outlet tube with an aperture ( 20 mm outer diameter ) that would be connected to a representative oesophagus . the steel cap also had a tubeless 20 mm aperture to allow the cylinder to be filled with fluid . once the cylinder was filled with fluid the aperture would be closed by means of a screw fixture . the cylinder and actuator were fixed to a stainless steel plate ( 102 mm 935 mm 25 mm ) to support the system during the simulated vomiting process . an authentic manikin head ( airway larry , simulaids , inc . ) was used for the head of the simulated vomiting system . airway larry is an adult airways management trainer , which simulates non - anaesthetised patients . the manikin has realistic anatomy and key structures including teeth , tongue , oral and nasal pharynx , larynx , epiglottis , arytenoids , false cords , true vocal cords , trachea , lungs , oesophagus and stomach . the lungs and stomach in airway larry were plastic attachments and not necessary for this research . the ends of the tubes representing the primary bronchi were blocked with 16 mm diameter stainless steel blanks fixed in place by hose clips . the tube representing the oesophagus from the manikin head was attached to the outlet tube on top of the cylinder and fixed by means of a hose clip . the piston pump and manikin head were then mounted to a wooden frame made from 18 mm thick plywood , which allowed the whole unit to be freestanding . on completion , the simulated vomiting system ( now termed vomiting larry ) stood 1.6 m from the floor to the top of the manikin head ( figure 1 ) . the simulated vomiting system ( vomiting larry ) b : blocked off bronchioles of airway larry c : outlet pipe connected to airway larry d : cylinder containing 1 l of fluid f : air inlet to push piston down h : air inlet to push piston up the simulated vomiting system was set up in a controlled atmosphere chamber ( cac ) . this chamber is a sealed 35 m unit with air inlet ducts in the ceiling and hepa - filtered air outlet air ducts in the floor below walking level . the walking floor level is a raised grid bed several inches above the air outlet ducts . the cac s air handling is controlled remotely and incorporates a temperature and relative humidity regulation system , which can be altered as required . in this instance the air handling was set to standard room temperature ( 25c ) and relative humidity ( 40% ) . the air handling system was then switched off and the cac isolated via damper closure prior to vomiting simulation so as to prevent any air movement from altering the distribution of the spray of droplets and aerosols produced from the system . in order to assess the distance travelled by the expelled fluid the cac floor was covered with safety vinyl followed by two layers of plastic sheeting , which were taped to the base of the walls of the cac so as to contain the fluid . a grid system was then prepared using masking tape to mark out 20 cm 20 cm squares . the simulated vomiting system was positioned mid - way along one wall of the chamber , facing into the chamber , which allowed an area of 3.1 m 2.6 m into which the fluid would be expelled . plastic tubing ( 5 mm inner diameter , 1.5 mm thick ) was connected to the laboratory plant air supply through a pressure regulator ( p3hea12esmbngb standard filter / regulator , parker hannifin corp ) and solenoid valve ( 5/2 mechanical valve port series vfm 350 , smc ) and finally attached to the actuator air inlet ports on the simulated vomiting system . to enable visibility of small splashes of projected fluid , 1 g of fluorescent powder ( tinopal , ciba inc . ) three tripods each mounting four black light uv bulbs ( t8 18w 600 mm , philips ) housed within uv filter cases ( custom made mug-2 uv filters , schott ag manufacturers ) were set up in three corners of the cac . once set up , the actuator was triggered via the solenoid valve switch , which forced air under the base of the piston pushing it up the length of the cylinder forcing fluid into the oesophageal tube of airway larry and out of the mouth . as the only published information on vomiting was possible volume ( 1 l , saetta and quinton , 1991 ) , potential distance covered ( 1.2 m , brimacombe and keller , 2006 ) and intragastric pressure required ( 10.93 kpa , iqbal et al , 2008 ) , several simulated vomiting trials were undertaken in order to ascertain the actual amount of pressure required for the bulk of the fluid to travel a distance of 1.2 m. post - simulated vomiting , the cac was left for 15 minutes to allow any aerosols produced to settle out before entering the chamber to photograph the spread of fluid across the grid floor . once this distance had been achieved , further experiments were undertaken to visually assess the extent of the spread of splash and droplets . the visual experiments were repeated in triplicate and photographs taken to identify distance of spread . projectile vomiting can occur without warning , and thus the simulated vomiting system was designed to represent a person projectile vomiting whilst in a standing position . the stomach of the system encompassed a plastic cylinder 3 mm thick , 95 mm in diameter to the outer edge and 300 mm in length to comfortably hold 1 l of fluid . the base of the cylinder was attached to a double acting actuator ( pneumatic ram , pra/182063/m/250 , norgren ltd . ) , which would operate in a piston pump fashion to force the fluid out of the cylinder by forcing compressed air in at the base of the actuator to push the piston up the shaft of the cylinder to force the fluid out . a stainless steel disc ( 91.5 mm diameter 15 mm thick ) was attached to the top of the piston head , i.e. at the top of the actuator . a rubber seal ( 6 mm thick ) was placed around the edge of the disc to ensure a snug fit into the cylinder and prevent fluid ( i.e. simulated vomit ) leaking from the cylinder into the actuator . two stainless steel cuffs ( 10 mm thick ) were located 100 mm from the top and bottom of the cylinder for support during simulated vomiting . the base and top of the cylinder were fitted with stainless steel plates ( 120 mm 120 mm 20 mm ) . the base plate comprised a centrally located hole ( 65 mm diameter ) to allow the piston of the actuator to move freely allowing the piston head to move up and down the cylinder . the top plate contained a 120 mm long stainless steel outlet tube with an aperture ( 20 mm outer diameter ) that would be connected to a representative oesophagus . the steel cap also had a tubeless 20 mm aperture to allow the cylinder to be filled with fluid . once the cylinder was filled with fluid the aperture would be closed by means of a screw fixture . the cylinder and actuator were fixed to a stainless steel plate ( 102 mm 935 mm 25 mm ) to support the system during the simulated vomiting process . an authentic manikin head ( airway larry , simulaids , inc . ) was used for the head of the simulated vomiting system . airway larry is an adult airways management trainer , which simulates non - anaesthetised patients . the manikin has realistic anatomy and key structures including teeth , tongue , oral and nasal pharynx , larynx , epiglottis , arytenoids , false cords , true vocal cords , trachea , lungs , oesophagus and stomach . the lungs and stomach in airway larry were plastic attachments and not necessary for this research . the ends of the tubes representing the primary bronchi were blocked with 16 mm diameter stainless steel blanks fixed in place by hose clips . the tube representing the oesophagus from the manikin head was attached to the outlet tube on top of the cylinder and fixed by means of a hose clip . the piston pump and manikin head were then mounted to a wooden frame made from 18 mm thick plywood , which allowed the whole unit to be freestanding . on completion , the simulated vomiting system ( now termed vomiting larry ) stood 1.6 m from the floor to the top of the manikin head ( figure 1 ) . the simulated vomiting system ( vomiting larry ) b : blocked off bronchioles of airway larry c : outlet pipe connected to airway larry d : cylinder containing 1 l of fluid f : air inlet to push piston down h : air inlet to push piston up the simulated vomiting system was set up in a controlled atmosphere chamber ( cac ) . this chamber is a sealed 35 m unit with air inlet ducts in the ceiling and hepa - filtered air outlet air ducts in the floor below walking level . the walking floor level is a raised grid bed several inches above the air outlet ducts . the cac s air handling is controlled remotely and incorporates a temperature and relative humidity regulation system , which can be altered as required . in this instance the air handling was set to standard room temperature ( 25c ) and relative humidity ( 40% ) . the air handling system was then switched off and the cac isolated via damper closure prior to vomiting simulation so as to prevent any air movement from altering the distribution of the spray of droplets and aerosols produced from the system . in order to assess the distance travelled by the expelled fluid the cac floor was covered with safety vinyl followed by two layers of plastic sheeting , which were taped to the base of the walls of the cac so as to contain the fluid . a grid system was then prepared using masking tape to mark out 20 cm 20 cm squares . the simulated vomiting system was positioned mid - way along one wall of the chamber , facing into the chamber , which allowed an area of 3.1 m 2.6 m into which the fluid would be expelled . plastic tubing ( 5 mm inner diameter , 1.5 mm thick ) was connected to the laboratory plant air supply through a pressure regulator ( p3hea12esmbngb standard filter / regulator , parker hannifin corp ) and solenoid valve ( 5/2 mechanical valve port series vfm 350 , smc ) and finally attached to the actuator air inlet ports on the simulated vomiting system . to enable visibility of small splashes of projected fluid , 1 g of fluorescent powder ( tinopal , ciba inc . ) three tripods each mounting four black light uv bulbs ( t8 18w 600 mm , philips ) housed within uv filter cases ( custom made mug-2 uv filters , schott ag manufacturers ) were set up in three corners of the cac . once set up , the actuator was triggered via the solenoid valve switch , which forced air under the base of the piston pushing it up the length of the cylinder forcing fluid into the oesophageal tube of airway larry and out of the mouth . as the only published information on vomiting was possible volume ( 1 l , saetta and quinton , 1991 ) , potential distance covered ( 1.2 m , brimacombe and keller , 2006 ) and intragastric pressure required ( 10.93 kpa , iqbal et al , 2008 ) , several simulated vomiting trials were undertaken in order to ascertain the actual amount of pressure required for the bulk of the fluid to travel a distance of 1.2 m. post - simulated vomiting , the cac was left for 15 minutes to allow any aerosols produced to settle out before entering the chamber to photograph the spread of fluid across the grid floor . once this distance had been achieved , further experiments were undertaken to visually assess the extent of the spread of splash and droplets . the visual experiments were repeated in triplicate and photographs taken to identify distance of spread . a simulated vomiting system ( vomiting larry ) was developed successfully ( figure 1 ) and used to assess the amount to which the local environment is contaminated after an episode of projectile vomiting . the initial 10.33 kpa intragastric pressure described by iqbal et al , ( 2008 ) was insufficient to move the piston of the actuator . considerable air pressure ( 800 kpa ) was required for this simulated projectile vomiting system to expel the main bulk of the fluid ( 1 l ) a distance of 1.2 m from the source , i.e. mouth of the system . film footage and photographs taken from three simulated projectile vomiting experiments carried out using 1 l of water ( plus fluorescent marker ) and 800 kpa of pressure to eject the bulk fluid 1.2 m highlighted fluid dynamics during vomiting , splash upon impact with the floor and total distance travelled by droplets . many droplets were released from vomiting larry prior to the appearance of the main bulk of the fluid ( figure 2a ) , which were projected forwards and outwards from the orifice . as the main bulk of the fluid exited the mouth , strings of fluid and droplets , some of which were directed back towards the system ( figure 2b ) . droplets tended to fall out as they lost momentum , which created a cloud of droplets beneath and around the main flow of the fluid ( figure 2b ) . during mid - flow , the bulk fluid appeared more directional , with less droplet formation ( figure 2c and d ) . towards the end of the simulated vomiting episode , the fluid travelled less far before fallout occurred , as the pressure decreased ( figure 2e ) . images ( a-e ) are sequential photographs of simulated vomiting taken 10 ms apart under uv light . a : release of droplets prior to main bulk fluid b : strings of fluid and droplet fallout d : less droplet formation e : fluid travelling less far towards end of simulation figure 3 highlights fluid rebound from the floor after the initial impact . splash and droplets were created by the rebounding fluid , which spread further still from the main impact site and/or collided with the contra flow of fluid still being projected from the mouth of the system to the floor . impacted fluid continued to move during and after simulated vomiting ( figure 3a ) . once settled , the main bulk of the fluid filled an area 1.2 m 1.6 m. for each experiment splash was identified on the wall directly opposite the front of the system and on the two adjacent walls covering a total distance > 3 m longitudinal spread and > 2.2 m lateral spread . the full extent to which the fluid was distributed is illustrated in figure 4 . examination of the expelled fluid on the floor of the cac revealed that only the main bulk of the liquid and major splashes were visible under standard white lighting . rebounding fluid ( highlighted at a ) from the initial impact during simulated vomiting ( scale : grid sections = 20 cm x 20 cm ) creates widespread splash . a : rebounding splash upon fluid impact with floor surface spread of splash post - simulated vomiting ( scale : grid sections = 20 cm x 20 cm ) this study describes the development of a novel simulated vomiting system ( vomiting larry ) , which will be used to assess further the contamination of the local environment after an episode of vomiting . this system can be used as a training aid for cleaning and disinfection practices as part of an infection prevention and control strategy . modifications to vomiting larry would also allow this system to simulate coughing and sneezing that could be used to investigate routes of transmission from individuals infected with a variety of pathogens ultimately increasing our knowledge of infection and its control . the development of vomiting larry has shown that splashes and droplets produced as a result of simulated projectile vomiting can travel a considerable distance . given that much of the smaller splashes and droplets were not visible under standard white lighting , it would be difficult to clean up all potentially contaminated splashes from surfaces that have become contaminated after an episode of vomiting from a symptomatic patient infected with an infectious agent such as norovirus solely based on visual observation of area affected . in confined spaces contamination will spread to vertical surfaces away from the obvious source of contamination . therefore , members of staff involved in cleaning surfaces after vomiting has occurred need to be aware of the likely surface area of contamination . it has been shown that many infectious particles can be contained within vomit ( caul , 1995 ) . vomiting larry could be employed to assess the true extent of dissemination of pathogens into the local environment through vomiting , by means of adding model surrogates of infectious agent such as norovirus to the system . this would demonstrate how well the agent survives the vomiting process , e.g. shearing forces and drying out , as well as identifying how far the viable agent could spread and whether it is in sufficient concentrations to be infectious upon contact with a susceptible host . information gathered from future research could then be built into a fluid model to highlight the dynamics of vomitus spread and associated pathogens as well as its impact in a range of settings . this study used 1 l of water as a vomitus substitute based on the research by saetta and quinton ( 1991 ) to allow for a worst - case scenario in terms of fluid spread due to being a lower density than vomit . it also utilised safety vinyl floor material covered with two layers of plastic sheeting , which would likely create a larger amount of splash and possibly achieve greater distances of travel compared with softer surfaces such as carpet . vomiting larry could be employed in further research to compare the spread of expelled fluid of differing volumes and viscosities as well as to assess differences in the amount of spread from rebounding droplets and splash when landing on alternative surfaces such as carpet . another key consideration in terms of environmental contamination and subsequent infection transmission is body position of the emetic individual , who may be lying down , bent over , kneeling , sitting , or standing . studies have described cases of infectious diseases being spread through vomiting in various settings such as restaurants and aeroplanes for instance where close proximity obstacles such as tables , seats and even fellow human beings would alter the trajectory of the vomitus flow ( widdowson et al , 2005 ; baker et al , 2010 ) . furniture and manikins could also be added into the cac to investigate how such obstacles affect the spread of projected fluid and therefore how this might affect clean up procedures and disease transmission . from the images and film footage gathered in this current study , simulated vomiting produced droplets and potentially aerosols . mathematical models based on epidemiological research have shown that norovirus transmission is possible via inhalation of infectious aerosols produced during an episode of vomiting from an infected individual ( leung et al , 2006 ) . the inhaled aerosols are then ingested by the susceptible host so as to elicit infection . the size and numbers of droplets and aerosols produced from vomiting , including projectile vomiting , remains unknown . a particle counter could be used to measure the numbers and size range of particles generated during simulated projectile vomiting . this would achieve a better understanding of the potential for infection transmission from infectious aerosols . the generally accepted process by which the body releases vomitus fluid from the stomach is by increasing the intragastric pressure through simultaneous contraction of the thoracic and abdominal muscles ( guyton and hall , 2011 ) . this essentially creates a pressure vessel of the stomach contents before the oesophageal sphincter opens to allow the release of stomach contents into the oesophagus and out of the mouth . this differs slightly from the piston pump arrangement of the simulator , which pushed the fluid out of the stomach into the oesophagus and out of the mouth . this mechanistic difference may be the reason for the considerable 800 kpa pressure requirement of vomiting larry to project fluid a distance of 1.2 m compared with that noted by iqbal et al ( 2008 ) . this system could be altered to incorporate a specialised and certified pressure vessel that specifically represented intragastric pressure of the stomach and contents as created by the thoracic and abdominal muscles , therefore more closely representing human physiology . in the system developed here , it is likely that increased pressure was required to overcome the mechanical weight and friction within the system , i.e. the weight of the piston , and friction created by the stainless steel piston rubbing against the inner walls of the plastic cylinder ( representative stomach ) . stiction , that is , the force required to move the fluid through the system ( comprised of a plastic cylinder stomach , and stainless steel and rubber oesophagus ) is also likely to be greater than that of the natural human system where the stomach , oesophageal and oral epidermal surfaces are flexible and well lubricated . it is also worth noting that although 800 kpa of pressure was measured at the supply source , pressure losses would occur through the system before reaching the piston . nonetheless , the simulated vomiting system ( vomiting larry ) described here successfully expelled 1 l of fluid 1.2 m as reported in the literature ( saetta and quinton , 1991 ; brimacombe and keller , 2006 ) and enabled the examination of the extent to which the local environment becomes contaminated after an episode of projectile vomiting .

infectious diseases such as norovirus can induce emesis ( vomiting ) , which can be of a projectile nature . although studies have been carried out on transmission , prevalence and decontamination of such micro - organisms within various environments , little is known about the extent to which the surrounding environment is contaminated when an individual vomits . this is an important consideration for infection control purposes . the aim of this study was to develop a simulated vomiting system ( vomiting larry ) to be used for assessing the extent to which projected fluid can contaminate the environment . vomiting larry was set up within a controlled atmosphere chamber ( cac ) facility at the health and safety laboratory ( hsl ) . simulated vomiting was undertaken using water as a vomitus substitute containing a fluorescent marker enabling small splashes , ordinarily missed , to be visualised using uv lighting . experiments revealed that splashes and droplets produced during an episode of projectile vomiting can travel great distances ( > 3 m forward spread and 2.6 m lateral spread ) . the research highlighted that small droplets can be hard to see and therefore cleaning all contaminated surfaces is difficult to achieve . evidence from this study suggests that areas of at least 7.8 m2 should be decontaminated following an episode of projectile vomiting .

Introduction Methods Development of Vomiting Larry Simulated projectile vomiting Results Discussion

the act of vomiting by an individual infected with an infectious disease such as norovirus disseminates the agent into the local environment . electron microscopic studies have suggested that as many as 310 norovirus particles could be distributed into the immediate surroundings following an episode of vomiting , based on a bolus of 30 ml i.e. the robust nature of many infectious agents allows them to survive in the environment for protracted periods of time , particularly when associated with organic matter such as skin squames , faeces and vomit . this increases the likelihood of infection transmission if they are not removed from the environment . cleaning and decontamination of surfaces contaminated with an infectious agent post - emesis is therefore an important aspect of infection control . research relating to the physiology of vomiting has been carried out , although most published studies relating to the mechanics of vomiting date back to the early 1900s . such studies have demonstrated that the mechanism of emesis in humans and other mammals is complex ( lumsden and holden , 1969 ) and is still controversial ( pickering and jones , 2002 ) . projectile vomiting ( sometimes referred to as forced vomiting ) often occurs suddenly and without warning , i.e. consequently an individual may have an episode of projectile vomiting at any time , e.g. based on the widely accepted text by guyton and hall ( 2011 ) , vomiting is initiated via a deep inspiratory breath , which allows the upper oesophageal sphincter to open , closing of the glottis and posterior nares , as well as a strong downward contraction of the diaphragm . the lower oesophageal sphincter at the top of the stomach then relaxes , as does the perioesophageal ( crural ) portion of the diaphragm required to release the fluid into the oesophagus and out of the mouth ( guyton and hall , 2011 ) . many early studies have shown reasonably conclusively that the role of the stomach wall during emesis is minimal ( haskell , 1924 ) . projectile vomiting is likely to have the biggest impact in terms of splash distribution and provides a worst - case scenario for cleaning and disinfection following vomiting . this study describes the development of a novel simulated vomiting system ( vomiting larry ) to simulate projectile vomiting . this system would then enable research into environmental contamination and aid development and implementation of infection control practices . in order to develop a simulated vomiting system , key pieces of information regarding vomiting were required . these were typical distances travelled by ejected vomitus fluid , the flow rate or intragastric pressure exerted during an episode of projectile vomiting , and the typical volumes of fluid produced . one study by brimacombe and keller ( 2006 ) established that a patient who had an episode of projectile vomiting during the testing of a laryngeal mask airway achieved a distance of 1.2 metres . evidence relating to the velocity or flow rate of emitted fluid during vomiting was not found . this study used a transnasally placed manometry catheter to measure intragastric pressure and found that the maximum intragastric pressure exerted during vomiting was 38.66 kpa , with a mean of 10.93 kpa after 40 episodes of vomiting from 10 volunteers . this research was used as a starting point in the development of the model with regards to pressure for the system , in an attempt to expel the main bulk of the fluid a distance of 1.2 metres . the volume of fluid produced through projectile vomiting is likely to vary from person to person and the volume of digester , and there are limited data on volumes of vomit emitted ; none have been identified for symptomatic norovirus patients . a study by saetta and quinton ( 1991 ) on the residual gastric content after gastric lavage and ipecacuanha - induced emesis in self - poisoned patients showed that the volumes of vomitus produced from 13 ipecacuanha - treated patients varied between 0.4 and 1.35 l. based on this information , it was decided that for this study , a volume of 1 l would be used . the simulated vomiting system was therefore required to hold 1 l of fluid , which was to be ejected in a similar fashion to that of a human during an episode of projectile vomiting . the bulk of the expelled fluid was to land 1.2 m from the outlet source possibly using a pressure of 10.93 kpa . projectile vomiting can occur without warning , and thus the simulated vomiting system was designed to represent a person projectile vomiting whilst in a standing position . , which would operate in a piston pump fashion to force the fluid out of the cylinder by forcing compressed air in at the base of the actuator to push the piston up the shaft of the cylinder to force the fluid out . at the top of the actuator . two stainless steel cuffs ( 10 mm thick ) were located 100 mm from the top and bottom of the cylinder for support during simulated vomiting . the base plate comprised a centrally located hole ( 65 mm diameter ) to allow the piston of the actuator to move freely allowing the piston head to move up and down the cylinder . the steel cap also had a tubeless 20 mm aperture to allow the cylinder to be filled with fluid . once the cylinder was filled with fluid the aperture would be closed by means of a screw fixture . the cylinder and actuator were fixed to a stainless steel plate ( 102 mm 935 mm 25 mm ) to support the system during the simulated vomiting process . was used for the head of the simulated vomiting system . airway larry is an adult airways management trainer , which simulates non - anaesthetised patients . the tube representing the oesophagus from the manikin head was attached to the outlet tube on top of the cylinder and fixed by means of a hose clip . the piston pump and manikin head were then mounted to a wooden frame made from 18 mm thick plywood , which allowed the whole unit to be freestanding . on completion , the simulated vomiting system ( now termed vomiting larry ) stood 1.6 m from the floor to the top of the manikin head ( figure 1 ) . the simulated vomiting system ( vomiting larry ) b : blocked off bronchioles of airway larry c : outlet pipe connected to airway larry d : cylinder containing 1 l of fluid f : air inlet to push piston down h : air inlet to push piston up the simulated vomiting system was set up in a controlled atmosphere chamber ( cac ) . the cac s air handling is controlled remotely and incorporates a temperature and relative humidity regulation system , which can be altered as required . in this instance the air handling was set to standard room temperature ( 25c ) and relative humidity ( 40% ) . in order to assess the distance travelled by the expelled fluid the cac floor was covered with safety vinyl followed by two layers of plastic sheeting , which were taped to the base of the walls of the cac so as to contain the fluid . the simulated vomiting system was positioned mid - way along one wall of the chamber , facing into the chamber , which allowed an area of 3.1 m 2.6 m into which the fluid would be expelled . plastic tubing ( 5 mm inner diameter , 1.5 mm thick ) was connected to the laboratory plant air supply through a pressure regulator ( p3hea12esmbngb standard filter / regulator , parker hannifin corp ) and solenoid valve ( 5/2 mechanical valve port series vfm 350 , smc ) and finally attached to the actuator air inlet ports on the simulated vomiting system . to enable visibility of small splashes of projected fluid , 1 g of fluorescent powder ( tinopal , ciba inc . ) three tripods each mounting four black light uv bulbs ( t8 18w 600 mm , philips ) housed within uv filter cases ( custom made mug-2 uv filters , schott ag manufacturers ) were set up in three corners of the cac . once set up , the actuator was triggered via the solenoid valve switch , which forced air under the base of the piston pushing it up the length of the cylinder forcing fluid into the oesophageal tube of airway larry and out of the mouth . as the only published information on vomiting was possible volume ( 1 l , saetta and quinton , 1991 ) , potential distance covered ( 1.2 m , brimacombe and keller , 2006 ) and intragastric pressure required ( 10.93 kpa , iqbal et al , 2008 ) , several simulated vomiting trials were undertaken in order to ascertain the actual amount of pressure required for the bulk of the fluid to travel a distance of 1.2 m. post - simulated vomiting , the cac was left for 15 minutes to allow any aerosols produced to settle out before entering the chamber to photograph the spread of fluid across the grid floor . once this distance had been achieved , further experiments were undertaken to visually assess the extent of the spread of splash and droplets . projectile vomiting can occur without warning , and thus the simulated vomiting system was designed to represent a person projectile vomiting whilst in a standing position . , which would operate in a piston pump fashion to force the fluid out of the cylinder by forcing compressed air in at the base of the actuator to push the piston up the shaft of the cylinder to force the fluid out . at the top of the actuator . two stainless steel cuffs ( 10 mm thick ) were located 100 mm from the top and bottom of the cylinder for support during simulated vomiting . the base plate comprised a centrally located hole ( 65 mm diameter ) to allow the piston of the actuator to move freely allowing the piston head to move up and down the cylinder . once the cylinder was filled with fluid the aperture would be closed by means of a screw fixture . the cylinder and actuator were fixed to a stainless steel plate ( 102 mm 935 mm 25 mm ) to support the system during the simulated vomiting process . was used for the head of the simulated vomiting system . airway larry is an adult airways management trainer , which simulates non - anaesthetised patients . the tube representing the oesophagus from the manikin head was attached to the outlet tube on top of the cylinder and fixed by means of a hose clip . the piston pump and manikin head were then mounted to a wooden frame made from 18 mm thick plywood , which allowed the whole unit to be freestanding . on completion , the simulated vomiting system ( now termed vomiting larry ) stood 1.6 m from the floor to the top of the manikin head ( figure 1 ) . the simulated vomiting system ( vomiting larry ) b : blocked off bronchioles of airway larry c : outlet pipe connected to airway larry d : cylinder containing 1 l of fluid f : air inlet to push piston down h : air inlet to push piston up the simulated vomiting system was set up in a controlled atmosphere chamber ( cac ) . the cac s air handling is controlled remotely and incorporates a temperature and relative humidity regulation system , which can be altered as required . in this instance the air handling was set to standard room temperature ( 25c ) and relative humidity ( 40% ) . in order to assess the distance travelled by the expelled fluid the cac floor was covered with safety vinyl followed by two layers of plastic sheeting , which were taped to the base of the walls of the cac so as to contain the fluid . the simulated vomiting system was positioned mid - way along one wall of the chamber , facing into the chamber , which allowed an area of 3.1 m 2.6 m into which the fluid would be expelled . plastic tubing ( 5 mm inner diameter , 1.5 mm thick ) was connected to the laboratory plant air supply through a pressure regulator ( p3hea12esmbngb standard filter / regulator , parker hannifin corp ) and solenoid valve ( 5/2 mechanical valve port series vfm 350 , smc ) and finally attached to the actuator air inlet ports on the simulated vomiting system . to enable visibility of small splashes of projected fluid , 1 g of fluorescent powder ( tinopal , ciba inc . ) three tripods each mounting four black light uv bulbs ( t8 18w 600 mm , philips ) housed within uv filter cases ( custom made mug-2 uv filters , schott ag manufacturers ) were set up in three corners of the cac . once set up , the actuator was triggered via the solenoid valve switch , which forced air under the base of the piston pushing it up the length of the cylinder forcing fluid into the oesophageal tube of airway larry and out of the mouth . as the only published information on vomiting was possible volume ( 1 l , saetta and quinton , 1991 ) , potential distance covered ( 1.2 m , brimacombe and keller , 2006 ) and intragastric pressure required ( 10.93 kpa , iqbal et al , 2008 ) , several simulated vomiting trials were undertaken in order to ascertain the actual amount of pressure required for the bulk of the fluid to travel a distance of 1.2 m. post - simulated vomiting , the cac was left for 15 minutes to allow any aerosols produced to settle out before entering the chamber to photograph the spread of fluid across the grid floor . once this distance had been achieved , further experiments were undertaken to visually assess the extent of the spread of splash and droplets . a simulated vomiting system ( vomiting larry ) was developed successfully ( figure 1 ) and used to assess the amount to which the local environment is contaminated after an episode of projectile vomiting . considerable air pressure ( 800 kpa ) was required for this simulated projectile vomiting system to expel the main bulk of the fluid ( 1 l ) a distance of 1.2 m from the source , i.e. film footage and photographs taken from three simulated projectile vomiting experiments carried out using 1 l of water ( plus fluorescent marker ) and 800 kpa of pressure to eject the bulk fluid 1.2 m highlighted fluid dynamics during vomiting , splash upon impact with the floor and total distance travelled by droplets . many droplets were released from vomiting larry prior to the appearance of the main bulk of the fluid ( figure 2a ) , which were projected forwards and outwards from the orifice . as the main bulk of the fluid exited the mouth , strings of fluid and droplets , some of which were directed back towards the system ( figure 2b ) . droplets tended to fall out as they lost momentum , which created a cloud of droplets beneath and around the main flow of the fluid ( figure 2b ) . towards the end of the simulated vomiting episode , the fluid travelled less far before fallout occurred , as the pressure decreased ( figure 2e ) . splash and droplets were created by the rebounding fluid , which spread further still from the main impact site and/or collided with the contra flow of fluid still being projected from the mouth of the system to the floor . impacted fluid continued to move during and after simulated vomiting ( figure 3a ) . once settled , the main bulk of the fluid filled an area 1.2 m 1.6 m. for each experiment splash was identified on the wall directly opposite the front of the system and on the two adjacent walls covering a total distance > 3 m longitudinal spread and > 2.2 m lateral spread . the full extent to which the fluid was distributed is illustrated in figure 4 . examination of the expelled fluid on the floor of the cac revealed that only the main bulk of the liquid and major splashes were visible under standard white lighting . rebounding fluid ( highlighted at a ) from the initial impact during simulated vomiting ( scale : grid sections = 20 cm x 20 cm ) creates widespread splash . a : rebounding splash upon fluid impact with floor surface spread of splash post - simulated vomiting ( scale : grid sections = 20 cm x 20 cm ) this study describes the development of a novel simulated vomiting system ( vomiting larry ) , which will be used to assess further the contamination of the local environment after an episode of vomiting . this system can be used as a training aid for cleaning and disinfection practices as part of an infection prevention and control strategy . modifications to vomiting larry would also allow this system to simulate coughing and sneezing that could be used to investigate routes of transmission from individuals infected with a variety of pathogens ultimately increasing our knowledge of infection and its control . the development of vomiting larry has shown that splashes and droplets produced as a result of simulated projectile vomiting can travel a considerable distance . given that much of the smaller splashes and droplets were not visible under standard white lighting , it would be difficult to clean up all potentially contaminated splashes from surfaces that have become contaminated after an episode of vomiting from a symptomatic patient infected with an infectious agent such as norovirus solely based on visual observation of area affected . it has been shown that many infectious particles can be contained within vomit ( caul , 1995 ) . vomiting larry could be employed to assess the true extent of dissemination of pathogens into the local environment through vomiting , by means of adding model surrogates of infectious agent such as norovirus to the system . shearing forces and drying out , as well as identifying how far the viable agent could spread and whether it is in sufficient concentrations to be infectious upon contact with a susceptible host . this study used 1 l of water as a vomitus substitute based on the research by saetta and quinton ( 1991 ) to allow for a worst - case scenario in terms of fluid spread due to being a lower density than vomit . it also utilised safety vinyl floor material covered with two layers of plastic sheeting , which would likely create a larger amount of splash and possibly achieve greater distances of travel compared with softer surfaces such as carpet . vomiting larry could be employed in further research to compare the spread of expelled fluid of differing volumes and viscosities as well as to assess differences in the amount of spread from rebounding droplets and splash when landing on alternative surfaces such as carpet . studies have described cases of infectious diseases being spread through vomiting in various settings such as restaurants and aeroplanes for instance where close proximity obstacles such as tables , seats and even fellow human beings would alter the trajectory of the vomitus flow ( widdowson et al , 2005 ; baker et al , 2010 ) . furniture and manikins could also be added into the cac to investigate how such obstacles affect the spread of projected fluid and therefore how this might affect clean up procedures and disease transmission . from the images and film footage gathered in this current study , simulated vomiting produced droplets and potentially aerosols . mathematical models based on epidemiological research have shown that norovirus transmission is possible via inhalation of infectious aerosols produced during an episode of vomiting from an infected individual ( leung et al , 2006 ) . a particle counter could be used to measure the numbers and size range of particles generated during simulated projectile vomiting . this would achieve a better understanding of the potential for infection transmission from infectious aerosols . the generally accepted process by which the body releases vomitus fluid from the stomach is by increasing the intragastric pressure through simultaneous contraction of the thoracic and abdominal muscles ( guyton and hall , 2011 ) . stiction , that is , the force required to move the fluid through the system ( comprised of a plastic cylinder stomach , and stainless steel and rubber oesophagus ) is also likely to be greater than that of the natural human system where the stomach , oesophageal and oral epidermal surfaces are flexible and well lubricated . nonetheless , the simulated vomiting system ( vomiting larry ) described here successfully expelled 1 l of fluid 1.2 m as reported in the literature ( saetta and quinton , 1991 ; brimacombe and keller , 2006 ) and enabled the examination of the extent to which the local environment becomes contaminated after an episode of projectile vomiting .

today , the application of information technology ( it ) has expanded rapidly in order to increase efficiency and productivity in most areas . research reveals high power of new it in increasing individuals health - related knowledge levels . at present information is data which has taken quantity matter in order to adapt with objectives and connect to work . the organization should recognize necessary information for work management ; otherwise , it will face a mass of confusing data . recognizing main axes of advance in organizations requires changes of it role from implicit into the objective or identifying its exact impacts to be able to use it in organization 's decisions . this is in fact related to the organization 's performance improvement or correction which can be dependent on it ; it provides managers an opportunity to control and co - ordinate complex structures using quick information processing which eventually leads to coherent and rapid management and better organizational performance . investment and identification of organizational performance based on information and the resulting products , is a new acceleration which is considered as the basis for mass organizational movement ; perhaps one of the most obvious results in this area is observed in employees faster and smoother access to required information which has been considered in most researches connected to information . today this is possible through using computers and some parts of middle management and leadership responsibilities . this way , employees will have access to more information and do their duties with a higher speed . with the advancement of science and technology in the field of computer and information systems , another significant point is the impact and role of human resources and organizational staff that are among the most critical organizational resources . therefore , for structural adaptation with information transformation and achievement of valuable and useful results in this area , human resources development is necessary in order to adapt readily to the outcomes of this era . capable , experienced , influential scientists and specialists are required to meet the needs of this age . undoubtedly , education and measurement of abilities will increase the rate of achievement toward expected results from application of it . access to information as an effective and critical tool and acquiring central and strategic information is an advantage in every organization , because higher , faster , more appropriate and efficient production depends on the use of information and its proper turnover . this is what majority of today 's organizations has defined as their working priority ; on the other hand , when managers equip organizational members with more information those people will feel capable and more likely to work with productivity and prosperity along with management demands . more information encourages individuals to experience self - order , self - control and higher confidence . obviously , the only possible way to deal with such challenges is employee empowerment , which means their development in all aspects and possible fields to be able to do all major tasks . in the present age , empowerment is known as a tool that enables managers to run organizations with various features such as different influential channels , development , reliance on horizontal and network structures , minimum distance between employees and managers , reduction of organizational attachment and use of it . empowerment leads to capable and motivated employees and helps managers to act properly and quickly in dynamics of the competitive environment and provide competitive advantages for the organization . it provides changes in organizations through which companies and organizations are more dependent on information , knowledge , training and decision - making of staff members . regarding the application of information and communication technologies at home and school along with organizations , recreational places and other sites , with the advent of technology , not only the structure of employee changes due to altered activities or modified components , but some new jobs will also be created . therefore , it is a powerful tool for data collection and processing into information . as a result , the application of it is expanding rapidly through organizations . management is easier in organizations which have high levels of technology and new information applications , because new tools will facilitate the ability of applying information . undeniable advantages of it can be observed in higher accuracy and speed of affairs , high quality , lower costs and higher client satisfaction which has encouraged organizations to establish and use such systems in order to survive , develop and achieve their objectives while they enjoy its competitive advantages at the same time . given the important role of employees as one of the main organizational resources , there is an urgent need to propel them toward it . therefore , the main question in this research is whether the use of it can empower employees and improve their performance . however , previous comments and remarks emphasized that information is a critical organizational element in today 's world . accordingly , one of the most important ways to empower employees is through their access to information . we used a sub - systematic method , which was divided into three phases : literature collection , assessing and selection . the literature was searched with the assessing , and selection of libraries , databases ( such as proquest , pubmed , science direct and scientific information database ) and also searches engines available at google , google scholar , books and conference proceedings with keywords including it , empowerment and employees in the searching areas of titles , keywords , abstracts and full texts . the preliminary search resulted in 85 articles , books and conference proceedings in which published between 1983 and 2013 during july 2013 . due to the large number of articles retrieved , only articles written in english , american and persian languages were considered because the researchers did not dominate on other languages . after a careful analysis of the content of each paper , the study was performed in order to select the most important professional empowerment dimensions using it . the word empowerment by oxford definition means giving power or the right of doing something to someone . employee empowerment is generally a contribute process applied to utilize all capacities of employees and to encourage them to increase their commitment to their job . some people believe that empowerment is a type of decentralization which requires assigning basic decision making to subordinates . furthermore , empowerment provides decision- making powers and lets employees perform according to their desire . , empowerment is regarded as one of the useful tools in promoting employees and increasing organizational effectiveness . in order to be successful in today 's changing environment , organizations need knowledge , ideas , energy and creativity of all employees , including those at the first level to top managers . therefore , investigating methods to increase efficiency and use employees maximum capabilities toward achieving organizational objectives has been a continuous challenge for managers and management scholars ; so that in recent years empowerment has changed into a typical management term . researchers have viewed empowerment from different perspectives : some concepts such as personal work control , independence in task performance , payment system associated with performance or payment based on performance , job enrichment , employee stock ownership and so on . these different views are not purely personal , but they are techniques which theorists and administrators use in order to create an empowering environment or facilitate empowering conditions . accordingly , different definitions have been provided on empowerment . for example , kinlow defines it as a process toward continuous improvement in organizational performance , which is possible through the expansion and development of influence based on individuals and team 's qualifications whose performance affects the organization 's general performance . empowerment does nt mean giving employees the right to vote , autonomy or even motivation ; despite a strong desire to do more work will result from empowerment . the long - term goal of empowerment is continuous improvement in organization 's overall performance , whereas its short - term objective is just application of existing qualifications . armstrong quotes conger describes the empowerment as a process of reinforcing employees qualifications through identifying those conditions that have caused their weakness and trying to deal with problems through formal actions , informal techniques and also providing information which is necessary for their efficiency . armstrong quotes blak and piterz have defined empowerment as power - sharing process between people who work in an organization . various ideas about empowerment dimensions given different views toward empowerment definitions and criterion , this research has been done based on the best and most comprehensive model which is more adaptable with employees present conditions in our country . according to this model that is called ardalan model , the most important dimensions of empowerment include : employees performance improvementoccupational freedom and independenceincreased decision - making responsibilityincreased employment opportunitiesenhanced a self - controlincreased staff knowledgeprofessional development of staff . employees performance improvement occupational freedom and independence increased decision - making responsibility increased employment opportunities enhanced a self - control increased staff knowledge professional development of staff . ardalan in his master 's thesis entitled investigating the effects of it on employees occupational empowerment , said that it increases employees knowledge and awareness leading to easier and more exact communications , less expenses and decrease in human error in the information process . today this is possible through using computers and some parts of middle management and leadership responsibilities . this way , employees will have access to more information and increase their knowledge . they will also come to better results . employees empowerment : golden key for human resources management , said that having an appropriate career is one of the basic constituent elements of citizenship whose importance is not less than the presence at elections . global developmental trends such as dramatic changes in demographic transition due to immigration , the emergence of new technologies and approaches in management have provided new horizons for employment planners . it has generated new employment opportunities and brought about fundamental changes in many other jobs . in 2012 , current jobs will be challenging and rewarding professions will be created . in the future , it components will be more co - ordinated and their influence on employment status and the relation between developing and developed countries will be more evident . not only has it created new professions which need special expertise in it but also affected other jobs so that the time required acquiring knowledge will be shorter . in some cases it also leads to more limited works . moreover , professions content which identifies the amount of individual 's challenge with the organization and task performance has a significant impact on job satisfaction , creating job opportunities , job productivity and personal status of employees in organizations and society and efficiency . principles , fundamentals and theories of organizational culture and climate said that since independence affects the individuals needs to engage in decision - making and refers to influence on the control of career achievements , occupational freedom , giving comments and the right to vote , using it and especially it systems will facilitate organization 's control and supervision through which managers supervision scope that is a limitation in traditional structures will expand . this leads to an increase in the number of middle managers and experts leading to less administrative levels . these facts show that it helps employees to be more independent and free . according to gudarzi , since the need for self - control and autonomy is a top scale , it is associated with an employees desire to engage in decision - making , identify objectives and work based on self - control and autonomous methods without others supervision . employees tend to accept responsibilities and act freely and hence it leads to an inner control system based on which individuals evaluate their own performance . sarrafizadeh in his research entitled it in organizations concludes that it facilitates managers roles and responsibilities . achieving required information in order to make decisions , more control and supervision on organization and processes , the ability to analyze conditions , analysis , programing and decision simulation are among it significant impacts . moreover , less time to respond and make decisions along with task assignment to lower levels will help managers to be away from daily routines and step toward designing and creating work opportunities . higher information about the organization and related responsibilities leads to better decisions which in turn results in more commitment in decision - making . lack of information is resolved through it and decision supporting tools . moreover , some of the greatest advantages of advanced it that have provided a higher communication between managers , staff and clients include : electronic message system , administrative information system and video conference . knowledge helps employees to have professional command of the organization and if we accept the popular expression that knowledge is the ability he refers to the quality and empowerment of human resources as important factors for organizations survival ; in other words , human resources are more important than new financial technologies . it means that the main difference between organizations refers to their knowledge , not the financial status . the role of efficient , capable and knowledgeable human resources in organizational achievement is undeniable and human resources are regarded as the most important , expensive and valuable organizational assets that can create a powerful organization . a powerful organization is a place where employees co - operate in different activities ; therefore , human resources help organizations productivity and eventually affect all the society . undoubtedly , every community 's prosperity is due to the development of human resources and thus administrators paying special attention to employees progress . urei yazdi quotes veten and kemron ( 2002 ) in a study entitled empowerment and authority assignment said that performance improvement means higher productivity , better quality , less expenses , less errors , higher speed , service providing , client satisfaction and following rules and regulations . on the other hand , it leads to minimize human errors in information processing , while providing service and client satisfaction increases . mazidabadi - farahani ( 2004 ) in his research entitled investigating it applications in employees occupational empowerment in the qom social security organization " concludes that it has empowered employees in the qom social security organization . in a research entitled effective strategies in empowerment of experts in shahre kord university of medical sciences identify occupational freedom , human resources effective management , motivation promotion , self - management and organizational learning promotion as influencing factors in empowering medical experts of chaharmahal va bakhtiari province . a study ( 2002 ) in america shows that it plays a significant role in the fast data collection , global and fast access to a broad range of health information , a quick evaluation of information , better communication among health experts and more awareness through access to various information sources . mcguire in a study in 2003 , states that it facilitates and speeds up data collection , categorization , access and exchange . it also helps in efficient management of resources , decreases inappropriate reception of patients and avoids repeated works leading to less organizational expenses . the word empowerment by oxford definition means giving power or the right of doing something to someone . employee empowerment is generally a contribute process applied to utilize all capacities of employees and to encourage them to increase their commitment to their job . some people believe that empowerment is a type of decentralization which requires assigning basic decision making to subordinates . furthermore , empowerment provides decision- making powers and lets employees perform according to their desire . , empowerment is regarded as one of the useful tools in promoting employees and increasing organizational effectiveness . in order to be successful in today 's changing environment , organizations need knowledge , ideas , energy and creativity of all employees , including those at the first level to top managers . therefore , investigating methods to increase efficiency and use employees maximum capabilities toward achieving organizational objectives has been a continuous challenge for managers and management scholars ; so that in recent years empowerment has changed into a typical management term . researchers have viewed empowerment from different perspectives : some concepts such as personal work control , independence in task performance , payment system associated with performance or payment based on performance , job enrichment , employee stock ownership and so on . these different views are not purely personal , but they are techniques which theorists and administrators use in order to create an empowering environment or facilitate empowering conditions . accordingly , different definitions have been provided on empowerment . for example , kinlow defines it as a process toward continuous improvement in organizational performance , which is possible through the expansion and development of influence based on individuals and team 's qualifications whose performance affects the organization 's general performance . empowerment does nt mean giving employees the right to vote , autonomy or even motivation ; despite a strong desire to do more work will result from empowerment . the long - term goal of empowerment is continuous improvement in organization 's overall performance , whereas its short - term objective is just application of existing qualifications . armstrong quotes conger describes the empowerment as a process of reinforcing employees qualifications through identifying those conditions that have caused their weakness and trying to deal with problems through formal actions , informal techniques and also providing information which is necessary for their efficiency . armstrong quotes blak and piterz have defined empowerment as power - sharing process between people who work in an organization . various ideas about empowerment dimensions given different views toward empowerment definitions and criterion , this research has been done based on the best and most comprehensive model which is more adaptable with employees present conditions in our country . according to this model that is called ardalan model , the most important dimensions of empowerment include : employees performance improvementoccupational freedom and independenceincreased decision - making responsibilityincreased employment opportunitiesenhanced a self - controlincreased staff knowledgeprofessional development of staff . employees performance improvement occupational freedom and independence increased decision - making responsibility increased employment opportunities enhanced a self - control increased staff knowledge professional development of staff . ardalan in his master 's thesis entitled investigating the effects of it on employees occupational empowerment , said that it increases employees knowledge and awareness leading to easier and more exact communications , less expenses and decrease in human error in the information process . today this is possible through using computers and some parts of middle management and leadership responsibilities . this way , employees will have access to more information and increase their knowledge . they will also come to better results . employees empowerment : golden key for human resources management , said that having an appropriate career is one of the basic constituent elements of citizenship whose importance is not less than the presence at elections . global developmental trends such as dramatic changes in demographic transition due to immigration , the emergence of new technologies and approaches in management have provided new horizons for employment planners . it has generated new employment opportunities and brought about fundamental changes in many other jobs . in 2012 , current jobs will be challenging and rewarding professions will be created . in the future , it components will be more co - ordinated and their influence on employment status and the relation between developing and developed countries will be more evident . not only has it created new professions which need special expertise in it but also affected other jobs so that the time required acquiring knowledge will be shorter . in some cases it also leads to more limited works . moreover , professions content which identifies the amount of individual 's challenge with the organization and task performance has a significant impact on job satisfaction , creating job opportunities , job productivity and personal status of employees in organizations and society and efficiency . principles , fundamentals and theories of organizational culture and climate said that since independence affects the individuals needs to engage in decision - making and refers to influence on the control of career achievements , occupational freedom , giving comments and the right to vote , using it and especially it systems will facilitate organization 's control and supervision through which managers supervision scope that is a limitation in traditional structures will expand . this leads to an increase in the number of middle managers and experts leading to less administrative levels . these facts show that it helps employees to be more independent and free . according to gudarzi , since the need for self - control and autonomy is a top scale , it is associated with an employees desire to engage in decision - making , identify objectives and work based on self - control and autonomous methods without others supervision . employees tend to accept responsibilities and act freely and hence it leads to an inner control system based on which individuals evaluate their own performance . sarrafizadeh in his research entitled it in organizations concludes that it facilitates managers roles and responsibilities . achieving required information in order to make decisions , more control and supervision on organization and processes , the ability to analyze conditions , analysis , programing and decision simulation are among it significant impacts . moreover , less time to respond and make decisions along with task assignment to lower levels will help managers to be away from daily routines and step toward designing and creating work opportunities . higher information about the organization and related responsibilities leads to better decisions which in turn results in more commitment in decision - making . lack of information is resolved through it and decision supporting tools . moreover , some of the greatest advantages of advanced it that have provided a higher communication between managers , staff and clients include : electronic message system , administrative information system and video conference . knowledge helps employees to have professional command of the organization and if we accept the popular expression that knowledge is the ability he refers to the quality and empowerment of human resources as important factors for organizations survival ; in other words , human resources are more important than new financial technologies . it means that the main difference between organizations refers to their knowledge , not the financial status . the role of efficient , capable and knowledgeable human resources in organizational achievement is undeniable and human resources are regarded as the most important , expensive and valuable organizational assets that can create a powerful organization . a powerful organization is a place where employees co - operate in different activities ; therefore , human resources help organizations productivity and eventually affect all the society . undoubtedly , every community 's prosperity is due to the development of human resources and thus administrators paying special attention to employees progress . urei yazdi quotes veten and kemron ( 2002 ) in a study entitled empowerment and authority assignment said that performance improvement means higher productivity , better quality , less expenses , less errors , higher speed , service providing , client satisfaction and following rules and regulations . on the other hand , it leads to minimize human errors in information processing , while providing service and client satisfaction increases . mazidabadi - farahani ( 2004 ) in his research entitled investigating it applications in employees occupational empowerment in the qom social security organization " concludes that it has empowered employees in the qom social security organization . in a research entitled effective strategies in empowerment of experts in shahre kord university of medical sciences identify occupational freedom , human resources effective management , motivation promotion , self - management and organizational learning promotion as influencing factors in empowering medical experts of chaharmahal va bakhtiari province . a study ( 2002 ) in america shows that it plays a significant role in the fast data collection , global and fast access to a broad range of health information , a quick evaluation of information , better communication among health experts and more awareness through access to various information sources . mcguire in a study in 2003 , states that it facilitates and speeds up data collection , categorization , access and exchange . it also helps in efficient management of resources , decreases inappropriate reception of patients and avoids repeated works leading to less organizational expenses . in fact , the first distinction between developed and developing countries is in production , programming , distribution and application of information . therefore , the it decreases human resources role and results in a systematic relation between human and machine . this may cause dissatisfaction due to work with a system and automation . on the other hand , employees are also an important organizational resource and it 's necessary and unavoidable to pay enough attention to them in order to achieve organizational objectives . however , one of the important ways of employees empowerment is education and access to information through topical technologies . it is one of tools and mechanisms that collect categorize and process information and provide the results . the main objective of empowerment is to decrease governmental hierarchy and emphasize on client needs ( instead of organizational needs ) . if more attachment and commitment is aimed , the traditional leadership chain between employees and managers needs to be changed . among the outcomes of employee empowerment , the following cases can be mentioned : employees broader control over surrounding environmentdecrease the role of top managers in organization 's partial affairsemployees overall understanding of whole organizational operations andemployees higher concentration of organizational values , tasks and objectives . employees broader control over surrounding environment decrease the role of top managers in organization 's partial affairs employees overall understanding of whole organizational operations and employees higher concentration of organizational values , tasks and objectives . moreover , the most important consequences of it application in employee empowerment include : increase employees job opportunitiesincrease decision - making responsibilities among employeesincrease employees independence and freedomincrease employees knowledge and awarenessminimize human errors in processing information and higher service providing which lead to client satisfaction and emphasized on the role of it in employees performance improvementit leads to a better performance accompanied by higher efficiency in service providing all of which will cause more satisfaction from fast and high - quality services . increase employees job opportunities increase decision - making responsibilities among employees increase employees independence and freedom increase employees knowledge and awareness minimize human errors in processing information and higher service providing which lead to client satisfaction and emphasized on the role of it in employees performance improvement it leads to a better performance accompanied by higher efficiency in service providing all of which will cause more satisfaction from fast and high - quality services .

information technology ( it ) is known as a valuable tool for information dissemination . today , information communication technology can be used as a powerful tool to improve employees quality and efficiency . the increasing development of technology - based tools and their adaptation speed with human requirements has led to a new form of the learning environment and creative , active and inclusive interaction . these days , information is one of the most important power resources in every organization and accordingly , acquiring information , especially central or strategic one can help organizations to build a power base and influence others . the aim of this study was to identify the most important criteria in job empowerment using it and also the advantages of assessing empowerment . this study was a narrative review . the literature was searched on databases and journals of springer , proquest , pubmed , science direct and scientific information database ) with keywords including it , empowerment and employees in the searching areas of titles , keywords , abstracts and full texts . the preliminary search resulted in 85 articles , books and conference proceedings in which published between 1983 and 2013 during july 2013 . after a careful analysis of the content of each paper , a total of 40 papers and books were selected based on their relevancy . according to ardalan model it plays a significant role in the fast data collection , global and fast access to a broad range of health information , a quick evaluation of information , better communication among health experts and more awareness through access to various information sources . it leads to a better performance accompanied by higher efficiency in service providing all of which will cause more satisfaction from fast and high - quality services .

INTRODUCTION METHODS RESULTS Definitions and dimensions of empowerment CONCLUSION

today , the application of information technology ( it ) has expanded rapidly in order to increase efficiency and productivity in most areas . research reveals high power of new it in increasing individuals health - related knowledge levels . at present information is data which has taken quantity matter in order to adapt with objectives and connect to work . the organization should recognize necessary information for work management ; otherwise , it will face a mass of confusing data . this is in fact related to the organization 's performance improvement or correction which can be dependent on it ; it provides managers an opportunity to control and co - ordinate complex structures using quick information processing which eventually leads to coherent and rapid management and better organizational performance . investment and identification of organizational performance based on information and the resulting products , is a new acceleration which is considered as the basis for mass organizational movement ; perhaps one of the most obvious results in this area is observed in employees faster and smoother access to required information which has been considered in most researches connected to information . this way , employees will have access to more information and do their duties with a higher speed . with the advancement of science and technology in the field of computer and information systems , another significant point is the impact and role of human resources and organizational staff that are among the most critical organizational resources . therefore , for structural adaptation with information transformation and achievement of valuable and useful results in this area , human resources development is necessary in order to adapt readily to the outcomes of this era . capable , experienced , influential scientists and specialists are required to meet the needs of this age . undoubtedly , education and measurement of abilities will increase the rate of achievement toward expected results from application of it . access to information as an effective and critical tool and acquiring central and strategic information is an advantage in every organization , because higher , faster , more appropriate and efficient production depends on the use of information and its proper turnover . this is what majority of today 's organizations has defined as their working priority ; on the other hand , when managers equip organizational members with more information those people will feel capable and more likely to work with productivity and prosperity along with management demands . in the present age , empowerment is known as a tool that enables managers to run organizations with various features such as different influential channels , development , reliance on horizontal and network structures , minimum distance between employees and managers , reduction of organizational attachment and use of it . empowerment leads to capable and motivated employees and helps managers to act properly and quickly in dynamics of the competitive environment and provide competitive advantages for the organization . it provides changes in organizations through which companies and organizations are more dependent on information , knowledge , training and decision - making of staff members . regarding the application of information and communication technologies at home and school along with organizations , recreational places and other sites , with the advent of technology , not only the structure of employee changes due to altered activities or modified components , but some new jobs will also be created . therefore , it is a powerful tool for data collection and processing into information . as a result , the application of it is expanding rapidly through organizations . management is easier in organizations which have high levels of technology and new information applications , because new tools will facilitate the ability of applying information . undeniable advantages of it can be observed in higher accuracy and speed of affairs , high quality , lower costs and higher client satisfaction which has encouraged organizations to establish and use such systems in order to survive , develop and achieve their objectives while they enjoy its competitive advantages at the same time . given the important role of employees as one of the main organizational resources , there is an urgent need to propel them toward it . however , previous comments and remarks emphasized that information is a critical organizational element in today 's world . accordingly , one of the most important ways to empower employees is through their access to information . we used a sub - systematic method , which was divided into three phases : literature collection , assessing and selection . the literature was searched with the assessing , and selection of libraries , databases ( such as proquest , pubmed , science direct and scientific information database ) and also searches engines available at google , google scholar , books and conference proceedings with keywords including it , empowerment and employees in the searching areas of titles , keywords , abstracts and full texts . the preliminary search resulted in 85 articles , books and conference proceedings in which published between 1983 and 2013 during july 2013 . after a careful analysis of the content of each paper , the study was performed in order to select the most important professional empowerment dimensions using it . the word empowerment by oxford definition means giving power or the right of doing something to someone . some people believe that empowerment is a type of decentralization which requires assigning basic decision making to subordinates . furthermore , empowerment provides decision- making powers and lets employees perform according to their desire . , empowerment is regarded as one of the useful tools in promoting employees and increasing organizational effectiveness . researchers have viewed empowerment from different perspectives : some concepts such as personal work control , independence in task performance , payment system associated with performance or payment based on performance , job enrichment , employee stock ownership and so on . these different views are not purely personal , but they are techniques which theorists and administrators use in order to create an empowering environment or facilitate empowering conditions . accordingly , different definitions have been provided on empowerment . for example , kinlow defines it as a process toward continuous improvement in organizational performance , which is possible through the expansion and development of influence based on individuals and team 's qualifications whose performance affects the organization 's general performance . empowerment does nt mean giving employees the right to vote , autonomy or even motivation ; despite a strong desire to do more work will result from empowerment . armstrong quotes conger describes the empowerment as a process of reinforcing employees qualifications through identifying those conditions that have caused their weakness and trying to deal with problems through formal actions , informal techniques and also providing information which is necessary for their efficiency . various ideas about empowerment dimensions given different views toward empowerment definitions and criterion , this research has been done based on the best and most comprehensive model which is more adaptable with employees present conditions in our country . according to this model that is called ardalan model , the most important dimensions of empowerment include : employees performance improvementoccupational freedom and independenceincreased decision - making responsibilityincreased employment opportunitiesenhanced a self - controlincreased staff knowledgeprofessional development of staff . employees performance improvement occupational freedom and independence increased decision - making responsibility increased employment opportunities enhanced a self - control increased staff knowledge professional development of staff . ardalan in his master 's thesis entitled investigating the effects of it on employees occupational empowerment , said that it increases employees knowledge and awareness leading to easier and more exact communications , less expenses and decrease in human error in the information process . today this is possible through using computers and some parts of middle management and leadership responsibilities . this way , employees will have access to more information and increase their knowledge . they will also come to better results . employees empowerment : golden key for human resources management , said that having an appropriate career is one of the basic constituent elements of citizenship whose importance is not less than the presence at elections . in the future , it components will be more co - ordinated and their influence on employment status and the relation between developing and developed countries will be more evident . not only has it created new professions which need special expertise in it but also affected other jobs so that the time required acquiring knowledge will be shorter . in some cases it also leads to more limited works . moreover , professions content which identifies the amount of individual 's challenge with the organization and task performance has a significant impact on job satisfaction , creating job opportunities , job productivity and personal status of employees in organizations and society and efficiency . principles , fundamentals and theories of organizational culture and climate said that since independence affects the individuals needs to engage in decision - making and refers to influence on the control of career achievements , occupational freedom , giving comments and the right to vote , using it and especially it systems will facilitate organization 's control and supervision through which managers supervision scope that is a limitation in traditional structures will expand . this leads to an increase in the number of middle managers and experts leading to less administrative levels . according to gudarzi , since the need for self - control and autonomy is a top scale , it is associated with an employees desire to engage in decision - making , identify objectives and work based on self - control and autonomous methods without others supervision . employees tend to accept responsibilities and act freely and hence it leads to an inner control system based on which individuals evaluate their own performance . achieving required information in order to make decisions , more control and supervision on organization and processes , the ability to analyze conditions , analysis , programing and decision simulation are among it significant impacts . higher information about the organization and related responsibilities leads to better decisions which in turn results in more commitment in decision - making . lack of information is resolved through it and decision supporting tools . moreover , some of the greatest advantages of advanced it that have provided a higher communication between managers , staff and clients include : electronic message system , administrative information system and video conference . knowledge helps employees to have professional command of the organization and if we accept the popular expression that knowledge is the ability he refers to the quality and empowerment of human resources as important factors for organizations survival ; in other words , human resources are more important than new financial technologies . the role of efficient , capable and knowledgeable human resources in organizational achievement is undeniable and human resources are regarded as the most important , expensive and valuable organizational assets that can create a powerful organization . a powerful organization is a place where employees co - operate in different activities ; therefore , human resources help organizations productivity and eventually affect all the society . undoubtedly , every community 's prosperity is due to the development of human resources and thus administrators paying special attention to employees progress . urei yazdi quotes veten and kemron ( 2002 ) in a study entitled empowerment and authority assignment said that performance improvement means higher productivity , better quality , less expenses , less errors , higher speed , service providing , client satisfaction and following rules and regulations . on the other hand , it leads to minimize human errors in information processing , while providing service and client satisfaction increases . mazidabadi - farahani ( 2004 ) in his research entitled investigating it applications in employees occupational empowerment in the qom social security organization " concludes that it has empowered employees in the qom social security organization . in a research entitled effective strategies in empowerment of experts in shahre kord university of medical sciences identify occupational freedom , human resources effective management , motivation promotion , self - management and organizational learning promotion as influencing factors in empowering medical experts of chaharmahal va bakhtiari province . a study ( 2002 ) in america shows that it plays a significant role in the fast data collection , global and fast access to a broad range of health information , a quick evaluation of information , better communication among health experts and more awareness through access to various information sources . mcguire in a study in 2003 , states that it facilitates and speeds up data collection , categorization , access and exchange . it also helps in efficient management of resources , decreases inappropriate reception of patients and avoids repeated works leading to less organizational expenses . furthermore , empowerment provides decision- making powers and lets employees perform according to their desire . , empowerment is regarded as one of the useful tools in promoting employees and increasing organizational effectiveness . in order to be successful in today 's changing environment , organizations need knowledge , ideas , energy and creativity of all employees , including those at the first level to top managers . researchers have viewed empowerment from different perspectives : some concepts such as personal work control , independence in task performance , payment system associated with performance or payment based on performance , job enrichment , employee stock ownership and so on . these different views are not purely personal , but they are techniques which theorists and administrators use in order to create an empowering environment or facilitate empowering conditions . accordingly , different definitions have been provided on empowerment . for example , kinlow defines it as a process toward continuous improvement in organizational performance , which is possible through the expansion and development of influence based on individuals and team 's qualifications whose performance affects the organization 's general performance . the long - term goal of empowerment is continuous improvement in organization 's overall performance , whereas its short - term objective is just application of existing qualifications . armstrong quotes conger describes the empowerment as a process of reinforcing employees qualifications through identifying those conditions that have caused their weakness and trying to deal with problems through formal actions , informal techniques and also providing information which is necessary for their efficiency . armstrong quotes blak and piterz have defined empowerment as power - sharing process between people who work in an organization . various ideas about empowerment dimensions given different views toward empowerment definitions and criterion , this research has been done based on the best and most comprehensive model which is more adaptable with employees present conditions in our country . according to this model that is called ardalan model , the most important dimensions of empowerment include : employees performance improvementoccupational freedom and independenceincreased decision - making responsibilityincreased employment opportunitiesenhanced a self - controlincreased staff knowledgeprofessional development of staff . employees performance improvement occupational freedom and independence increased decision - making responsibility increased employment opportunities enhanced a self - control increased staff knowledge professional development of staff . ardalan in his master 's thesis entitled investigating the effects of it on employees occupational empowerment , said that it increases employees knowledge and awareness leading to easier and more exact communications , less expenses and decrease in human error in the information process . today this is possible through using computers and some parts of middle management and leadership responsibilities . this way , employees will have access to more information and increase their knowledge . employees empowerment : golden key for human resources management , said that having an appropriate career is one of the basic constituent elements of citizenship whose importance is not less than the presence at elections . it has generated new employment opportunities and brought about fundamental changes in many other jobs . in the future , it components will be more co - ordinated and their influence on employment status and the relation between developing and developed countries will be more evident . in some cases it also leads to more limited works . moreover , professions content which identifies the amount of individual 's challenge with the organization and task performance has a significant impact on job satisfaction , creating job opportunities , job productivity and personal status of employees in organizations and society and efficiency . principles , fundamentals and theories of organizational culture and climate said that since independence affects the individuals needs to engage in decision - making and refers to influence on the control of career achievements , occupational freedom , giving comments and the right to vote , using it and especially it systems will facilitate organization 's control and supervision through which managers supervision scope that is a limitation in traditional structures will expand . this leads to an increase in the number of middle managers and experts leading to less administrative levels . according to gudarzi , since the need for self - control and autonomy is a top scale , it is associated with an employees desire to engage in decision - making , identify objectives and work based on self - control and autonomous methods without others supervision . employees tend to accept responsibilities and act freely and hence it leads to an inner control system based on which individuals evaluate their own performance . sarrafizadeh in his research entitled it in organizations concludes that it facilitates managers roles and responsibilities . achieving required information in order to make decisions , more control and supervision on organization and processes , the ability to analyze conditions , analysis , programing and decision simulation are among it significant impacts . higher information about the organization and related responsibilities leads to better decisions which in turn results in more commitment in decision - making . lack of information is resolved through it and decision supporting tools . moreover , some of the greatest advantages of advanced it that have provided a higher communication between managers , staff and clients include : electronic message system , administrative information system and video conference . knowledge helps employees to have professional command of the organization and if we accept the popular expression that knowledge is the ability he refers to the quality and empowerment of human resources as important factors for organizations survival ; in other words , human resources are more important than new financial technologies . the role of efficient , capable and knowledgeable human resources in organizational achievement is undeniable and human resources are regarded as the most important , expensive and valuable organizational assets that can create a powerful organization . a powerful organization is a place where employees co - operate in different activities ; therefore , human resources help organizations productivity and eventually affect all the society . undoubtedly , every community 's prosperity is due to the development of human resources and thus administrators paying special attention to employees progress . urei yazdi quotes veten and kemron ( 2002 ) in a study entitled empowerment and authority assignment said that performance improvement means higher productivity , better quality , less expenses , less errors , higher speed , service providing , client satisfaction and following rules and regulations . on the other hand , it leads to minimize human errors in information processing , while providing service and client satisfaction increases . mazidabadi - farahani ( 2004 ) in his research entitled investigating it applications in employees occupational empowerment in the qom social security organization " concludes that it has empowered employees in the qom social security organization . in a research entitled effective strategies in empowerment of experts in shahre kord university of medical sciences identify occupational freedom , human resources effective management , motivation promotion , self - management and organizational learning promotion as influencing factors in empowering medical experts of chaharmahal va bakhtiari province . a study ( 2002 ) in america shows that it plays a significant role in the fast data collection , global and fast access to a broad range of health information , a quick evaluation of information , better communication among health experts and more awareness through access to various information sources . mcguire in a study in 2003 , states that it facilitates and speeds up data collection , categorization , access and exchange . it also helps in efficient management of resources , decreases inappropriate reception of patients and avoids repeated works leading to less organizational expenses . in fact , the first distinction between developed and developing countries is in production , programming , distribution and application of information . on the other hand , employees are also an important organizational resource and it 's necessary and unavoidable to pay enough attention to them in order to achieve organizational objectives . however , one of the important ways of employees empowerment is education and access to information through topical technologies . it is one of tools and mechanisms that collect categorize and process information and provide the results . the main objective of empowerment is to decrease governmental hierarchy and emphasize on client needs ( instead of organizational needs ) . among the outcomes of employee empowerment , the following cases can be mentioned : employees broader control over surrounding environmentdecrease the role of top managers in organization 's partial affairsemployees overall understanding of whole organizational operations andemployees higher concentration of organizational values , tasks and objectives . employees broader control over surrounding environment decrease the role of top managers in organization 's partial affairs employees overall understanding of whole organizational operations and employees higher concentration of organizational values , tasks and objectives . moreover , the most important consequences of it application in employee empowerment include : increase employees job opportunitiesincrease decision - making responsibilities among employeesincrease employees independence and freedomincrease employees knowledge and awarenessminimize human errors in processing information and higher service providing which lead to client satisfaction and emphasized on the role of it in employees performance improvementit leads to a better performance accompanied by higher efficiency in service providing all of which will cause more satisfaction from fast and high - quality services . increase employees job opportunities increase decision - making responsibilities among employees increase employees independence and freedom increase employees knowledge and awareness minimize human errors in processing information and higher service providing which lead to client satisfaction and emphasized on the role of it in employees performance improvement it leads to a better performance accompanied by higher efficiency in service providing all of which will cause more satisfaction from fast and high - quality services .

physical inactivity among adolescents is believed to play a key role in their risk for obesity . while reducing sedentary behavior is an often - recommended strategy to prevent and treat obesity among adolescents [ 1 , 2 ] , studies aiming to understand associations between sedentary behavior and weight have almost exclusively focused on adolescents ' television use [ 37 ] , or the broader concept of screen time , which incorporates other technology - based activities such as computer and video game use [ 810 ] . this predominant attention to television use and screen time has called into question whether other types of sedentary behavior may displace physical activity ( pa ) and prompt unhealthful dietary intake , contributing to excess weight gain among adolescents . adolescent girls are a particularly important population in which to understand the associations between a range of sedentary behaviors and pa and weight as adolescent girls experience a significant decline in pa as they move through the adolescent years [ 12 , 13 ] and are less likely to participate in vigorous pa than adolescent boys . while watching television is a common leisure - time activity among adolescents , many adolescent girls report frequently participating in sedentary behaviors that are not screen - based yet still may pose a risk for excess weight gain , such as sitting and hanging around with friends , talking on the phone , and listening to music [ 11 , 15 , 16 ] . additionally , for a significant proportion of adolescent girls , television , computer , and/or video game use are not their favored sedentary activities , and , therefore , these girls spend little time participating in screen - based activities but a great deal of time engaging in other sedentary behaviors [ 11 , 15 ] . while there is increasing evidence that adolescent girls engage in a number of different sedentary activities that account for a large proportion of their day , only a small number of studies have utilized a comprehensive view of adolescent girls ' sedentary behavior to examine associations between these behaviors and girls ' pa , dietary intake , or weight status [ 1721 ] , and findings from these studies have been somewhat contradictory . for example , leatherdale observed that adolescent girls who were physically active were more likely to engage in communication time , including talking on the phone , texting , and instant messaging , as well as frequently watching tv and movies and playing video or computer games . . observed that girls who spent the majority of their leisure time participating in social activities including talking on the phone , hanging out , and talking with friends tended to watch relatively little television and were unlikely to meet recommendations for daily pa . additional studies are needed with diverse samples of adolescent girls to better understand girls ' sedentary behavior preferences , as well as whether participation in these behaviors is associated with girls ' risk for obesity . in light of the need for research examining associations between a wide range of sedentary behaviors and adolescent girls ' weight and weight - related behaviors , the current study aims to describe the frequency and duration of participation in a variety of recreational sedentary behaviors ( e.g. watching television , hanging around , talking on the phone , and internet use ) and identify associations between these recreational sedentary behaviors and body mass index ( bmi ) , pa , and dietary behaviors , among a sociodemographically diverse population of overweight , obese , and/or sedentary adolescent girls . recreational sedentary behaviors were selected as a focus because they characterize girls ' use of their discretionary time as opposed to necessary sedentary pursuits such as doing homework and sleep . study findings can aid in the identification of behavioral targets for interventions aiming to decrease sedentary time and promote pa and healthful dietary intake among sedentary adolescent girls , who are at risk for obesity and related health outcomes . data were drawn from the baseline evaluation of new moves , a school - based intervention that aimed to prevent obesity and other weight - related problems among adolescent girls . the population included 283 adolescent girls in grades 9 through 12 ( mean age = 15.8 years ; sd = 1.2 ) . twelve schools from 1 urban and 6 suburban school districts offered the new moves program during either the 2007/2008 or 2008/2009 school years . to recruit girls to participate in the study , the new moves intervention was described in the school course catalogue and on posters located around the school ; recruitment materials were designed to appeal to girls who were currently overweight or obese and/or were inactive and not comfortable being physically active , but who had a desire to be healthier . a short screening questionnaire developed for the current study was used to assess girls ' frequency and duration of pa / exercise and their frequency of use of disordered eating behaviors . four girls were excluded from the intervention because of high levels of pa ( 1 hour / day ) . the study sample was sociodemographically diverse with 28% of girls identifying as african / american black , 28% white , 16% hispanic , 18% asian , and 11% of mixed or other race / ethnicity . approximately , half of the girls were of normal weight status while 18% were overweight ( age- and gender - adjusted bmi percentile between the 85th and 95th percentile ) , and 28% were obese ( bmi percentile 95th ) . across the 12 schools , between 22 and 92% of students were eligible for free or reduced - price school breakfast and lunch , and in 8 of the 12 schools over 55% of the students were eligible for free or reduced - price school breakfast and lunch . girls completed baseline data collection during either the end of spring semester or beginning of fall semester preceding their participation in new moves . data collection occurred either at the university of minnesota 's general clinical research center or at the participants ' schools . trained study staff oversaw all data collection with groups of up to 15 girls at a time . staff reviewed all survey instruments for clarity and completeness at the time of the data collection . two staff members edited survey instruments , and the data were entered and double checked for accuracy . the study was approved by the university of minnesota 's institutional review board and by each participating school district . girls ' recreational sedentary behavior and pa were measured using the 3-day physical activity recall ( 3dpar ) . the 3dpar assessed the sedentary behaviors and physical activities that study participants engaged in during each half hour time block between 6 am and midnight on the three days previous to the day of data collection . girls completed the 3dpar on a monday , tuesday , or wednesday in order to capture at least 1 weekday and 1 weekend day . in order to complete the 3dpar , participants were provided with a list of 65 common sedentary behaviors and physical activities and were asked to select the activity that they participated in for the majority of every 30-minute block . girls ' participation in recreational sedentary behavior was determined by the average daily number of 30-minute blocks in which girls ' reported participating in the following activities : hanging around ; listening to music ( sitting ) ; playing video games ; surfing the internet , instant messaging , emailing , shopping online ; reading ; watching tv or movies ; talking on the phone [ 16 , 23 , 24 ] . to assess the frequency of girls ' total pa and moderate - to - vigorous physical activity ( mvpa ) , for each block of pa reported girls noted whether their exertion level while engaging in that activity was light , moderate , hard , or very hard . for every activity at each exertion level , a corresponding metabolic equivalent ( met ) value was identified using the compendium of physical activities [ 25 , 26 ] . total pa was defined as a per day average number of blocks for which any pa was reported . mvpa was defined as the per day average number of 30-minute blocks for which physical activities with a met value greater than or equal to 3 were recorded [ 27 , 28 ] . the 3dpar has been shown to be a valid measure of mvpa as compared to accelerometry and among adolescent girls had a 2-day test - retest reliability of r = 0.71 and r = 0.77 for mvpa and vigorous activity , respectively . for recreational sedentary behaviors , total pa , and mvpa , girls ' weekend and weekday behavior was weighted to reflect 2 weekend days and 5 weekdays and then divided by 7 to produce a daily average . the previous - day physical activity recall instrument , which is the same measure as the 3dpar but collects one day of data regarding both sedentary behaviors and physical activities , has been determined to be a valid measure of screen time when compared to pedometer - measured activity . trained study staff measured girls ' body weight using a tanita body composition analyzer tbf-300a ( tanita corporation of america , arlington heights , ill , usa ) and height using a portable stadiometer . girls ' weight was measured twice , and both measurements were recorded to the nearest 0.1 kg . if between the two measurements there was a discrepancy of greater than 0.5 kg , the two measurements were repeated . similarly , girls ' height was measured twice , and both measurements were recorded to the nearest 0.1 cm . if there was a discrepancy of greater than 0.5 cm , the two measurements were repeated . the multiple measurements were then averaged to produce a single height and weight for each participant . bmi was calculated using the formula : weight in kilograms divided by height in meters squared , and bmi was transformed to age- and gender - specific z - scores according to the cdc 's bmi - for - age and -gender growth charts . girls ' fruit and vegetable intake was assessed using the questions , thinking back over the past week , how many servings of fruit thinking back over the past week , how many servings of vegetables did you usually eat on a typical day ? a serving would be 1/2 cup of cooked vegetables or 1 cup of raw vegetables . do not include potatoes or french fries . response options for both questions included none , less than 1 serving , 1 serving , 2 servings , 3 servings , 4 servings , and 5 or more servings [ 31 , 32 ] . two - week test - retest correlations for these items were 0.67 for fruit intake and 0.68 for vegetable intake . girls ' intake of soft drinks was assessed with the following item : over the past month , how often did you drink regular soda pop ( not diet ) ? response options included never , less than once a week , 1 - 2 times per week , 3 - 4 times per week , 5 - 6 times per week , 1 time per day , 2 times per day , 3 times per day , 4 times per day , 5 or more times per day . girls ' weekly intake of fast food was assessed with the question , in the past week , how often did you eat something from a fast food restaurant ( like mcdonald 's , burger king , etc . ) ? response options included , 0 times , 1 time , 2 times , 3 times , 4 - 5 times , 6 - 7 times , more than 7 times . this survey item was adapted from a previously existing measure and had a 2-week test - retest correlation of r = 0.81 . girls who completed a 3dpar on monday through wednesday and , therefore , reported their sedentary behaviors and physical activities for at least one weekday and one weekend day were included in the current study . girls who completed the 3dpar on thursday or friday were excluded as they did not recall any weekend days . due to this selection criterion , the analytic sample for the current study represents 79.4% of all girls who participated in the new moves evaluation trial ( 283/356 ) . day of completion of the 3dpar was determined by the availability of the general clinical research center , not characteristics of the girls , therefore , reducing the potential for selection bias . descriptive statistics including the percent of girls reporting any participation in each of the recreational sedentary behaviors , and the median blocks of participation in each behavior for both the whole sample and the sample of girls who reported participating in each behavior were calculated for both weekdays and weekend days . to examine cross - sectional associations between girls ' participation in each of the recreational sedentary behaviors and bmi z - scores , pa , and dietary habits , three levels of girls ' daily participation in each behavior the low and high categories were created by dichotomizing the sample of girls who participated in each behavior at the median level of participation . to determine the three levels of girls ' combined participation in all of the assessed recreational sedentary behaviors ( low , moderate , and high ) , the summary variable was divided into tertiles . ancova was used to calculate the mean level of the outcome variable for each of the three behavior participation levels , adjusted for girls ' race / ethnicity and age . school was included as a random effect to account for potential clustering of girls ' sedentary behavior by school . the p values presented are based on an f statistic with 2 degrees of freedom and a tukey - kramer adjusted p value was used to identify sources of differences between the adjusted means . sas 9.2 ( sas institute , cary , nc , usa ) was used for all analyses . watching television , hanging around , talking on the phone , and listening to music were the most common recreational sedentary behaviors reported by study participants ( table 1 ) . over half of girls reported watching television and/or hanging around on both weekdays and weekend days . playing video games was the most infrequently reported of the assessed recreational sedentary behaviors with 8% of girls reporting playing video games on weekdays and 10% reporting playing video games on weekend days . overall , almost all girls reported engaging in at least one of the recreational sedentary behaviors examined in this study on both weekdays and weekend days . among those who reported participating in any recreational sedentary behavior , the median number of blocks of recreational sedentary behavior was 8.0 blocks on weekdays and 10.8 blocks on weekend days . several of the recreational sedentary behaviors were associated with lower pa and mvpa , and less healthful dietary intake , in analyses adjusting for girls ' ethnicity / race and age ( table 2 ) . time spent watching tv was inversely associated with total pa and mvpa ( p = 0.03 for both ) . girls who spent the most time watching tv during the recall period also reported the lowest fruit and vegetable intake ( 3.8 servings per day ) while girls who did not report any tv use reported consuming 5.3 servings of fruits and vegetables per day ( p = 0.02 ) . high amounts of time spent hanging around , the second most commonly reported activity , were associated with less time spent in total pa ( p = 0.006 ) , higher soft drink intake ( p = 0.001 ) , and eating fast food more frequently ( p = 0.006 ) . for example , girls who reported the greatest amount of time hanging around during the recall period consumed fast food 1 time more per week on average compared to girls who did not report any hanging around . amount of time spent talking on the phone was positively associated with soft drink intake ( p = 0.03 ) but was not inversely associated with pa , whereas internet use was inversely associated with both total pa and mvpa ( p = 0.03 and p = 0.003 , resp . ) . reading was the only recreational sedentary behavior associated with healthful dietary behaviors ; girls who reported high amounts of reading reported lower soft drink and fast food intake ( p = 0.02 and 0.04 , resp . ) . higher total recreational sedentary behavior time was associated with lower mvpa , pa , and fruit and vegetable intake , as well as higher soft drink intake . girls ' bmi z - scores did not significantly differ by their recreational sedentary behavior participation . results of this study highlight both the variety of sedentary behaviors in which adolescent girls frequently participate , as well as the potential for many of these behaviors , alone or in combination , to contribute to lower pa levels and less healthful dietary intake . findings from the current study align with those examining sedentary behavior patterns among adolescent girls [ 19 , 35 ] , especially those of gorely et al . , who found that watching television and social sedentary activities , such as talking on the phone , were quite common among adolescent girls in the united kingdom . meanwhile , video game use and to a lesser extent internet use was relatively uncommon . these consistent results across two different populations of girls suggest that messages about limiting internet and video game time may not be highly relevant to the majority of adolescent girls . interventions may be more successful at reducing sedentary behavior by capitalizing on girls ' desire to engage in social activities including talking on the phone and hanging around with friends . additionally , in the current sample , internet use may be relatively low due to the lower socioeconomic status of many girls ' families . therefore , if internet - based activities are an essential component of future interventions with diverse communities , care should be taken to ensure that study participants have consistent access to the internet . in the current study , many girls reported spending a large amount of time hanging around , which was associated with low levels of pa and poor dietary intake . a small number of other studies have also observed that many adolescent girls spend a significant amount of their day hanging around [ 16 , 36 , 37 ] although these studies did not examine whether hanging around was associated with other weight - related behaviors . during data collection , girls completing the 3dpar were encouraged by study staff to not utilize the hanging around code unless they truly were not engaging in any other activity . therefore , this time likely represents highly unstructured time and provides an opportunity for intervention . future interventions aiming to increase adolescent girls ' pa or improve their dietary intake may benefit from working with girls to substitute unstructured time spent hanging around with other enjoyable social activities that are more physically active or expose girls to healthier food choices . girls may be especially receptive to this type of intervention activity because it does not emphasize limiting enjoyable sedentary behaviors such as watching television but focuses on finding ways to spend more time during the day with friends participating in enjoyable activities . while several of the recreational sedentary behaviors examined in the current study were associated with lower levels of physical activity and less healthful dietary habits , no associations were observed between recreational sedentary behavior and girls ' bmi z - score . this finding is consistent with other studies that have observed no association or a small association between participation in sedentary behavior and adolescents ' weight status [ 38 , 39 ] . one potential explanation for the lack of associations observed in the current study and others is that excess weight gain may be due to the accumulation of several less healthful behaviors ( e.g. , energy - dense diet , inadequate sleep , sedentary lifestyle ) , and , therefore , only weak associations are observed when examining the role of sedentary behaviors in weight status in isolation of other behaviors . for example , a recent study has observed that obesity risk was higher among adolescents who engaged in both low levels of physical activity and high levels of screen time . additionally , sedentary behaviors may only be associated with obesity risk among some individuals , such as those with a genetic susceptibility to obesity , leading to the observation of null associations between sedentary behavior and weight among the general population . despite the inconsistency of associations reported in observational studies , sedentary behavior is still an important intervention target , and studies that have aimed to reduce sedentary activity have successfully led to decreases in adolescents ' weight status [ 4143 ] . a strength of the current study is its use of a sociodemographically diverse population of adolescent girls who were either currently overweight or obese , or at high risk for overweight due to having a highly sedentary lifestyle . there is a great need to develop relevant and enjoyable programs that encourage greater participation in healthful pa and healthy eating behaviors among this population . however , this focus on a specific , underserved population does limit the generalizability of study findings , and the sedentary behavior patterns of highly physically active adolescent girls may be different than what was observed in this study population . an additional strength of this study is the use of the 3dpar , which allowed for an understanding of the context of girls ' sedentary time . while objective measures of pa and sedentary behavior , such as accelerometry , play an important role in obtaining an unbiased assessment of pa , use of self - report measures that record the specific activities that study participants are engaging in can provide great richness to the understanding of how individuals are spending their time . a limitation of the 3dpar is that participants are only able to report a single activity that they participate in for the majority of each 30-minute block . therefore multitasking , which adolescents do frequently when watching television , or behaviors that participants engaged in for less than 15 minutes , were not recorded with this instrument . additionally , girls completed the 3dpar using a set list of common activities . because this list was not comprehensive , activities that may be increasingly common , such as texting , were not captured by the instrument . however , it is important to note that girls rarely asked how to categorize texting during the data collection process , as they were often texting for short amounts of time throughout the day and not for the majority of a 30-minute time period . girls who did ask how to code their texting time were told to categorize it with internet use / instant messaging , which may have inflated the assessment of the amount of time girls spent on the computer . future studies utilizing the 3dpar are encouraged to conduct extensive pilot testing of the measure and adapt the activity options provided based on the popular activities among the intended study population . adolescent girls participate in a number of recreational sedentary behaviors for a significant portion of their free time . understanding these patterns of behavior , and associations between recreational sedentary behaviors and pa and dietary habits , is informative to the development of interventions aiming to decrease adolescents ' risk for overweight and obesity . findings from the current study suggest that interventions developed to engage adolescent girls may benefit from incorporating messages and behavioral targets that address recreational sedentary behaviors such as hanging around , talking on the phone , and listening to music , in addition to the often - targeted watching television . future research in this area is also needed to understand whether girls ' participation in specific recreational behaviors contributes to decreases in pa and poor dietary intake , or whether the behaviors co - occur and are markers of a less healthful lifestyle in total . additionally , in light of the lack of associations observed in the current study between recreational sedentary behavior and bmi , and the inconsistent findings between sedentary behavior and weight observed elsewhere in the literature [ 38 , 45 ] , further investigations of the role of television , screen time , and other common recreational sedentary behaviors in adolescents ' weight and excess weight gain are a priority .

most studies of sedentary behavior have focused on television use or screen time . this study aims to examine adolescent girls ' participation in a variety of recreational sedentary behaviors ( e.g. , talking on the phone and hanging around ) , and their association with physical activity ( pa ) , dietary behaviors , and body mass index . data were from a sample of 283 adolescent girls . recreational sedentary behavior , pa , and dietary behaviors were self - reported , and girls ' height and weight were measured . over 95% of girls engaged in at least one recreational sedentary behavior during the recall period . watching television and hanging around were the most common behaviors . watching television , using the internet , and hanging around were associated with less pa ; watching television , hanging around , and talking on the phone were associated with less healthful dietary behaviors . no associations were found with body mass index . interventions may benefit from capitalizing on and intervening upon girls ' common recreational sedentary behaviors .

1. Introduction 2. Methods 3. Results and Discussion 4. Conclusions

physical inactivity among adolescents is believed to play a key role in their risk for obesity . while reducing sedentary behavior is an often - recommended strategy to prevent and treat obesity among adolescents [ 1 , 2 ] , studies aiming to understand associations between sedentary behavior and weight have almost exclusively focused on adolescents ' television use [ 37 ] , or the broader concept of screen time , which incorporates other technology - based activities such as computer and video game use [ 810 ] . this predominant attention to television use and screen time has called into question whether other types of sedentary behavior may displace physical activity ( pa ) and prompt unhealthful dietary intake , contributing to excess weight gain among adolescents . adolescent girls are a particularly important population in which to understand the associations between a range of sedentary behaviors and pa and weight as adolescent girls experience a significant decline in pa as they move through the adolescent years [ 12 , 13 ] and are less likely to participate in vigorous pa than adolescent boys . while watching television is a common leisure - time activity among adolescents , many adolescent girls report frequently participating in sedentary behaviors that are not screen - based yet still may pose a risk for excess weight gain , such as sitting and hanging around with friends , talking on the phone , and listening to music [ 11 , 15 , 16 ] . additionally , for a significant proportion of adolescent girls , television , computer , and/or video game use are not their favored sedentary activities , and , therefore , these girls spend little time participating in screen - based activities but a great deal of time engaging in other sedentary behaviors [ 11 , 15 ] . while there is increasing evidence that adolescent girls engage in a number of different sedentary activities that account for a large proportion of their day , only a small number of studies have utilized a comprehensive view of adolescent girls ' sedentary behavior to examine associations between these behaviors and girls ' pa , dietary intake , or weight status [ 1721 ] , and findings from these studies have been somewhat contradictory . for example , leatherdale observed that adolescent girls who were physically active were more likely to engage in communication time , including talking on the phone , texting , and instant messaging , as well as frequently watching tv and movies and playing video or computer games . observed that girls who spent the majority of their leisure time participating in social activities including talking on the phone , hanging out , and talking with friends tended to watch relatively little television and were unlikely to meet recommendations for daily pa . additional studies are needed with diverse samples of adolescent girls to better understand girls ' sedentary behavior preferences , as well as whether participation in these behaviors is associated with girls ' risk for obesity . in light of the need for research examining associations between a wide range of sedentary behaviors and adolescent girls ' weight and weight - related behaviors , the current study aims to describe the frequency and duration of participation in a variety of recreational sedentary behaviors ( e.g. watching television , hanging around , talking on the phone , and internet use ) and identify associations between these recreational sedentary behaviors and body mass index ( bmi ) , pa , and dietary behaviors , among a sociodemographically diverse population of overweight , obese , and/or sedentary adolescent girls . recreational sedentary behaviors were selected as a focus because they characterize girls ' use of their discretionary time as opposed to necessary sedentary pursuits such as doing homework and sleep . study findings can aid in the identification of behavioral targets for interventions aiming to decrease sedentary time and promote pa and healthful dietary intake among sedentary adolescent girls , who are at risk for obesity and related health outcomes . data were drawn from the baseline evaluation of new moves , a school - based intervention that aimed to prevent obesity and other weight - related problems among adolescent girls . the population included 283 adolescent girls in grades 9 through 12 ( mean age = 15.8 years ; sd = 1.2 ) . a short screening questionnaire developed for the current study was used to assess girls ' frequency and duration of pa / exercise and their frequency of use of disordered eating behaviors . the study sample was sociodemographically diverse with 28% of girls identifying as african / american black , 28% white , 16% hispanic , 18% asian , and 11% of mixed or other race / ethnicity . approximately , half of the girls were of normal weight status while 18% were overweight ( age- and gender - adjusted bmi percentile between the 85th and 95th percentile ) , and 28% were obese ( bmi percentile 95th ) . across the 12 schools , between 22 and 92% of students were eligible for free or reduced - price school breakfast and lunch , and in 8 of the 12 schools over 55% of the students were eligible for free or reduced - price school breakfast and lunch . girls completed baseline data collection during either the end of spring semester or beginning of fall semester preceding their participation in new moves . trained study staff oversaw all data collection with groups of up to 15 girls at a time . two staff members edited survey instruments , and the data were entered and double checked for accuracy . girls ' recreational sedentary behavior and pa were measured using the 3-day physical activity recall ( 3dpar ) . the 3dpar assessed the sedentary behaviors and physical activities that study participants engaged in during each half hour time block between 6 am and midnight on the three days previous to the day of data collection . girls completed the 3dpar on a monday , tuesday , or wednesday in order to capture at least 1 weekday and 1 weekend day . in order to complete the 3dpar , participants were provided with a list of 65 common sedentary behaviors and physical activities and were asked to select the activity that they participated in for the majority of every 30-minute block . girls ' participation in recreational sedentary behavior was determined by the average daily number of 30-minute blocks in which girls ' reported participating in the following activities : hanging around ; listening to music ( sitting ) ; playing video games ; surfing the internet , instant messaging , emailing , shopping online ; reading ; watching tv or movies ; talking on the phone [ 16 , 23 , 24 ] . to assess the frequency of girls ' total pa and moderate - to - vigorous physical activity ( mvpa ) , for each block of pa reported girls noted whether their exertion level while engaging in that activity was light , moderate , hard , or very hard . for every activity at each exertion level , a corresponding metabolic equivalent ( met ) value was identified using the compendium of physical activities [ 25 , 26 ] . mvpa was defined as the per day average number of 30-minute blocks for which physical activities with a met value greater than or equal to 3 were recorded [ 27 , 28 ] . the 3dpar has been shown to be a valid measure of mvpa as compared to accelerometry and among adolescent girls had a 2-day test - retest reliability of r = 0.71 and r = 0.77 for mvpa and vigorous activity , respectively . for recreational sedentary behaviors , total pa , and mvpa , girls ' weekend and weekday behavior was weighted to reflect 2 weekend days and 5 weekdays and then divided by 7 to produce a daily average . the previous - day physical activity recall instrument , which is the same measure as the 3dpar but collects one day of data regarding both sedentary behaviors and physical activities , has been determined to be a valid measure of screen time when compared to pedometer - measured activity . trained study staff measured girls ' body weight using a tanita body composition analyzer tbf-300a ( tanita corporation of america , arlington heights , ill , usa ) and height using a portable stadiometer . girls ' weight was measured twice , and both measurements were recorded to the nearest 0.1 kg . if between the two measurements there was a discrepancy of greater than 0.5 kg , the two measurements were repeated . similarly , girls ' height was measured twice , and both measurements were recorded to the nearest 0.1 cm . the multiple measurements were then averaged to produce a single height and weight for each participant . bmi was calculated using the formula : weight in kilograms divided by height in meters squared , and bmi was transformed to age- and gender - specific z - scores according to the cdc 's bmi - for - age and -gender growth charts . girls ' fruit and vegetable intake was assessed using the questions , thinking back over the past week , how many servings of fruit thinking back over the past week , how many servings of vegetables did you usually eat on a typical day ? do not include potatoes or french fries . response options for both questions included none , less than 1 serving , 1 serving , 2 servings , 3 servings , 4 servings , and 5 or more servings [ 31 , 32 ] . girls ' intake of soft drinks was assessed with the following item : over the past month , how often did you drink regular soda pop ( not diet ) ? girls ' weekly intake of fast food was assessed with the question , in the past week , how often did you eat something from a fast food restaurant ( like mcdonald 's , burger king , etc . ) this survey item was adapted from a previously existing measure and had a 2-week test - retest correlation of r = 0.81 . girls who completed a 3dpar on monday through wednesday and , therefore , reported their sedentary behaviors and physical activities for at least one weekday and one weekend day were included in the current study . descriptive statistics including the percent of girls reporting any participation in each of the recreational sedentary behaviors , and the median blocks of participation in each behavior for both the whole sample and the sample of girls who reported participating in each behavior were calculated for both weekdays and weekend days . to examine cross - sectional associations between girls ' participation in each of the recreational sedentary behaviors and bmi z - scores , pa , and dietary habits , three levels of girls ' daily participation in each behavior the low and high categories were created by dichotomizing the sample of girls who participated in each behavior at the median level of participation . to determine the three levels of girls ' combined participation in all of the assessed recreational sedentary behaviors ( low , moderate , and high ) , the summary variable was divided into tertiles . ancova was used to calculate the mean level of the outcome variable for each of the three behavior participation levels , adjusted for girls ' race / ethnicity and age . school was included as a random effect to account for potential clustering of girls ' sedentary behavior by school . watching television , hanging around , talking on the phone , and listening to music were the most common recreational sedentary behaviors reported by study participants ( table 1 ) . over half of girls reported watching television and/or hanging around on both weekdays and weekend days . playing video games was the most infrequently reported of the assessed recreational sedentary behaviors with 8% of girls reporting playing video games on weekdays and 10% reporting playing video games on weekend days . overall , almost all girls reported engaging in at least one of the recreational sedentary behaviors examined in this study on both weekdays and weekend days . among those who reported participating in any recreational sedentary behavior , the median number of blocks of recreational sedentary behavior was 8.0 blocks on weekdays and 10.8 blocks on weekend days . several of the recreational sedentary behaviors were associated with lower pa and mvpa , and less healthful dietary intake , in analyses adjusting for girls ' ethnicity / race and age ( table 2 ) . time spent watching tv was inversely associated with total pa and mvpa ( p = 0.03 for both ) . girls who spent the most time watching tv during the recall period also reported the lowest fruit and vegetable intake ( 3.8 servings per day ) while girls who did not report any tv use reported consuming 5.3 servings of fruits and vegetables per day ( p = 0.02 ) . high amounts of time spent hanging around , the second most commonly reported activity , were associated with less time spent in total pa ( p = 0.006 ) , higher soft drink intake ( p = 0.001 ) , and eating fast food more frequently ( p = 0.006 ) . for example , girls who reported the greatest amount of time hanging around during the recall period consumed fast food 1 time more per week on average compared to girls who did not report any hanging around . amount of time spent talking on the phone was positively associated with soft drink intake ( p = 0.03 ) but was not inversely associated with pa , whereas internet use was inversely associated with both total pa and mvpa ( p = 0.03 and p = 0.003 , resp . ) . reading was the only recreational sedentary behavior associated with healthful dietary behaviors ; girls who reported high amounts of reading reported lower soft drink and fast food intake ( p = 0.02 and 0.04 , resp . ) higher total recreational sedentary behavior time was associated with lower mvpa , pa , and fruit and vegetable intake , as well as higher soft drink intake . girls ' bmi z - scores did not significantly differ by their recreational sedentary behavior participation . results of this study highlight both the variety of sedentary behaviors in which adolescent girls frequently participate , as well as the potential for many of these behaviors , alone or in combination , to contribute to lower pa levels and less healthful dietary intake . findings from the current study align with those examining sedentary behavior patterns among adolescent girls [ 19 , 35 ] , especially those of gorely et al . , who found that watching television and social sedentary activities , such as talking on the phone , were quite common among adolescent girls in the united kingdom . these consistent results across two different populations of girls suggest that messages about limiting internet and video game time may not be highly relevant to the majority of adolescent girls . interventions may be more successful at reducing sedentary behavior by capitalizing on girls ' desire to engage in social activities including talking on the phone and hanging around with friends . additionally , in the current sample , internet use may be relatively low due to the lower socioeconomic status of many girls ' families . therefore , if internet - based activities are an essential component of future interventions with diverse communities , care should be taken to ensure that study participants have consistent access to the internet . in the current study , many girls reported spending a large amount of time hanging around , which was associated with low levels of pa and poor dietary intake . a small number of other studies have also observed that many adolescent girls spend a significant amount of their day hanging around [ 16 , 36 , 37 ] although these studies did not examine whether hanging around was associated with other weight - related behaviors . during data collection , girls completing the 3dpar were encouraged by study staff to not utilize the hanging around code unless they truly were not engaging in any other activity . future interventions aiming to increase adolescent girls ' pa or improve their dietary intake may benefit from working with girls to substitute unstructured time spent hanging around with other enjoyable social activities that are more physically active or expose girls to healthier food choices . girls may be especially receptive to this type of intervention activity because it does not emphasize limiting enjoyable sedentary behaviors such as watching television but focuses on finding ways to spend more time during the day with friends participating in enjoyable activities . while several of the recreational sedentary behaviors examined in the current study were associated with lower levels of physical activity and less healthful dietary habits , no associations were observed between recreational sedentary behavior and girls ' bmi z - score . this finding is consistent with other studies that have observed no association or a small association between participation in sedentary behavior and adolescents ' weight status [ 38 , 39 ] . one potential explanation for the lack of associations observed in the current study and others is that excess weight gain may be due to the accumulation of several less healthful behaviors ( e.g. , energy - dense diet , inadequate sleep , sedentary lifestyle ) , and , therefore , only weak associations are observed when examining the role of sedentary behaviors in weight status in isolation of other behaviors . for example , a recent study has observed that obesity risk was higher among adolescents who engaged in both low levels of physical activity and high levels of screen time . additionally , sedentary behaviors may only be associated with obesity risk among some individuals , such as those with a genetic susceptibility to obesity , leading to the observation of null associations between sedentary behavior and weight among the general population . despite the inconsistency of associations reported in observational studies , sedentary behavior is still an important intervention target , and studies that have aimed to reduce sedentary activity have successfully led to decreases in adolescents ' weight status [ 4143 ] . a strength of the current study is its use of a sociodemographically diverse population of adolescent girls who were either currently overweight or obese , or at high risk for overweight due to having a highly sedentary lifestyle . there is a great need to develop relevant and enjoyable programs that encourage greater participation in healthful pa and healthy eating behaviors among this population . however , this focus on a specific , underserved population does limit the generalizability of study findings , and the sedentary behavior patterns of highly physically active adolescent girls may be different than what was observed in this study population . an additional strength of this study is the use of the 3dpar , which allowed for an understanding of the context of girls ' sedentary time . while objective measures of pa and sedentary behavior , such as accelerometry , play an important role in obtaining an unbiased assessment of pa , use of self - report measures that record the specific activities that study participants are engaging in can provide great richness to the understanding of how individuals are spending their time . therefore multitasking , which adolescents do frequently when watching television , or behaviors that participants engaged in for less than 15 minutes , were not recorded with this instrument . however , it is important to note that girls rarely asked how to categorize texting during the data collection process , as they were often texting for short amounts of time throughout the day and not for the majority of a 30-minute time period . girls who did ask how to code their texting time were told to categorize it with internet use / instant messaging , which may have inflated the assessment of the amount of time girls spent on the computer . future studies utilizing the 3dpar are encouraged to conduct extensive pilot testing of the measure and adapt the activity options provided based on the popular activities among the intended study population . adolescent girls participate in a number of recreational sedentary behaviors for a significant portion of their free time . understanding these patterns of behavior , and associations between recreational sedentary behaviors and pa and dietary habits , is informative to the development of interventions aiming to decrease adolescents ' risk for overweight and obesity . findings from the current study suggest that interventions developed to engage adolescent girls may benefit from incorporating messages and behavioral targets that address recreational sedentary behaviors such as hanging around , talking on the phone , and listening to music , in addition to the often - targeted watching television . future research in this area is also needed to understand whether girls ' participation in specific recreational behaviors contributes to decreases in pa and poor dietary intake , or whether the behaviors co - occur and are markers of a less healthful lifestyle in total . additionally , in light of the lack of associations observed in the current study between recreational sedentary behavior and bmi , and the inconsistent findings between sedentary behavior and weight observed elsewhere in the literature [ 38 , 45 ] , further investigations of the role of television , screen time , and other common recreational sedentary behaviors in adolescents ' weight and excess weight gain are a priority .

trypanosoma cruzi ( t. cruzi ) is the etiological agent of chagas disease ( cd ) . it is estimated that 8 million people are infected worldwide . in the endemic area of south and central america , cd is transmitted through contact with the feces of the triatomine bug ( the kissing bug ) . when taking a blood meal from a human , the bug defecates on the skin where t. cruzi can enter the wound or the mucosal membrane by scratching . effective vector - control programs have greatly decreased disease transmission in these areas [ 2 , 3 ] . however , cd was brought to north america , europe , and asia by infected individuals , through migration in recent years . in nonendemic area , cd is transmitted through blood transfusion , organ transplantation , and congenital transmission . during the t. cruzi infection process , the parasite interacts with a wide range of host immunological and metabolic factors . in the past decade , special attention was given to the close relationship between t. cruzi infection and host lipid metabolism . several research groups have uncovered the interaction between t. cruzi and players in the host cholesterol transport and storage system such as macrophage [ 57 ] , adipocytes , low density lipoprotein ( ldl ) , and high density lipoprotein ( hdl ) [ 911 ] . the molecular landscape and impact of these relationships in t. cruzi infection and pathogenesis , as well as host immunological responses and inflammatory reactions , will be reviewed in this paper . although the majority of infected individuals are asymptomatic while carrying the life - long infection , some develop severe symptoms upon infection . during the acute stage , infected individuals may develop unspecific symptoms such as fever , nausea , diarrhea , and rash , as well as severe symptoms such as a raised inflammatory lesion at the site of parasite entry ( chagoma ) , unilateral periorbital edema ( romana 's sign ) , lymphadenopathy , and hepatosplenomegaly . the majority of patients survive the acute stage and enter the prolonged indeterminate stage without overt symptoms of disease , which lasts for life . however , thirty percent of patients develop chronic cd , which includes grave symptoms such as megaesophagus , megacolon , and chronic heart disease . t. cruzi has a complex life cycle and undergoes several transformations during the infection process . it transforms into metacyclic trypomastigote in the hind gut of the vector which is then defecated to infect human host . once in the host , the metacyclic form infects a wide range of phagocytic ( i.e. , monocytes , neutrophils , mast cells , and macrophages ) and nonphagocytic cells ( i.e. , epithelial cells , endothelial cells , fibroblasts , and mesenchymal cells ) . upon infection , trypomastigotes transform into intracellular amastigotes and divide by binary fission . once the division process is complete , amastigotes transform back into blood trypomastigotes which escape the cell to infect neighbouring cells or enter the blood circulation . lipid bodies ( lb ) , also named lipid droplets or adiposomes , are lipid - rich organelles existing in almost all organisms . unlike other organelles the core of the lipid body is rich in neutral lipids , mainly triacylglycerol and sterol esters , as well as other putative membranous structures . historically , lipid bodies were thought to function in neutral lipid storage and transport ; however , recent research has uncovered their importance in regulation of host immune responses . lipid bodies are involved in the formation of paracrine mediator eicosanoids in cells involved in inflammatory processes [ 16 , 17 ] . the number of lipid bodies in leukocytes increases in response to a variety of inflammatory conditions , such as atherosclerosis and mycobacterial infections [ 18 , 19 ] . during acute t. cruzi infection , host macrophages are strongly activated and will inhibit parasite replication . it has been demonstrated that activated murine macrophages are capable of killing the parasites in vitro [ 57 ] . the macrophage inhibition of parasite replication also correlated positively with increases in the oxidative burst activity , tumor necrosis factor - alpha production ( tnf- ) , and nitric oxide secretion . macrophages from more resistant c57/bl6 mice strain also secreted higher tnf- in the in vivo experiments compared to macrophages from the susceptible strains , such as c3h and balb . in macrophage - depleted t. cruzi infected rats , myocardial parasite load as well as blood parasitemia was significantly increased compared to control . when irradiate rats , which have very low numbers of t and b lymphocytes , were treated with recombinant interferon- ( ifn- ) , which classically activates host macrophages , t. cruzi parasite load was significantly reduced . however , the roles of macrophage in t. cruzi infection may not be as simple as previously thought . melo showed that , during acute t. cruzi infection , there is a prominent increase in the number of lipid bodies in macrophages . it was further demonstrated that the induction of lipid body formation during t. cruzi infection was toll - like receptor ( tlr-2 ) dependent and was enhanced by the uptake of apoptotic cells , which causes macrophage to interact with v3 integrin and activates tgf--dependent lipid body formation [ 27 , 28 ] . increased levels of tgf- are known to cause phagocytic cells to become permissive to t. cruzi infection [ 29 , 30 ] ( figure 1(a ) ) . increased lipid body formation also led to increased eicosanoid prostaglandin e2 ( pge2 ) production in inflammatory macrophages prostaglandins are known to inhibit tnf- and ifn- production , while enhancing tgf- secretion [ 3133 ] . although the impact of pge2 release in t. cruzi infection is contradictory , the release of pge2 was correlated in resistance against certain strains of t. cruzi infection . in addition , treatment with nonsteroidal anti - inflammatory drugs ( nsaids ) or cyclooxygenase ( cox ) inhibitors was able to modulate lipid body formation and decrease pge2 production , which led to decreased parasite growth in macrophages [ 27 , 36 ] . furthermore , these newly formed lipid bodies also varied significantly in size and light density , which indicated the structural participation of these organelles in immune responses to t. cruzi infection . the structural alterations of lb in macrophages may be related to the different lipid compositions in the organelle , stage of new lb formation , or fluctuation of the arachidonate production and concentration . ultrastructural investigation revealed that the newly formed lipid bodies are localized in close proximity to macrophage phagolysosomes or even within these structures . this suggests that lipid bodies may interact with the phagolysosomes during acute t. cruzi infection . lipid bodies are known to provide nutrients to intracellular parasites such as leishmania chagasi , which are located in the phagolysosome . the relationship between lipid body and phagolysosome can also be beneficial to the host . as reviewed by melo et al . , lipids recruited during lipid body formation , such as arachidonic acid ( aa ) , are able to activate actin assembly , phagosome - lysosome fusion , and phagosome maturation [ 40 , 41 ] . in addition , these lipids can activate phagosomal nicotinamide adenine dinucleotide phosphate- ( nadph- ) oxidase , which leads to pathogen elimination . the implication of the close localization of lipid bodies and phagolysosome in t. cruzi infected macrophages needs to be further investigated . it is comprised of a wide range of cell types including adipocytes , pericytes , monocytes , macrophages , and endothelial cells . more than 95% of adipocyte cell mass is lipid droplets where triglycerides and cholesterol esters are stored ; however , it was recently uncovered that the functions of adipose tissue include not only energy storage , but also metabolic regulation , neuroendocrine , and immune regulations . adipose tissue is home to a variety of adipokines , such as adiponectin , leptin , and resistin , which are prominent regulators of lipid homeostasis and immunological functions . recently , metabolic dysfunction was linked to cd pathogenesis by the observation that there are greater incidences of diabetes in t. cruzi infected individuals . later research showed decreased insulin level and dysregulated glucose responses among cd patients [ 50 , 51 ] , which further demonstrated the dysregulation of energy metabolism in these patients . it was also shown that chemically induced diabetic mice as well as genetically predisposed db / db diabetic mice with defective leptin receptors had higher parasitemia and mortality after t. cruzi infection , which suggests that the dysregulation of host metabolism may be beneficial to parasitic survival in the host . mice infected with t. cruzi showed symptoms of hypoglycemia during the acute stage of infection ; however , insulin sensitivity was unaltered . levels of adiponectin and leptin were significantly reduced in t. cruzi infected mice , which further suggest the altered state of glucose regulation and possible adipocyte involvement in disease progression . adiponectin is the only adipokine secreted exclusively by adipocytes and is strongly associated with insulin resistance and hyperglycemia . high parasite load was detected in the adipose tissue at the chronic stage , 300 days postinfection , as measured by quantitative polymerase chain reaction ( qpcr ) . decreased levels of adiponectin in the plasma and adipose tissue of infected mice were also observed during the chronic stage . microscopic investigation revealed the preferred localization of t. cruzi in the brown fat of adipose tissue , where lipid bodies are higher in number and smaller in size compared to white adipocytes . these findings suggest that adipose tissues may serve as the parasitic reservoir during chronic infection and adipokine synthesis was disrupted possibly due to the infection . observations that t. cruzi parasite is present in the adipose tissue biopsy of chronically infected human patients have further confirmed the finding that adipose tissue is the reservoir of chronic t. cruzi infection . several follow - up studies have also shown the susceptible nature of adipocytes to t. cruzi infection [ 8 , 56 ] . in vitro infection of cultured adipocytes with t. cruzi revealed that a panel of proinflammatory cytokines was upregulated ; these include il-1 , ifn- , tnf- , chemokine ligand ( ccl2 ) , ccl5 , and c - x - c motif chemokine 10 ( cxcl10 ) the expressions of tlr-2 and 9 are also upregulated . other pathways , such as notch , extracellular signaling - regulated kinases ( erk ) , and phosphoinositide-3-kinases ( pi3k ) , were also activated . it was shown that both erk and pi3k pathways were activated upon t. cruzi infection [ 57 , 58 ] . furthermore , ppar- is highly expressed in adipose tissue and , along with adiponectin , exerts anti - inflammatory effect . levels of peroxisome proliferator - activated receptor ( ppar- ) were decreased in the infected cells , which may have led to the decreased secretion of adiponectin and increased inflammatory reactions . these findings suggest that infection of adipocytes with t. cruzi may contribute to the systemic proinflammatory immune responses as well as metabolic dysregulation ( figure 1(b ) ) . in summary , recent research has revealed that adipose tissue may be the most important reservoir for t. cruzi chronic infection and these infected adipocytes display a proinflammatory phenotype . altered activation profile of several kinase pathways in adipose tissues may also contribute to host metabolic dysregulation . it is clear that chronic t. cruzi infection displays tissue tropism ; however the evolutionary benefits of t. cruzi residing in adipocytes are unknown . t. cruzi may utilize the lipid stores within the adipocytes for its multiplication and survival . it is also possible that t. cruzi chooses adipocytes for its prolonged life - span . further research is needed to unravel the biological processes behind the relationship between t. cruzi and adipocytes . t. cruzi glycoprotein 85 ( gp85)/trans - sialidase is similar to that of viral and bacterial neuraminidases . however , unlike other neuraminidases , upon hydrolysis of -linked sialic acid from glycoconjugates on cell surfaces , t. cruzi trans - sialidase transfers the sialic acid onto parasitic receptors . the expression and activity of trans - sialidase are developmentally regulated and are present at about the same extent in epimastigotes and trypomastigotes . trans - sialidase is known to be involved in trypomastigote cell adhesion and invasion process by interacting with a wide range of ligands , such as laminin , fibronectin , and collagen [ 6265 ] . inhibition of t. cruzi trans - sialidase by specific antibodies led to the increased rate of infection . cholesterol transport chains are the major components of maintaining host lipid homeostasis and lipoproteins are essential players in these pathways . lipoproteins are categorized based on their density and protein content into high density lipoproteins ( hdl , density 1.6031.210 ) , low density lipoproteins ( ldl , density 1.0191.603 ) , intermediate density lipoproteins ( idl , density 1.0061.019 ) , very low density lipoproteins ( vldl , density 0.951.006 ) , and chylomicrons ( density < 0.95 ) . all lipoproteins allow the transport of hydrophobic lipid contents , such as cholesterol , triglycerides , and phospholipids , within the hydrophilic blood circulation system . ldl is characterized by the presence of a single copy of apolipoprotein b-100 ( apo b-100 ) molecule on its surface . it is generated from liver - derived vldl by a process mediated by lipoprotein lipase and hepatic lipase as well as lipid exchange proteins [ 67 , 68 ] . ldl has been shown to be a potent inhibitor of t. cruzi trans - sialidase and enhances the infection of human fibroblasts in vitro in a dose - dependent manner . the enhanced infection rate seen upon the addition of ldl in vitro is comparable to that of the enhancement caused by trans - sialidase inhibition . ldl particles were seen covering the parasite cellular surface of t. cruzi trypomastigotes , but not amastigotes . the localization of ldl particles correlates with trans - sialidase localization on the parasite surface and suggests that ldl may directly inhibit t. cruzi surface trans - sialidase to enhance rate of infection ( figure 1(c ) ) . immunoelectron microscopy showed that trans - sialidase expression is most concentrated in the flagellar pocket region , which suggested that despite ldl inhibition of t. cruzi trans - sialidase , trans - sialidase may also facilitate ldl endocytosis by the parasite . this organelle in the parasite provides support for metacyclogenesis from epimastigotes to trypomastigotes [ 71 , 72 ] . ldl particles were also found in the t. cruzi membrane enclosed vesicles and reservosome within the parasite . ldl may be stored and processed in the reservosome for usage during this transformation and infection process . similar process of ldl uptake was also demonstrated in leishmania amazonensis , a parasite closely related to t. cruzi in the trypanosomatidae family . another important molecule in the ldl metabolic cycle is the ldl receptor ( ldlr ) . ldlr plays an essential role in the internalization of circulating ldl in the host liver and peripheral cells . a significant amount of cholesterol is delivered to these organs via the interaction of ldl - ldlr . ldlr also facilitates the endocytosis of a variety of other ligands , such as proteinases and proteinase - inhibitor complexes , as well as interacting with cytoplasmic adaptor proteins which have signaling transduction functions . the expression of ldlr by the host cell is regulated by a wide range of lipid metabolic and immune regulatory stimuli , such as intracellular cholesterol level , oxysterols , various growth factors , and cytokines [ 78 , 79 ] . demonstrated that , in human mesangial cells , increased levels of tnf- , tgf- , and il1- caused increased transcription of ldlr . ldlr was previously shown to be a potential host receptor for hepatitis c virus ( hcv ) and other flaviviridae viruses [ 81 , 82 ] . activation of ldlr facilitates the recruitment of lysosomes to the parasitophorous vacuole , which leads to the internalization of t. cruzi into the cytoplasm . disruption of ldlr by genetic knockout resulted in 62% reduction in t. cruzi infection , which suggests ldlr is essential for t. cruzi cell invasion process ( figure 1(c ) ) . furthermore , upregulation of ldlr expression was also seen in the heart of t. cruzi infected cd1 mice . moreover , in toxoplasma gondii infection , ldlr functions to uptake ldl particles and support intracellular parasite growth . it is recently demonstrated that t. cruzi interaction with ldl receptor leads to the increased accumulation of ldl - cholesterol in host tissue in both acute and chronic cd . alterations in the micro- and macrovascular circulations and atherosclerosis - like symptoms are commonly seen in cardiomyopathic patients [ 86 , 87 ] . reported that t. cruzi infection in combination with a high cholesterol diet can induce early symptoms of atherosclerosis in mice [ 88 , 89 ] . it is known that ldl particles are transported across the endothelium and become trapped in the matrix of arterial wall cells , which leads to the production of highly cytotoxic oxidized ldl and subsequently activates inflammatory pathways , such as nfb . the interaction of t. cruzi with ldlr may increase host susceptibility to atherosclerosis and arterial pathology . in addition to the parasite interaction with ldl and ldlr , t. cruzi also interacts with hdl ( originally named cruzin in t. cruzi research ) , the major component of the reverse cholesterol transport pathway . hdl is a complex , multistructured particle consisting of two layers of phospholipids that are held together by two molecules of apolipoprotein a - i ( apo a - i ) . the main function of hdl is to remove excess cholesterol from peripheral tissues and return it to the liver for storage and excretion . other functions of hdl also include inhibiting ldl oxidation , platelet aggregation and coagulations , and endothelial inflammation , as well as promoting endothelial nitric oxide production and prostacyclin bioavailability [ 93 , 94 ] . cruzi interaction , hdl was shown to bind to and inhibit t. cruzi trypomastigotes trans - sialidase activity [ 11 , 95 ] . interestingly , this interaction is specific for t. cruzi and was not found in trypanosoma rangeli , an infectious agent nonpathogenic to human hosts . t. cruzi and t. rangeli overlap geographically , share antigenic protein , and are able to infect the same triatominae vector and vertebrate hosts . hdl inhibition of t. cruzi trans - sialidase functions in a dose - dependent manner through a reversible noncompetitive mechanism . trans - sialidase occurs in less than 5 min and lasts more than 120 min . more importantly , hdl inhibition of t. cruzi trans - sialidase enhances parasite infection in vitro . recently , weizong et al . have discovered similar interaction between apo a - i and dengue virus . the research group showed that apo a - i is associated with the virus particles and preincubation of dengue virus with hdl enhances viral infection through a scavenger receptor - bi- ( sr - bi- ) mediated mechanism . these findings may also provide a possible mechanism for the enhancement of t. cruzi infection by hdl ( figure 1(e ) ) . furthermore , our research has shown that , during the intracellular amastigote stage of infection , groups infected in the presence of hdl had lower number of intracellular parasites than groups without hdl ( q. miao & m. ndao , personal communication ) . it is possible that hdl inhibition of t. cruzi trans - sialidase led to the decreased rate of trypomastigotes escaping from the parasitophorous vacuole and delaying the process of trypomastigote transformation . in the t. cruzi epimastigote t. brucei ( african trypanosome ) is closely related to t. cruzi ( american trypanosome ) in evolutionary lineage and shares a high level of biological resemblance . in the interaction of hdl with t. brucei , hdl is named trypanolytic factor ( tlf ) , because endocytosis of certain hdl subspecies , which contain haptoglobin - related protein ( hpr , tlf-1 ) and apolipoprotein l - i ( apo l - i , tlf-2 ) , causes lysis of t. b. brucei and protects mammalian hosts from infection . the interaction between hdl and t. cruzi was recently reinforced by the discovery that the major structural component of hdl , apolipoprotein a - i ( apo a - i , full - length 28.1 kda ) , is truncated into fragments ( 24.7 , 13.6 , 10.3 , and 9.3 kda ) in sera of t. cruzi infected patients . apo a - i ( 243 amino acids ) accounts for ~75% of hdl protein content . both the n- and the c - termini of apo a - i are involved in lipid binding functions [ 103105 ] . the central domain of the apo a - i protein is involved in the activation of lecithin - cholesterol acyltransferase ( lcat ) , which is responsible for the esterification and storage of cholesterol within hdl particles . minor changes in the apo a - i amino acid sequence or structure could seriously affect hdl function . therefore , apo a - i truncation seen in t. cruzi infection may contribute to the dysregulation of host lipid metabolism . however , the unique truncation pattern seen in these patients has high discriminatory power between infected and uninfected patients and can be used as t. cruzi diagnostic biomarkers [ 101 , 108 , 109 ] . our research has revealed that the series of apo a - i truncations was facilitated by the major cysteine protease of t. cruzi , cruzipain , which is also known as gp 57/51 or cruzain . this protease which belongs to the mammalian papain superfamily is known to cleave immunoglobulin class g proteins [ 110 , 111 ] . cruzipain has an essential function in the invasion and survival processes of t. cruzi and is expressed in all developmental stages of the parasite life cycle . at each stage , cruzipain is differentially located within the parasite to carry out stage specific functions [ 112 , 113 ] . in the t. cruzi trypomastigote form , cruzipain is located on the parasite surface , flagellar pocket , and lysosome - like structure [ 114 , 115 ] . it was also shown that cruzipain was only able to cleave apo a - i at an acidic ph , which suggests that the cleavage may take place within acidic environments . furthermore , cruzipain from parasite surface ( figure 2(a ) ) and cruzipain within the lysosome - like structure ( figure 2(b ) ) are both required in order to produce the truncation pattern . it is interesting to note that the localization of cruzipain highly resembles that of trans - sialidase . therefore , it is possible that hdl is both endocytosed by trypomastigotes and bound to the surface of the parasite via trans - sialidase . during the infection process , the parasite bound hdl is cleaved by cruzipain in the acidic parasitophorous vacuole . with the emerging evidence , it is becoming obvious that t. cruzi exploits the complex cholesterol transport system via a variety of molecules such as ldl , ldl - r , and hdl . the results of these interactions seem to all lead to the establishment of t. cruzi infection and chagas disease chronicity . the role of host lipid metabolism in response to infectious agents is drawing increasing attention . this review may aid in deeper understanding of t. cruzi interacting with host lipid metabolism with a more systematic approach , as well as the role of lipids in t. cruzi pathogenesis . we have clearly illustrated that t. cruzi interacts with several specific factors in host lipid metabolism . further research in these interactions and the role of lipids in t. cruzi pathogenesis will be highly useful in the future .

trypanosoma cruzi is the causative agent of chagas disease . approximately 8 million people are thought to be affected worldwide . several players in host lipid metabolism have been implicated in t. cruzi - host interactions in recent research , including macrophages , adipocytes , low density lipoprotein ( ldl ) , low density lipoprotein receptor ( ldlr ) , and high density lipoprotein ( hdl ) . all of these factors are required to maintain host lipid homeostasis and are intricately connected via several metabolic pathways . we reviewed the interaction of t. cruzi with each of the relevant host components , in order to further understand the roles of host lipid metabolism in t. cruzi infection . this review sheds light on the potential impact of t. cruzi infection on the status of host lipid homeostasis .

1. Introduction 2. 3. 4. 5. Conclusion

trypanosoma cruzi ( t. cruzi ) is the etiological agent of chagas disease ( cd ) . it is estimated that 8 million people are infected worldwide . in the endemic area of south and central america , cd is transmitted through contact with the feces of the triatomine bug ( the kissing bug ) . when taking a blood meal from a human , the bug defecates on the skin where t. cruzi can enter the wound or the mucosal membrane by scratching . however , cd was brought to north america , europe , and asia by infected individuals , through migration in recent years . in nonendemic area , cd is transmitted through blood transfusion , organ transplantation , and congenital transmission . during the t. cruzi infection process , the parasite interacts with a wide range of host immunological and metabolic factors . in the past decade , special attention was given to the close relationship between t. cruzi infection and host lipid metabolism . several research groups have uncovered the interaction between t. cruzi and players in the host cholesterol transport and storage system such as macrophage [ 57 ] , adipocytes , low density lipoprotein ( ldl ) , and high density lipoprotein ( hdl ) [ 911 ] . the molecular landscape and impact of these relationships in t. cruzi infection and pathogenesis , as well as host immunological responses and inflammatory reactions , will be reviewed in this paper . during the acute stage , infected individuals may develop unspecific symptoms such as fever , nausea , diarrhea , and rash , as well as severe symptoms such as a raised inflammatory lesion at the site of parasite entry ( chagoma ) , unilateral periorbital edema ( romana 's sign ) , lymphadenopathy , and hepatosplenomegaly . however , thirty percent of patients develop chronic cd , which includes grave symptoms such as megaesophagus , megacolon , and chronic heart disease . t. cruzi has a complex life cycle and undergoes several transformations during the infection process . it transforms into metacyclic trypomastigote in the hind gut of the vector which is then defecated to infect human host . , monocytes , neutrophils , mast cells , and macrophages ) and nonphagocytic cells ( i.e. , epithelial cells , endothelial cells , fibroblasts , and mesenchymal cells ) . lipid bodies ( lb ) , also named lipid droplets or adiposomes , are lipid - rich organelles existing in almost all organisms . unlike other organelles the core of the lipid body is rich in neutral lipids , mainly triacylglycerol and sterol esters , as well as other putative membranous structures . historically , lipid bodies were thought to function in neutral lipid storage and transport ; however , recent research has uncovered their importance in regulation of host immune responses . during acute t. cruzi infection , host macrophages are strongly activated and will inhibit parasite replication . the macrophage inhibition of parasite replication also correlated positively with increases in the oxidative burst activity , tumor necrosis factor - alpha production ( tnf- ) , and nitric oxide secretion . in macrophage - depleted t. cruzi infected rats , myocardial parasite load as well as blood parasitemia was significantly increased compared to control . when irradiate rats , which have very low numbers of t and b lymphocytes , were treated with recombinant interferon- ( ifn- ) , which classically activates host macrophages , t. cruzi parasite load was significantly reduced . however , the roles of macrophage in t. cruzi infection may not be as simple as previously thought . melo showed that , during acute t. cruzi infection , there is a prominent increase in the number of lipid bodies in macrophages . it was further demonstrated that the induction of lipid body formation during t. cruzi infection was toll - like receptor ( tlr-2 ) dependent and was enhanced by the uptake of apoptotic cells , which causes macrophage to interact with v3 integrin and activates tgf--dependent lipid body formation [ 27 , 28 ] . increased levels of tgf- are known to cause phagocytic cells to become permissive to t. cruzi infection [ 29 , 30 ] ( figure 1(a ) ) . although the impact of pge2 release in t. cruzi infection is contradictory , the release of pge2 was correlated in resistance against certain strains of t. cruzi infection . furthermore , these newly formed lipid bodies also varied significantly in size and light density , which indicated the structural participation of these organelles in immune responses to t. cruzi infection . the structural alterations of lb in macrophages may be related to the different lipid compositions in the organelle , stage of new lb formation , or fluctuation of the arachidonate production and concentration . this suggests that lipid bodies may interact with the phagolysosomes during acute t. cruzi infection . , lipids recruited during lipid body formation , such as arachidonic acid ( aa ) , are able to activate actin assembly , phagosome - lysosome fusion , and phagosome maturation [ 40 , 41 ] . the implication of the close localization of lipid bodies and phagolysosome in t. cruzi infected macrophages needs to be further investigated . it is comprised of a wide range of cell types including adipocytes , pericytes , monocytes , macrophages , and endothelial cells . more than 95% of adipocyte cell mass is lipid droplets where triglycerides and cholesterol esters are stored ; however , it was recently uncovered that the functions of adipose tissue include not only energy storage , but also metabolic regulation , neuroendocrine , and immune regulations . adipose tissue is home to a variety of adipokines , such as adiponectin , leptin , and resistin , which are prominent regulators of lipid homeostasis and immunological functions . recently , metabolic dysfunction was linked to cd pathogenesis by the observation that there are greater incidences of diabetes in t. cruzi infected individuals . later research showed decreased insulin level and dysregulated glucose responses among cd patients [ 50 , 51 ] , which further demonstrated the dysregulation of energy metabolism in these patients . it was also shown that chemically induced diabetic mice as well as genetically predisposed db / db diabetic mice with defective leptin receptors had higher parasitemia and mortality after t. cruzi infection , which suggests that the dysregulation of host metabolism may be beneficial to parasitic survival in the host . mice infected with t. cruzi showed symptoms of hypoglycemia during the acute stage of infection ; however , insulin sensitivity was unaltered . levels of adiponectin and leptin were significantly reduced in t. cruzi infected mice , which further suggest the altered state of glucose regulation and possible adipocyte involvement in disease progression . adiponectin is the only adipokine secreted exclusively by adipocytes and is strongly associated with insulin resistance and hyperglycemia . microscopic investigation revealed the preferred localization of t. cruzi in the brown fat of adipose tissue , where lipid bodies are higher in number and smaller in size compared to white adipocytes . observations that t. cruzi parasite is present in the adipose tissue biopsy of chronically infected human patients have further confirmed the finding that adipose tissue is the reservoir of chronic t. cruzi infection . several follow - up studies have also shown the susceptible nature of adipocytes to t. cruzi infection [ 8 , 56 ] . in vitro infection of cultured adipocytes with t. cruzi revealed that a panel of proinflammatory cytokines was upregulated ; these include il-1 , ifn- , tnf- , chemokine ligand ( ccl2 ) , ccl5 , and c - x - c motif chemokine 10 ( cxcl10 ) the expressions of tlr-2 and 9 are also upregulated . other pathways , such as notch , extracellular signaling - regulated kinases ( erk ) , and phosphoinositide-3-kinases ( pi3k ) , were also activated . it was shown that both erk and pi3k pathways were activated upon t. cruzi infection [ 57 , 58 ] . levels of peroxisome proliferator - activated receptor ( ppar- ) were decreased in the infected cells , which may have led to the decreased secretion of adiponectin and increased inflammatory reactions . these findings suggest that infection of adipocytes with t. cruzi may contribute to the systemic proinflammatory immune responses as well as metabolic dysregulation ( figure 1(b ) ) . in summary , recent research has revealed that adipose tissue may be the most important reservoir for t. cruzi chronic infection and these infected adipocytes display a proinflammatory phenotype . it is clear that chronic t. cruzi infection displays tissue tropism ; however the evolutionary benefits of t. cruzi residing in adipocytes are unknown . t. cruzi may utilize the lipid stores within the adipocytes for its multiplication and survival . it is also possible that t. cruzi chooses adipocytes for its prolonged life - span . further research is needed to unravel the biological processes behind the relationship between t. cruzi and adipocytes . t. cruzi glycoprotein 85 ( gp85)/trans - sialidase is similar to that of viral and bacterial neuraminidases . however , unlike other neuraminidases , upon hydrolysis of -linked sialic acid from glycoconjugates on cell surfaces , t. cruzi trans - sialidase transfers the sialic acid onto parasitic receptors . the expression and activity of trans - sialidase are developmentally regulated and are present at about the same extent in epimastigotes and trypomastigotes . trans - sialidase is known to be involved in trypomastigote cell adhesion and invasion process by interacting with a wide range of ligands , such as laminin , fibronectin , and collagen [ 6265 ] . inhibition of t. cruzi trans - sialidase by specific antibodies led to the increased rate of infection . cholesterol transport chains are the major components of maintaining host lipid homeostasis and lipoproteins are essential players in these pathways . lipoproteins are categorized based on their density and protein content into high density lipoproteins ( hdl , density 1.6031.210 ) , low density lipoproteins ( ldl , density 1.0191.603 ) , intermediate density lipoproteins ( idl , density 1.0061.019 ) , very low density lipoproteins ( vldl , density 0.951.006 ) , and chylomicrons ( density < 0.95 ) . all lipoproteins allow the transport of hydrophobic lipid contents , such as cholesterol , triglycerides , and phospholipids , within the hydrophilic blood circulation system . ldl has been shown to be a potent inhibitor of t. cruzi trans - sialidase and enhances the infection of human fibroblasts in vitro in a dose - dependent manner . the enhanced infection rate seen upon the addition of ldl in vitro is comparable to that of the enhancement caused by trans - sialidase inhibition . ldl particles were seen covering the parasite cellular surface of t. cruzi trypomastigotes , but not amastigotes . the localization of ldl particles correlates with trans - sialidase localization on the parasite surface and suggests that ldl may directly inhibit t. cruzi surface trans - sialidase to enhance rate of infection ( figure 1(c ) ) . immunoelectron microscopy showed that trans - sialidase expression is most concentrated in the flagellar pocket region , which suggested that despite ldl inhibition of t. cruzi trans - sialidase , trans - sialidase may also facilitate ldl endocytosis by the parasite . ldl particles were also found in the t. cruzi membrane enclosed vesicles and reservosome within the parasite . similar process of ldl uptake was also demonstrated in leishmania amazonensis , a parasite closely related to t. cruzi in the trypanosomatidae family . another important molecule in the ldl metabolic cycle is the ldl receptor ( ldlr ) . a significant amount of cholesterol is delivered to these organs via the interaction of ldl - ldlr . the expression of ldlr by the host cell is regulated by a wide range of lipid metabolic and immune regulatory stimuli , such as intracellular cholesterol level , oxysterols , various growth factors , and cytokines [ 78 , 79 ] . demonstrated that , in human mesangial cells , increased levels of tnf- , tgf- , and il1- caused increased transcription of ldlr . ldlr was previously shown to be a potential host receptor for hepatitis c virus ( hcv ) and other flaviviridae viruses [ 81 , 82 ] . activation of ldlr facilitates the recruitment of lysosomes to the parasitophorous vacuole , which leads to the internalization of t. cruzi into the cytoplasm . disruption of ldlr by genetic knockout resulted in 62% reduction in t. cruzi infection , which suggests ldlr is essential for t. cruzi cell invasion process ( figure 1(c ) ) . furthermore , upregulation of ldlr expression was also seen in the heart of t. cruzi infected cd1 mice . moreover , in toxoplasma gondii infection , ldlr functions to uptake ldl particles and support intracellular parasite growth . it is recently demonstrated that t. cruzi interaction with ldl receptor leads to the increased accumulation of ldl - cholesterol in host tissue in both acute and chronic cd . reported that t. cruzi infection in combination with a high cholesterol diet can induce early symptoms of atherosclerosis in mice [ 88 , 89 ] . the interaction of t. cruzi with ldlr may increase host susceptibility to atherosclerosis and arterial pathology . in addition to the parasite interaction with ldl and ldlr , t. cruzi also interacts with hdl ( originally named cruzin in t. cruzi research ) , the major component of the reverse cholesterol transport pathway . hdl is a complex , multistructured particle consisting of two layers of phospholipids that are held together by two molecules of apolipoprotein a - i ( apo a - i ) . other functions of hdl also include inhibiting ldl oxidation , platelet aggregation and coagulations , and endothelial inflammation , as well as promoting endothelial nitric oxide production and prostacyclin bioavailability [ 93 , 94 ] . cruzi interaction , hdl was shown to bind to and inhibit t. cruzi trypomastigotes trans - sialidase activity [ 11 , 95 ] . interestingly , this interaction is specific for t. cruzi and was not found in trypanosoma rangeli , an infectious agent nonpathogenic to human hosts . t. cruzi and t. rangeli overlap geographically , share antigenic protein , and are able to infect the same triatominae vector and vertebrate hosts . hdl inhibition of t. cruzi trans - sialidase functions in a dose - dependent manner through a reversible noncompetitive mechanism . more importantly , hdl inhibition of t. cruzi trans - sialidase enhances parasite infection in vitro . these findings may also provide a possible mechanism for the enhancement of t. cruzi infection by hdl ( figure 1(e ) ) . it is possible that hdl inhibition of t. cruzi trans - sialidase led to the decreased rate of trypomastigotes escaping from the parasitophorous vacuole and delaying the process of trypomastigote transformation . in the t. cruzi epimastigote t. brucei ( african trypanosome ) is closely related to t. cruzi ( american trypanosome ) in evolutionary lineage and shares a high level of biological resemblance . in the interaction of hdl with t. brucei , hdl is named trypanolytic factor ( tlf ) , because endocytosis of certain hdl subspecies , which contain haptoglobin - related protein ( hpr , tlf-1 ) and apolipoprotein l - i ( apo l - i , tlf-2 ) , causes lysis of t. b. brucei and protects mammalian hosts from infection . the interaction between hdl and t. cruzi was recently reinforced by the discovery that the major structural component of hdl , apolipoprotein a - i ( apo a - i , full - length 28.1 kda ) , is truncated into fragments ( 24.7 , 13.6 , 10.3 , and 9.3 kda ) in sera of t. cruzi infected patients . both the n- and the c - termini of apo a - i are involved in lipid binding functions [ 103105 ] . the central domain of the apo a - i protein is involved in the activation of lecithin - cholesterol acyltransferase ( lcat ) , which is responsible for the esterification and storage of cholesterol within hdl particles . therefore , apo a - i truncation seen in t. cruzi infection may contribute to the dysregulation of host lipid metabolism . however , the unique truncation pattern seen in these patients has high discriminatory power between infected and uninfected patients and can be used as t. cruzi diagnostic biomarkers [ 101 , 108 , 109 ] . our research has revealed that the series of apo a - i truncations was facilitated by the major cysteine protease of t. cruzi , cruzipain , which is also known as gp 57/51 or cruzain . cruzipain has an essential function in the invasion and survival processes of t. cruzi and is expressed in all developmental stages of the parasite life cycle . in the t. cruzi trypomastigote form , cruzipain is located on the parasite surface , flagellar pocket , and lysosome - like structure [ 114 , 115 ] . furthermore , cruzipain from parasite surface ( figure 2(a ) ) and cruzipain within the lysosome - like structure ( figure 2(b ) ) are both required in order to produce the truncation pattern . therefore , it is possible that hdl is both endocytosed by trypomastigotes and bound to the surface of the parasite via trans - sialidase . with the emerging evidence , it is becoming obvious that t. cruzi exploits the complex cholesterol transport system via a variety of molecules such as ldl , ldl - r , and hdl . the results of these interactions seem to all lead to the establishment of t. cruzi infection and chagas disease chronicity . the role of host lipid metabolism in response to infectious agents is drawing increasing attention . this review may aid in deeper understanding of t. cruzi interacting with host lipid metabolism with a more systematic approach , as well as the role of lipids in t. cruzi pathogenesis . we have clearly illustrated that t. cruzi interacts with several specific factors in host lipid metabolism . further research in these interactions and the role of lipids in t. cruzi pathogenesis will be highly useful in the future .

an association between premorbid intelligence and later morbidity , including coronary heart disease ( chd ) , has been established in longitudinal studies.14 however , no previous study has investigated whether intelligence affects prognosis of chd . according to the hypothesis that higher intelligence affects health outcomes through a better ability to prevent and manage disease,57 those with higher intelligence should be at lower risk for recurrence and early death after a chd incidence . for instance , poor compliance in medication for secondary prevention cardiovascular diseases ( cvd ) is a frequent problem ; several prospective studies have found that most patients stop taking at least one prescribed medication during the first year after an event.8 intelligence has previously been found to predict medication persistence in a population at risk for cvd.6 the aim of this study was to investigate if lower premorbid intelligence is associated with recurrence among men diagnosed with chd . in addition , we wanted to investigate the association of intelligence with case - fatality and all - cause mortality in this group . we used the swedish conscription cohort of 19691970 , which consists of 49 231 men born in 19491951 . this cohort contains information on physical condition , health behaviours and intelligence obtained by medical examinations , questionnaires and cognitive assessment . record linkage with national registers provided data on morbidity , mortality and measures of socioeconomic factors in adulthood . the study was based on data from a nationwide , mandatory examination of 49 321 swedish men who were conscripted for military service in 19691970 , at age 1820 . the background of the swedish conscription examination and the measurements included have been presented in detail elsewhere.9 10 only 23% of all swedish men were exempted from conscription at that time , in most cases due to severe mental or physical disability . ninety - eight per cent of all men conscripted in 1969 and 1970 were born in 19491951 ; the remaining 2% were born before 1949 and are excluded in order to increase homogeneity . data from national registers were linked to the conscription data and anonymised by statistics sweden . this study includes those 2186 men ( 4.4% ) who obtained a first diagnosis of chd ( see below ) between 1991 and 2007 , age about 4056 , for whom information was available on all variables in this study ( figure 1 ) . ethical approval was obtained from the regional ethical review board in stockholm , decision reference number 2004/5:9639/5 and 2010/604:32 . intelligence was assessed at conscription in 19691970 by four subtests measuring verbal , logic - inductive and visuospatial ability and technical comprehension . the cognitive assessment has been described in detail elsewhere.4 11 12 the results were converted to standard - nine ( stanine ) scales with a normal distribution for each subtest , with scores 19 . these were combined and transformed into a new stanine scale as a global measure of general ability , corresponding to approximate iq bands of : < 74 , 7481 , 8289 , 9095 , 96104 , 105110 , 111118 , 119126 , > 126.13 of the men , 49 262 ( 99.9% ) had an intelligence score in the data . diagnoses of first and recurrent chd ( swedish version of international classification of disease ninth edition ( icd-9 ) up to 1996 , codes 410414 ; thereafter icd-10 , i20-i25 ) were obtained from the swedish national hospital discharge register , and the national cause of death register for out - of - hospital deaths in chd , which are held by the national board of health and welfare . in sweden , men with a first event of chd registered between 1 january 1991 and 31 december 2007 were included in the analyses . follow - up started when a man was diagnosed with chd , at any time during 19912007 , and ended at the date of whichever occurred first : a second fatal or non - fatal event of chd recorded in the national hospital or death registers ( with a minimum of 28 days after the first event ; diagnoses and deaths within 28 days were regarded as reflecting the first event ) , emigration , death from other causes , or 31 december 2008 . the possible follow - up time therefore ranged from 1 to 17 years depending on the time of the first event . first chd events leading to death out of hospital and recorded in the cause of death register but not in hospital registers were included in analyses of case - fatality at baseline , but only men surviving their first chd event were included in follow - up analyses . case - fatality in first chd was defined as death with any underlying cause registered within 28 days of the first hospital admission for chd , as is common in case - fatality research , or chd as underlying cause in case of out - of - hospital death . mortality with icd - codes 390459 ( icd-9 ) or i00-i99 ( icd-10 ) as underlying causes , that is , all cvd , were classified as cvd mortality . an overview of all covariates , including additional information about the variables and any categorisations , is presented in the online supplementary table s1 . at conscription , all men underwent a 2-day examination during which health measures were obtained including weight and length , from which body mass index ( bmi , kg / m ) was calculated , and blood pressure . information on cvd mortality in parents before age 65 was included as an indicator of genetic risk for early cvd . socioeconomic position in childhood ( at age about 10 ) and adulthood ( at age about 40 ) was registered in the national population and housing censuses . the original six and eight occupation - based socioeconomic groups4 were collapsed into three and four , respectively , to retain statistical power . information on income at about 40 years of age ( in 1990 ) and marital status in the year of the first chd event , or the preceding year in case of missing data , was obtained from the longitudinal database of education , income and occupation ( louise ) . education is highly correlated with intelligence and was not included in the main analyses due to the risk for multicollinearity ( correlation about 0.55 in this cohort14 ) and overadjustment , but was added in an additional analysis . since intelligence is associated with various forms of morbidity and possibly the severity of conditions , comorbid conditions and complications might account for any differences in outcome between iq categories . hence , diagnoses obtained from the hospital discharge register , either registered as concomitant diagnoses at discharge after the first chd event or as primary or concomitant diagnoses up to 28 days after or in the 7 years preceding the event , in correspondence to an extension of the ontario ami prediction rule applied by rasmussen et al,15 were included as either comorbid conditions or complications ( diagnoses and corresponding icd codes are shown in online supplementary table s1 ) . depression was also included among comorbid conditions since it is also predictive of recurrence in chd.16 intelligence was modelled in three groups : low ( stanine score 13 ) , medium ( 46 ) and high ( 79 ) , using the highest performing group as reference . descriptive statistics of iq level and covariates were calculated for the full cohort and in subsamples based on men with a first chd 19912007 ( table 1 ) , and of covariates for the three iq categories ( table 2 ) . ors for case - fatality among men with a first chd event 19912007 were estimated by the logistic regression model . among men surviving a first chd event , hrs for a second chd event and for all - cause and cvd mortality , during the period 19912008 in relation to level of intelligence at conscription proportionality of hazards was assessed in log - log survival plots and no major violations against the proportionality assumption were found . after adjustment for age at first chd ( model 1 ) , early - life factors that were considered as potential confounders were added to the model ( model 2 ) , followed by factors measured at conscription or later ( model 3 ) . an additional analysis added education in three levels : 9 , 912 and 12 completed years . additional analyses restricted to myocardial infarction ( mi ; icd-9 410 , icd-10 i2122 ) , applied to both the inclusion criteria ( first event ) and outcome ( recurrence ) , were performed . possible interaction by adult socioeconomic position17 was examined in stratified analyses in groups of white - collar or blue - collar occupations . also , due to the higher mortality among people with lower intelligence , which might cause an underestimation of the iq - recurrence association , we performed an analysis combining recurrences and deaths from any causes as end points . characteristics of men with first chd 19912008 and in the full cohort of conscripts * not within 28 days of first chd . single household at the year or preceding year of first chd event in the samples ; single household in 1990 in the full cohort for comparison . diabetes with complications , pulmonary oedema , acute or chronic renal failure , cerebrovascular disease , malignancy and depression . bmi , body mass index ; chd , coronary heart disease ; cvd , cardiovascular diseases . distribution of risk factors in iq categories among men in the study sample * occupation and income in 1990 , at age about 40 . diabetes with complications , pulmonary oedema , acute or chronic renal failure , cerebrovascular disease , malignancy and depression . bmi , body mass index ; chd , coronary heart disease ; cvd , cardiovascular diseases . intelligence was assessed at conscription in 19691970 by four subtests measuring verbal , logic - inductive and visuospatial ability and technical comprehension . the cognitive assessment has been described in detail elsewhere.4 11 12 the results were converted to standard - nine ( stanine ) scales with a normal distribution for each subtest , with scores 19 . these were combined and transformed into a new stanine scale as a global measure of general ability , corresponding to approximate iq bands of : < 74 , 7481 , 8289 , 9095 , 96104 , 105110 , 111118 , 119126 , > 126.13 of the men , 49 262 ( 99.9% ) had an intelligence score in the data . diagnoses of first and recurrent chd ( swedish version of international classification of disease ninth edition ( icd-9 ) up to 1996 , codes 410414 ; thereafter icd-10 , i20-i25 ) were obtained from the swedish national hospital discharge register , and the national cause of death register for out - of - hospital deaths in chd , which are held by the national board of health and welfare . in sweden , hospital care is publically financed and available to all citizens . men with a first event of chd registered between 1 january 1991 and 31 december 2007 follow - up started when a man was diagnosed with chd , at any time during 19912007 , and ended at the date of whichever occurred first : a second fatal or non - fatal event of chd recorded in the national hospital or death registers ( with a minimum of 28 days after the first event ; diagnoses and deaths within 28 days were regarded as reflecting the first event ) , emigration , death from other causes , or 31 december 2008 . the possible follow - up time therefore ranged from 1 to 17 years depending on the time of the first event . first chd events leading to death out of hospital and recorded in the cause of death register but not in hospital registers were included in analyses of case - fatality at baseline , but only men surviving their first chd event were included in follow - up analyses . case - fatality in first chd was defined as death with any underlying cause registered within 28 days of the first hospital admission for chd , as is common in case - fatality research , or chd as underlying cause in case of out - of - hospital death . mortality with icd - codes 390459 ( icd-9 ) or i00-i99 ( icd-10 ) as underlying causes , that is , all cvd , were classified as cvd mortality . an overview of all covariates , including additional information about the variables and any categorisations , is presented in the online supplementary table s1 . at conscription , all men underwent a 2-day examination during which health measures were obtained including weight and length , from which body mass index ( bmi , kg / m ) was calculated , and blood pressure . information on cvd mortality in parents before age 65 was included as an indicator of genetic risk for early cvd . socioeconomic position in childhood ( at age about 10 ) and adulthood ( at age about 40 ) was registered in the national population and housing censuses . the original six and eight occupation - based socioeconomic groups4 were collapsed into three and four , respectively , to retain statistical power . information on income at about 40 years of age ( in 1990 ) and marital status in the year of the first chd event , or the preceding year in case of missing data , was obtained from the longitudinal database of education , income and occupation ( louise ) . education is highly correlated with intelligence and was not included in the main analyses due to the risk for multicollinearity ( correlation about 0.55 in this cohort14 ) and overadjustment , but was added in an additional analysis . since intelligence is associated with various forms of morbidity and possibly the severity of conditions , comorbid conditions and complications might account for any differences in outcome between iq categories . hence , diagnoses obtained from the hospital discharge register , either registered as concomitant diagnoses at discharge after the first chd event or as primary or concomitant diagnoses up to 28 days after or in the 7 years preceding the event , in correspondence to an extension of the ontario ami prediction rule applied by rasmussen et al,15 were included as either comorbid conditions or complications ( diagnoses and corresponding icd codes are shown in online supplementary table s1 ) . depression was also included among comorbid conditions since it is also predictive of recurrence in chd.16 intelligence was modelled in three groups : low ( stanine score 13 ) , medium ( 46 ) and high ( 79 ) , using the highest performing group as reference . descriptive statistics of iq level and covariates were calculated for the full cohort and in subsamples based on men with a first chd 19912007 ( table 1 ) , and of covariates for the three iq categories ( table 2 ) . ors for case - fatality among men with a first chd event 19912007 were estimated by the logistic regression model . among men surviving a first chd event , hrs for a second chd event and for all - cause and cvd mortality , during the period 19912008 in relation to level of intelligence at conscription proportionality of hazards was assessed in log - log survival plots and no major violations against the proportionality assumption were found . after adjustment for age at first chd ( model 1 ) , early - life factors that were considered as potential confounders were added to the model ( model 2 ) , followed by factors measured at conscription or later ( model 3 ) . an additional analysis added education in three levels : 9 , 912 and 12 completed years . additional analyses restricted to myocardial infarction ( mi ; icd-9 410 , icd-10 i2122 ) , applied to both the inclusion criteria ( first event ) and outcome ( recurrence ) , were performed . possible interaction by adult socioeconomic position17 was examined in stratified analyses in groups of white - collar or blue - collar occupations . also , due to the higher mortality among people with lower intelligence , which might cause an underestimation of the iq - recurrence association , we performed an analysis combining recurrences and deaths from any causes as end points . characteristics of men with first chd 19912008 and in the full cohort of conscripts * not within 28 days of first chd . single household at the year or preceding year of first chd event in the samples ; single household in 1990 in the full cohort for comparison . diabetes with complications , pulmonary oedema , acute or chronic renal failure , cerebrovascular disease , malignancy and depression . bmi , body mass index ; chd , coronary heart disease ; cvd , cardiovascular diseases . distribution of risk factors in iq categories among men in the study sample * occupation and income in 1990 , at age about 40 . diabetes with complications , pulmonary oedema , acute or chronic renal failure , cerebrovascular disease , malignancy and depression . bmi , body mass index ; chd , coronary heart disease ; cvd , cardiovascular diseases . data were available for 2156 men with a first chd in 19912007 and for 1923 men who survived for follow - up through 2008 ( 7543.6 person years , mean 3.9 years of follow - up time ) . men with chd had lower iq score , a greater proportion of cvd risk factors ( measured at conscription ) , manual occupations or no registered occupation in 1990 and lower income compared to the full cohort ( table 1 ) . men who died from other causes than chd during follow - up had a lower iq score and a poorer cvd risk factor profile , more disadvantageous socioeconomic conditions and more often comorbid diagnoses or complications in the hospital records , compared to the total group of men with chd . men with a second ( recurrent ) event did not differ substantially from the total group of men with chd concerning the factors included . among the 1923 ( 89% ) men who survived for 28 days after their first chd event , 902 ( 47% ) had a recurrent event during follow - up of which 17 were fatal . sixty - one per cent of the recurrent events occurred within 1 year after the first . of the first - event survivors , 146 ( 7.6% ) died from any cause . of the deceased during follow - up , 37 ( 25% ) had no recurrent chd event , and 86 ( 59% ) died from other causes than chd . among the 74 men who died from cvd , 51 ( 69% ) had a recurrent chd event prior to the fatal event . table 2 shows the prevalence and distribution of risk factors in the three iq categories . the prevalence of smoking and risk use of alcohol , and average bmi and systolic blood pressure , increased somewhat with decreasing iq category . non - manual employees had higher iq to a greater extent while manual employees more often had lower iq . parent 's early cvd death , diastolic blood pressure , and comorbidity and complications were not associated with iq . table 3 shows the associations of intelligence at conscription with case - fatality at first chd event , and mortality from cvd and all causes during follow - up among 28-day survivors of the first event . there was a tendency of higher case - fatality with decreasing iq level but it was entirely explained by cvd risk factors and , in particular , socioeconomic and marital status . men with medium and low iq had a clearly higher all - cause mortality rate than men with high iq . after adjusting for confounders the point estimates were attenuated , reaching just below the conventional level of statistical significance . iq and case - fatality at first chd event 19912008 and subsequent mortality high iq is the reference in all analyses . * bmi , smoking , risk use of alcohol , systolic blood pressure , occupational class , income , single household and comorbidity / complications . bmi , body mass index ; chd , coronary heart disease ; cvd , cardiovascular diseases . table 4 shows that intelligence in adolescence was not associated with recurrence among men with chd in this cohort , neither during the full 17 years follow - up period nor in the analysis limited to 2 years follow - up . all of the estimates , crude or adjusted , were close to 1.0 and none reached conventional levels of statistical significance . adjusting for education had no impact on the risk for chd recurrence ( data available on request ) . restricting the study group to men with mi showed a similar pattern , although statistical power was low due to the smaller number of events . iq and recurrence among survivors of a first chd event 19912008 high iq is the reference in all analyses . bmi , smoking , risk use of alcohol , systolic blood pressure , occupational class , income , single household and comorbidity / complications . bmi , body mass index ; chd , coronary heart disease ; cvd , cardiovascular diseases . there was no clear evidence of interaction by socioeconomic position in the stratified analyses ( data available on request ) . there was no difference between blue - collar and white - collar occupations at the medium iq level , while the hr point estimate was slightly higher at the low iq level among white - collar employees compared to blue - collar workers , but statistical power was low due to the small number of events in the stratified groups . analyses stratified by age ( 4050 and 5158 years at first chd ) did not show any statistically significant differences either . in an analysis combining recurrent events and deaths from all causes ( 37 deaths ) as end points , the hrs were slightly higher compared to the main analysis ( age adjusted : medium iq 1.08 , 0.921.28 ; low iq 1.22 , 1.011.47 ; fully adjusted : medium iq 1.02 , 0.871.21 ; low iq 1.12 , 0.921.37 ) . including fatal first events in the analysis yielded similar results ( data available on request ) . in this group of swedish middle - aged men with chd , we found no association of premorbid intelligence with chd recurrence . by contrast , the risk of later all - cause and cvd mortality was higher among men with medium or low premorbid iq compared to men in the highest iq category , in line with findings in the normal population.13 these associations were attenuated after adjustment for traditional cvd risk factors , comorbidity , socioeconomic factors and marital status . several studies in various cohorts have found associations between intelligence measured early in life and later cvd morbidity , cvd mortality and all - cause mortality in general populations,24 18 19 and we found that the risk for mortality from cvd or all causes increased with decreasing iq also among middle - aged men with chd . yet , we are not aware of any previous studies investigating the role of premorbid intelligence in prognosis after a chd event . studies that have included health behaviours in models of the association between intelligence and later morbidity and mortality , such as smoking and alcohol consumption,2023 have been inconsistent and at most found little attenuation from these factors , implying that health behaviours are not important mediators . the prognostic value of health literacy , which is related to general intelligence,24 on adherence to treatment in cvd and other conditions such as diabetes has been inconsistent in review studies.25 26 one of the hypotheses often put forward to explain the association of iq with later morbidity and mortality is via the ability to manage one 's own health condition.27 28 some have indeed found associations between higher intelligence and health behaviours , for instance quitting smoking,29 30 having a healthy diet,31 32 physical exercise32 and persisting with prescribed medications6 ( but null findings are also reported33 34 ) . gottfredson argues that those with higher intelligence more easily understand and adapt to health messages , and that iq - related differences in health outcomes should increase rather than attenuate with increasing health information and medical support.7 lifestyle interventions and pharmacological treatment are central in secondary prevention of cvd,35 and differences in adherence to medications can indeed have a rapid impact . in a prospective study on patients with acute mi , non - adherence to medication 1 month after the event was associated with increased mortality within 1 year ; even stopping one of three prescribed medications was associated with an 80180% higher risk depending on medication type.36 intelligence has been found to be associated with medication persistence in a randomised controlled trial among people aged 5572 with an elevated risk for future cvd ( indicated by the ankle brachial index).6 in a cohort of danish men , lower iq measured in adolescence ( lowest vs highest tertile ) was associated with low persistence with prescribed antihypertensives , defined as not refilling prescriptions for at least a year up to age 54 , among the 1571 men obtaining such prescriptions from age 41 . given these previous findings , associations of iq with medication adherence would be expected to cause differences also between iq categories in chd recurrence . moreover , men in the present study with high iq at conscription had lower prevalence of smoking and risk use of alcohol , and lower bmi and systolic blood pressure , and more advantageous socioeconomic conditions than men with medium or low iq . despite these differences , there were no differences between iq levels regarding the risk of a second chd event during follow - up . one possible explanation is that medical treatment and rehabilitation is given according to patients needs , which might mitigate inequalities based on individual as well as socioeconomic factors . the attention , care and support given by medical professionals might also outweigh individual differences . furthermore , this is a relatively young group of men with diagnosed chd at an early age ( 4158 ) when factors not related to life style , for example genetic factors , might be more influential and thus more difficult for the individual to affect.37 in addition , a form of selection bias might occur if men with high intelligence developed chd due to unmeasured factors associated with better or worse prognosis . such bias can distort causal inference in any study restricted to already sick individuals.38 although we found no differences between iq levels in regards to recurrence , it is possible that the higher mortality rate among men with medium and low iq , compared to men with high iq , reflected a long - term effect of poorer disease management and lower adherence to treatment . most deaths occurred long after recurrent events of chd , so there is a time difference between the outcomes . it is also possible that factors that were present already early in life27 28 that were not captured by the covariates in our data contributed to the long - term differences in mortality between intelligence levels . factors early in life have been found to predict cvd and mortality later in life.39 40 moreover , lifestyle risk factors such as high bmi and elevated blood pressure in adolescence4 and socioeconomic factors in adulthood3 4 19 have been found to partly explain associations of early iq with cvd and mortality later in life . here , we found that high bmi , blood pressure and smoking in late adolescence also attenuated the associations of low iq with case - fatality and long - term mortality after first chd , along with socioeconomic and social factors measured in middle age . parent 's early death from cvd and socioeconomic position in the men 's childhood did not explain the associations , in line with previous findings on associations of intelligence with morbidity and mortality in general populations.24 19 in this nearly complete cohort of all swedish men born in 19491951 , intelligence was measured in adolescence long before chd developed , which minimised the risk for reverse causation , that is , precursors of chd affecting cognitive ability . information on chd events , comorbid diagnoses , mortality and socioeconomic factors were obtained from national records using the unique identification number held by all citizens , yielding reliable data and a virtually complete follow - up . however , patients who did not stay overnight at the hospital or had a silent event that did not lead to hospitalisation were not recorded in our data , and we had no clinical information on severity of the disease . moreover , the participants were followed until about 58 years of age when chd is still quite rare and the number of cases was consequently small , limiting statistical power . also , the results can not necessarily be generalised to chd later in life , to women , or to countries with a dissimilar healthcare system . there is a strong but far from perfect correlation between measures of iq early and iq later in life,41 and , therefore , our exposure may not fully capture iq during the follow - up . moreover , those with higher intelligence might have had less severe events , in concordance with the association between intelligence and health . finally , our adjustment for comorbidities was not perfect since we could adjust only for comorbidities requiring hospitalisations . one of the hypotheses often put forward to explain the association of iq with later morbidity and mortality is via the ability to manage one 's own health condition.27 28 some have indeed found associations between higher intelligence and health behaviours , for instance quitting smoking,29 30 having a healthy diet,31 32 physical exercise32 and persisting with prescribed medications6 ( but null findings are also reported33 34 ) . gottfredson argues that those with higher intelligence more easily understand and adapt to health messages , and that iq - related differences in health outcomes should increase rather than attenuate with increasing health information and medical support.7 lifestyle interventions and pharmacological treatment are central in secondary prevention of cvd,35 and differences in adherence to medications can indeed have a rapid impact . in a prospective study on patients with acute mi , non - adherence to medication 1 month after the event was associated with increased mortality within 1 year ; even stopping one of three prescribed medications was associated with an 80180% higher risk depending on medication type.36 intelligence has been found to be associated with medication persistence in a randomised controlled trial among people aged 5572 with an elevated risk for future cvd ( indicated by the ankle brachial index).6 in a cohort of danish men , lower iq measured in adolescence ( lowest vs highest tertile ) was associated with low persistence with prescribed antihypertensives , defined as not refilling prescriptions for at least a year up to age 54 , among the 1571 men obtaining such prescriptions from age 41 . given these previous findings , associations of iq with medication adherence would be expected to cause differences also between iq categories in chd recurrence . moreover , men in the present study with high iq at conscription had lower prevalence of smoking and risk use of alcohol , and lower bmi and systolic blood pressure , and more advantageous socioeconomic conditions than men with medium or low iq . despite these differences , there were no differences between iq levels regarding the risk of a second chd event during follow - up . one possible explanation is that medical treatment and rehabilitation is given according to patients needs , which might mitigate inequalities based on individual as well as socioeconomic factors . the attention , care and support given by medical professionals might also outweigh individual differences . furthermore , this is a relatively young group of men with diagnosed chd at an early age ( 4158 ) when factors not related to life style , for example genetic factors , might be more influential and thus more difficult for the individual to affect.37 in addition , a form of selection bias might occur if men with high intelligence developed chd due to unmeasured factors associated with better or worse prognosis . such bias can distort causal inference in any study restricted to already sick individuals.38 although we found no differences between iq levels in regards to recurrence , it is possible that the higher mortality rate among men with medium and low iq , compared to men with high iq , reflected a long - term effect of poorer disease management and lower adherence to treatment . most deaths occurred long after recurrent events of chd , so there is a time difference between the outcomes . it is also possible that factors that were present already early in life27 28 that were not captured by the covariates in our data contributed to the long - term differences in mortality between intelligence levels . factors early in life have been found to predict cvd and mortality later in life.39 40 moreover , lifestyle risk factors such as high bmi and elevated blood pressure in adolescence4 and socioeconomic factors in adulthood3 4 19 have been found to partly explain associations of early iq with cvd and mortality later in life . here , we found that high bmi , blood pressure and smoking in late adolescence also attenuated the associations of low iq with case - fatality and long - term mortality after first chd , along with socioeconomic and social factors measured in middle age . parent 's early death from cvd and socioeconomic position in the men 's childhood did not explain the associations , in line with previous findings on associations of intelligence with morbidity and mortality in general populations.24 19 in this nearly complete cohort of all swedish men born in 19491951 , intelligence was measured in adolescence long before chd developed , which minimised the risk for reverse causation , that is , precursors of chd affecting cognitive ability . information on chd events , comorbid diagnoses , mortality and socioeconomic factors were obtained from national records using the unique identification number held by all citizens , yielding reliable data and a virtually complete follow - up . however , patients who did not stay overnight at the hospital or had a silent event that did not lead to hospitalisation were not recorded in our data , and we had no clinical information on severity of the disease . moreover , the participants were followed until about 58 years of age when chd is still quite rare and the number of cases was consequently small , limiting statistical power . also , the results can not necessarily be generalised to chd later in life , to women , or to countries with a dissimilar healthcare system . there is a strong but far from perfect correlation between measures of iq early and iq later in life,41 and , therefore , our exposure may not fully capture iq during the follow - up . moreover , those with higher intelligence might have had less severe events , in concordance with the association between intelligence and health . finally , our adjustment for comorbidities was not perfect since we could adjust only for comorbidities requiring hospitalisations . among middle - aged swedish men with chd , premorbid intelligence was not associated with the risk for recurrence . by contrast , lower intelligence was associated with higher case - fatality rate at first event and higher mortality during 17 years of follow - up . although lower intelligence is a risk factor for morbidity and mortality in a life course perspective , we found no evidence of lower intelligence having a negative effect in secondary prevention of chd . what is already known on this subjecthigher intelligence early in life is associated with decreased risk for coronary heart disease ( chd ) and mortality but its prognostic importance in patients with chd is unknown.differences in the ability to manage health conditions has been suggested as one explanation for the association between intelligence and health outcomes . what this study addsin this cohort of 1923 men , obtaining their first chd diagnosis in middle age , lower intelligence was associated with all - cause mortality but not with the risk of recurrence.we found no evidence of intelligence level being an important factor in secondary prevention of chd . higher intelligence early in life is associated with decreased risk for coronary heart disease ( chd ) and mortality but its prognostic importance in patients with chd is unknown . differences in the ability to manage health conditions has been suggested as one explanation for the association between intelligence and health outcomes . in this cohort of 1923 men , obtaining their first chd diagnosis in middle age , lower intelligence was associated with all - cause mortality but not with the risk of recurrence . we found no evidence of intelligence level being an important factor in secondary prevention of chd .

backgroundlower intelligence early in life is associated with increased risks for coronary heart disease ( chd ) and mortality . intelligence level might affect compliance to treatment but its prognostic importance in patients with chd is unknown.methodsa cohort of 1923 swedish men with a measure of intelligence from mandatory military conscription in 19691970 at age 1820 , who were diagnosed with chd 19912007 , were followed to the end of 2008 . primary outcome : recurrent chd event . secondary outcome : case fatality from the first event , cardiovascular and all - cause mortality . national registers provided information on chd events , comorbidity , mortality and socioeconomic factors.resultsthe fully adjusted hrs for recurrent chd for medium and low intelligence , compared with high intelligence , were 0.98 , ( 95% cis 0.83 to 1.16 ) and 1.09 ( 0.89 to 1.34 ) , respectively . the risks were increased for cardiovascular and all - cause mortality with lower intelligence , but were attenuated in the fully adjusted models ( fully adjusted hrs for cardiovascular mortality 1.92 ( 0.94 to 3.94 ) and 1.98 ( 0.89 to 4.37 ) , respectively ; for all - cause mortality 1.63 ( 1.00 to 2.65 ) and 1.62 ( 0.94 to 2.78 ) , respectively ) . there was no increased risk for case - fatality at the first event ( fully adjusted ors 1.06 ( 0.73 to 1.55 ) and 0.97 ( 0.62 to 1.50 ) , respectively).conclusionsalthough we found lower intelligence to be associated with increased mortality in middle - aged men with chd , there was no evidence for its possible effect on recurrence in chd .

Introduction Methods Exposure: intelligence Outcome: recurrent CHD Case fatality and mortality Covariates Statistical analyses Results Discussion Interpretation of the results Strengths and limitations Conclusion Supplementary Material

an association between premorbid intelligence and later morbidity , including coronary heart disease ( chd ) , has been established in longitudinal studies.14 however , no previous study has investigated whether intelligence affects prognosis of chd . for instance , poor compliance in medication for secondary prevention cardiovascular diseases ( cvd ) is a frequent problem ; several prospective studies have found that most patients stop taking at least one prescribed medication during the first year after an event.8 intelligence has previously been found to predict medication persistence in a population at risk for cvd.6 the aim of this study was to investigate if lower premorbid intelligence is associated with recurrence among men diagnosed with chd . in addition , we wanted to investigate the association of intelligence with case - fatality and all - cause mortality in this group . record linkage with national registers provided data on morbidity , mortality and measures of socioeconomic factors in adulthood . the study was based on data from a nationwide , mandatory examination of 49 321 swedish men who were conscripted for military service in 19691970 , at age 1820 . diagnoses of first and recurrent chd ( swedish version of international classification of disease ninth edition ( icd-9 ) up to 1996 , codes 410414 ; thereafter icd-10 , i20-i25 ) were obtained from the swedish national hospital discharge register , and the national cause of death register for out - of - hospital deaths in chd , which are held by the national board of health and welfare . in sweden , men with a first event of chd registered between 1 january 1991 and 31 december 2007 were included in the analyses . follow - up started when a man was diagnosed with chd , at any time during 19912007 , and ended at the date of whichever occurred first : a second fatal or non - fatal event of chd recorded in the national hospital or death registers ( with a minimum of 28 days after the first event ; diagnoses and deaths within 28 days were regarded as reflecting the first event ) , emigration , death from other causes , or 31 december 2008 . first chd events leading to death out of hospital and recorded in the cause of death register but not in hospital registers were included in analyses of case - fatality at baseline , but only men surviving their first chd event were included in follow - up analyses . case - fatality in first chd was defined as death with any underlying cause registered within 28 days of the first hospital admission for chd , as is common in case - fatality research , or chd as underlying cause in case of out - of - hospital death . information on income at about 40 years of age ( in 1990 ) and marital status in the year of the first chd event , or the preceding year in case of missing data , was obtained from the longitudinal database of education , income and occupation ( louise ) . education is highly correlated with intelligence and was not included in the main analyses due to the risk for multicollinearity ( correlation about 0.55 in this cohort14 ) and overadjustment , but was added in an additional analysis . hence , diagnoses obtained from the hospital discharge register , either registered as concomitant diagnoses at discharge after the first chd event or as primary or concomitant diagnoses up to 28 days after or in the 7 years preceding the event , in correspondence to an extension of the ontario ami prediction rule applied by rasmussen et al,15 were included as either comorbid conditions or complications ( diagnoses and corresponding icd codes are shown in online supplementary table s1 ) . depression was also included among comorbid conditions since it is also predictive of recurrence in chd.16 intelligence was modelled in three groups : low ( stanine score 13 ) , medium ( 46 ) and high ( 79 ) , using the highest performing group as reference . descriptive statistics of iq level and covariates were calculated for the full cohort and in subsamples based on men with a first chd 19912007 ( table 1 ) , and of covariates for the three iq categories ( table 2 ) . ors for case - fatality among men with a first chd event 19912007 were estimated by the logistic regression model . among men surviving a first chd event , hrs for a second chd event and for all - cause and cvd mortality , during the period 19912008 in relation to level of intelligence at conscription proportionality of hazards was assessed in log - log survival plots and no major violations against the proportionality assumption were found . additional analyses restricted to myocardial infarction ( mi ; icd-9 410 , icd-10 i2122 ) , applied to both the inclusion criteria ( first event ) and outcome ( recurrence ) , were performed . also , due to the higher mortality among people with lower intelligence , which might cause an underestimation of the iq - recurrence association , we performed an analysis combining recurrences and deaths from any causes as end points . single household at the year or preceding year of first chd event in the samples ; single household in 1990 in the full cohort for comparison . bmi , body mass index ; chd , coronary heart disease ; cvd , cardiovascular diseases . bmi , body mass index ; chd , coronary heart disease ; cvd , cardiovascular diseases . diagnoses of first and recurrent chd ( swedish version of international classification of disease ninth edition ( icd-9 ) up to 1996 , codes 410414 ; thereafter icd-10 , i20-i25 ) were obtained from the swedish national hospital discharge register , and the national cause of death register for out - of - hospital deaths in chd , which are held by the national board of health and welfare . men with a first event of chd registered between 1 january 1991 and 31 december 2007 follow - up started when a man was diagnosed with chd , at any time during 19912007 , and ended at the date of whichever occurred first : a second fatal or non - fatal event of chd recorded in the national hospital or death registers ( with a minimum of 28 days after the first event ; diagnoses and deaths within 28 days were regarded as reflecting the first event ) , emigration , death from other causes , or 31 december 2008 . first chd events leading to death out of hospital and recorded in the cause of death register but not in hospital registers were included in analyses of case - fatality at baseline , but only men surviving their first chd event were included in follow - up analyses . case - fatality in first chd was defined as death with any underlying cause registered within 28 days of the first hospital admission for chd , as is common in case - fatality research , or chd as underlying cause in case of out - of - hospital death . information on income at about 40 years of age ( in 1990 ) and marital status in the year of the first chd event , or the preceding year in case of missing data , was obtained from the longitudinal database of education , income and occupation ( louise ) . education is highly correlated with intelligence and was not included in the main analyses due to the risk for multicollinearity ( correlation about 0.55 in this cohort14 ) and overadjustment , but was added in an additional analysis . hence , diagnoses obtained from the hospital discharge register , either registered as concomitant diagnoses at discharge after the first chd event or as primary or concomitant diagnoses up to 28 days after or in the 7 years preceding the event , in correspondence to an extension of the ontario ami prediction rule applied by rasmussen et al,15 were included as either comorbid conditions or complications ( diagnoses and corresponding icd codes are shown in online supplementary table s1 ) . depression was also included among comorbid conditions since it is also predictive of recurrence in chd.16 intelligence was modelled in three groups : low ( stanine score 13 ) , medium ( 46 ) and high ( 79 ) , using the highest performing group as reference . descriptive statistics of iq level and covariates were calculated for the full cohort and in subsamples based on men with a first chd 19912007 ( table 1 ) , and of covariates for the three iq categories ( table 2 ) . ors for case - fatality among men with a first chd event 19912007 were estimated by the logistic regression model . among men surviving a first chd event , hrs for a second chd event and for all - cause and cvd mortality , during the period 19912008 in relation to level of intelligence at conscription proportionality of hazards was assessed in log - log survival plots and no major violations against the proportionality assumption were found . additional analyses restricted to myocardial infarction ( mi ; icd-9 410 , icd-10 i2122 ) , applied to both the inclusion criteria ( first event ) and outcome ( recurrence ) , were performed . also , due to the higher mortality among people with lower intelligence , which might cause an underestimation of the iq - recurrence association , we performed an analysis combining recurrences and deaths from any causes as end points . single household at the year or preceding year of first chd event in the samples ; single household in 1990 in the full cohort for comparison . bmi , body mass index ; chd , coronary heart disease ; cvd , cardiovascular diseases . bmi , body mass index ; chd , coronary heart disease ; cvd , cardiovascular diseases . men with chd had lower iq score , a greater proportion of cvd risk factors ( measured at conscription ) , manual occupations or no registered occupation in 1990 and lower income compared to the full cohort ( table 1 ) . men who died from other causes than chd during follow - up had a lower iq score and a poorer cvd risk factor profile , more disadvantageous socioeconomic conditions and more often comorbid diagnoses or complications in the hospital records , compared to the total group of men with chd . men with a second ( recurrent ) event did not differ substantially from the total group of men with chd concerning the factors included . table 3 shows the associations of intelligence at conscription with case - fatality at first chd event , and mortality from cvd and all causes during follow - up among 28-day survivors of the first event . men with medium and low iq had a clearly higher all - cause mortality rate than men with high iq . iq and case - fatality at first chd event 19912008 and subsequent mortality high iq is the reference in all analyses . bmi , body mass index ; chd , coronary heart disease ; cvd , cardiovascular diseases . table 4 shows that intelligence in adolescence was not associated with recurrence among men with chd in this cohort , neither during the full 17 years follow - up period nor in the analysis limited to 2 years follow - up . bmi , body mass index ; chd , coronary heart disease ; cvd , cardiovascular diseases . there was no difference between blue - collar and white - collar occupations at the medium iq level , while the hr point estimate was slightly higher at the low iq level among white - collar employees compared to blue - collar workers , but statistical power was low due to the small number of events in the stratified groups . in this group of swedish middle - aged men with chd , we found no association of premorbid intelligence with chd recurrence . by contrast , the risk of later all - cause and cvd mortality was higher among men with medium or low premorbid iq compared to men in the highest iq category , in line with findings in the normal population.13 these associations were attenuated after adjustment for traditional cvd risk factors , comorbidity , socioeconomic factors and marital status . several studies in various cohorts have found associations between intelligence measured early in life and later cvd morbidity , cvd mortality and all - cause mortality in general populations,24 18 19 and we found that the risk for mortality from cvd or all causes increased with decreasing iq also among middle - aged men with chd . the prognostic value of health literacy , which is related to general intelligence,24 on adherence to treatment in cvd and other conditions such as diabetes has been inconsistent in review studies.25 26 one of the hypotheses often put forward to explain the association of iq with later morbidity and mortality is via the ability to manage one 's own health condition.27 28 some have indeed found associations between higher intelligence and health behaviours , for instance quitting smoking,29 30 having a healthy diet,31 32 physical exercise32 and persisting with prescribed medications6 ( but null findings are also reported33 34 ) . in a prospective study on patients with acute mi , non - adherence to medication 1 month after the event was associated with increased mortality within 1 year ; even stopping one of three prescribed medications was associated with an 80180% higher risk depending on medication type.36 intelligence has been found to be associated with medication persistence in a randomised controlled trial among people aged 5572 with an elevated risk for future cvd ( indicated by the ankle brachial index).6 in a cohort of danish men , lower iq measured in adolescence ( lowest vs highest tertile ) was associated with low persistence with prescribed antihypertensives , defined as not refilling prescriptions for at least a year up to age 54 , among the 1571 men obtaining such prescriptions from age 41 . furthermore , this is a relatively young group of men with diagnosed chd at an early age ( 4158 ) when factors not related to life style , for example genetic factors , might be more influential and thus more difficult for the individual to affect.37 in addition , a form of selection bias might occur if men with high intelligence developed chd due to unmeasured factors associated with better or worse prognosis . such bias can distort causal inference in any study restricted to already sick individuals.38 although we found no differences between iq levels in regards to recurrence , it is possible that the higher mortality rate among men with medium and low iq , compared to men with high iq , reflected a long - term effect of poorer disease management and lower adherence to treatment . factors early in life have been found to predict cvd and mortality later in life.39 40 moreover , lifestyle risk factors such as high bmi and elevated blood pressure in adolescence4 and socioeconomic factors in adulthood3 4 19 have been found to partly explain associations of early iq with cvd and mortality later in life . here , we found that high bmi , blood pressure and smoking in late adolescence also attenuated the associations of low iq with case - fatality and long - term mortality after first chd , along with socioeconomic and social factors measured in middle age . parent 's early death from cvd and socioeconomic position in the men 's childhood did not explain the associations , in line with previous findings on associations of intelligence with morbidity and mortality in general populations.24 19 in this nearly complete cohort of all swedish men born in 19491951 , intelligence was measured in adolescence long before chd developed , which minimised the risk for reverse causation , that is , precursors of chd affecting cognitive ability . information on chd events , comorbid diagnoses , mortality and socioeconomic factors were obtained from national records using the unique identification number held by all citizens , yielding reliable data and a virtually complete follow - up . in a prospective study on patients with acute mi , non - adherence to medication 1 month after the event was associated with increased mortality within 1 year ; even stopping one of three prescribed medications was associated with an 80180% higher risk depending on medication type.36 intelligence has been found to be associated with medication persistence in a randomised controlled trial among people aged 5572 with an elevated risk for future cvd ( indicated by the ankle brachial index).6 in a cohort of danish men , lower iq measured in adolescence ( lowest vs highest tertile ) was associated with low persistence with prescribed antihypertensives , defined as not refilling prescriptions for at least a year up to age 54 , among the 1571 men obtaining such prescriptions from age 41 . furthermore , this is a relatively young group of men with diagnosed chd at an early age ( 4158 ) when factors not related to life style , for example genetic factors , might be more influential and thus more difficult for the individual to affect.37 in addition , a form of selection bias might occur if men with high intelligence developed chd due to unmeasured factors associated with better or worse prognosis . such bias can distort causal inference in any study restricted to already sick individuals.38 although we found no differences between iq levels in regards to recurrence , it is possible that the higher mortality rate among men with medium and low iq , compared to men with high iq , reflected a long - term effect of poorer disease management and lower adherence to treatment . factors early in life have been found to predict cvd and mortality later in life.39 40 moreover , lifestyle risk factors such as high bmi and elevated blood pressure in adolescence4 and socioeconomic factors in adulthood3 4 19 have been found to partly explain associations of early iq with cvd and mortality later in life . here , we found that high bmi , blood pressure and smoking in late adolescence also attenuated the associations of low iq with case - fatality and long - term mortality after first chd , along with socioeconomic and social factors measured in middle age . parent 's early death from cvd and socioeconomic position in the men 's childhood did not explain the associations , in line with previous findings on associations of intelligence with morbidity and mortality in general populations.24 19 in this nearly complete cohort of all swedish men born in 19491951 , intelligence was measured in adolescence long before chd developed , which minimised the risk for reverse causation , that is , precursors of chd affecting cognitive ability . information on chd events , comorbid diagnoses , mortality and socioeconomic factors were obtained from national records using the unique identification number held by all citizens , yielding reliable data and a virtually complete follow - up . among middle - aged swedish men with chd , premorbid intelligence was not associated with the risk for recurrence . by contrast , lower intelligence was associated with higher case - fatality rate at first event and higher mortality during 17 years of follow - up . although lower intelligence is a risk factor for morbidity and mortality in a life course perspective , we found no evidence of lower intelligence having a negative effect in secondary prevention of chd . what is already known on this subjecthigher intelligence early in life is associated with decreased risk for coronary heart disease ( chd ) and mortality but its prognostic importance in patients with chd is unknown.differences in the ability to manage health conditions has been suggested as one explanation for the association between intelligence and health outcomes . what this study addsin this cohort of 1923 men , obtaining their first chd diagnosis in middle age , lower intelligence was associated with all - cause mortality but not with the risk of recurrence.we found no evidence of intelligence level being an important factor in secondary prevention of chd . higher intelligence early in life is associated with decreased risk for coronary heart disease ( chd ) and mortality but its prognostic importance in patients with chd is unknown . in this cohort of 1923 men , obtaining their first chd diagnosis in middle age , lower intelligence was associated with all - cause mortality but not with the risk of recurrence . we found no evidence of intelligence level being an important factor in secondary prevention of chd .

the runt domain ( rd ) is the dna - binding domain of p53 type transcription factors known as core binding factor ( cbf ) subunits . the cbfs are expressed by the three related genes runx1 , runx2 and runx3 ( 1 ) . the three runx genes are important for developmental processes ; runx1 is important for hematopoiesis , runx2 is essential for skeletal development ( osteogenesis ) and runx3 is required for the development of gastric epithelium ( 24 ) . runx1 and runx2 also have been shown to be important for vascular development or angiogenesis and their activity is augmented by angiogenic growth factors ( 5,6 ) . mutation of the runx1 gene is associated with human leukemias , whereas mutation of the runx3 gene is involved with the development and progression of gastric cancers and testicular yolk sac tumor ( 1,7,8 ) . mutation of the runx2 gene is associated with the inherited human skeletal disorder known as cleidocranial dysplasia ( 9 ) . a related protein , known as cbf , binds to the rd of the cbfs and enhances the dna - binding affinity of the rd . the rd is the site of both dna binding and heterodimerization with cbf. because of the involvement of cbfs in many developmental processes and cancerous diseases , understanding rd dna and rd cbf interactions may shed light on the control of these processes . in addition , the involvement of runx genes with angiogenesis also makes the rd a potential target in the development and treatment of many types of cancers . the 3d structures of the rd in the free state , bound to dna , cbf and both dna and cbf have been determined using nuclear magnetic resonance ( nmr ) spectroscopy and/or x - ray crystallography ( 1014 ) . biochemical and biophysical studies have been performed characterizing the binding of rd to dna in the presence and absence of cbf ( 15,16 ) . in the presence of cbf , the dissociation constant ( kd ) for rd dna decreases 6- to 10-fold ( 16 ) . several studies have identified rd residues important for both dna and cbf binding via mutational analysis ( 1719 ) . the mutational analysis usually involved mutations to alanine and analysis of its effect on rd dna and rd cbf-binding activities , as determined using yeast hybrid assay analysis . in addition , a recent nmr - based study has identified a variety of residues involved in the cooperativity associated with cbf binding to rd ( 20 ) . while these important studies have given insights into the role of individual residues to rd dna and rd cbf interactions , the interpretation of the mutational analysis may be complicated by the influence of the mutation on protein stability , protein folding and other localized changes , especially at protein protein interfaces , while structural and dynamic information from nmr and x - ray studies can not yield energetic information at an atomic level of detail . computational approaches can yield important details about the interaction of rd with dna and cbf ( 21 ) . for example , the energetic contribution of individual residues to both dna and cbf binding can be determined . by doing such analysis in systems with all wild - type residues present , the risk of mutation induced conformational change , including changes in protein stability , is avoided . computational analysis can also account for the contribution of all atoms in a given residue , including the backbone atoms , whereas mutational analysis can only detect the importance of side chain atoms . thus , computational analysis can fill gaps in understanding the rd dna , rd cbf and cbfrd dna ternary complex interactions . in the present work , the rd was subjected to molecular dynamic ( md ) simulations in the free state and in several complexes . complexes include the binary rd dna and rd cbf complexes , and the cbfrd dna ternary complex . in addition , two dna simulations and a cbf monomer simulation were performed . from the md simulations , in combination with free energy component analysis ( 2224 ) based on the generalized - born model ( 25,26 ) , overall binding energies , desolvation effects and detailed interaction energies these results , along with previous structural analysis and observations , were used to interpret a variety of experimental data involving rd dna and rd cbf interactions ( 1619,27 ) . md simulations and all calculations were performed using the program charmm ( 28 ) . the all - hydrogen charmm22 protein ( 29 ) and charmm27 nucleic acid ( 30,31 ) , the tip3p water model ( 32 ) , along with published sodium and chloride parameters were used ( 33 ) . coordinates from the cbfrd dna ternary complex x - ray crystal structure ( pdb accession code 1h9d ) ( 13 ) were used as starting structures of the seven simulation systems ( supplementary table s1 ) . the only exception was the second dna simulation , which was initiated with the dna in the canonical b form ( 34 ) , as obtained from the program quanta ( accelrys inc . ) . in the crystal structure terminal residues 5053 and 179183 in rd are not observed experimentally as are residues 7179 of cbf. accordingly , in the simulations these residues were omitted . omission of residues 7179 of cbf , which are spatially removed from the rd in the experimental structure ( figure 1 ) , was performed by creating a peptide bond between residues 70 and 80 and relaxing the structure during the minimization and md simulation protocols described below . root - mean - square ( rms ) difference analysis of the residues adjacent to the deleted residues in cbf ( i.e. residues 6670 and 8084 ) showed significant structural changes to occur only in the one or two residues directly adjacent to the deleted residues ( supplementary table s2 ) . the dimensions of each complex ( solute ) were determined , and a box of solvent was prepared to overlay the solute . the edge of the solvent was extended to a minimum of 8 beyond the solute in each dimension . then each solute was overlaid with the solvent water box that also contained either sodium or chloride ions , depending on the total charge of the solute . all water molecules whose oxygen atom was within 2.5 of any solute non - hydrogen atom were then deleted and excess ions were deleted to make the systems electrically neutral . all subsequent calculations were performed using periodic boundary conditions , with images generated using the crystal module of charmm . equilibration of each simulation system was carried out by performing 500 steps of steepest descent energy minimization followed by a 10 ps md nvt simulation with harmonic restraints of 5 kcal/(mol ) on the solute non - hydrogen atoms . additional minimization using the adopted basis newton raphson ( abnr ) algorithm was performed for 100 steps without any restraints . the systems were then subjected to a 5 ns md simulation in the npt ensemble at 300 k with the leap frog integrator , and a time step of 0.002 ps . particle mesh ewald ( pme ) was used to treat long - range electrostatic interactions ( 37 ) . pme calculations were carried out using real space electrostatic and lennard jones interaction cutoffs at 10 , with utilization of heuristic non - bond list update out to 12 and smoothing of the lj interactions from 8 to 10 using the force switch method ( 38 ) . coordinates were saved every 5 ps , and by the end of the 4.5 ns production md simulation , 900 sets of structural coordinates were available for analysis . structural figures were generated using the program vmd ( 39 ) and rendered by raster3d ( 40 ) . calculation of binding free energies via free energy component analysis : binding free energies , gbind , were calculated as follows : 1gbind = gcomplexgpartneragpartnerb , where the overall free energy of each complex was described using ( 24 ) 2g = emm+gsolvationtsmm . in equation 2 emm describes the molecular mechanical energy of the system consisting of all components in the charmm potential energy function , as shown in the following equation 3 : 3emm = ebond+eangle+eurey - bradley+edihedral+eimproper+evdw+eelec . for complexes , emm , can be separated into interaction energy , eie and strain , estrain components : 4emm , complex = eie+estrain , where eie is the sum of the vdw and electrostatic interaction energy between individual molecules in a complex and estrain is the sum of emm terms for each molecule in the complex ( i.e. all mm energy contributions for each species ignoring contributions from the other molecules in the complex ) . the gsolvation term describes the solvation free energy of a system and includes both electrostatic , gelec and non - polar , gnp , components . the electrostatic portion of the solvation free energy was calculated using the generalized - born molecular volume ( gbmv ) method ( 25,26 ) . the non - polar portion of the solvation energy ( i.e. due to cavity formation and hydrophobicity ) was calculated using the still equation , enp = sa , where is an empirical atomic solvation parameter , 7.2 cal / , and sa is the solvent accessible surface area calculated with a solvent probe radius of 1.4 ( 25,41 ) . the final term in equation 2 , tsmm , describes the entropic part of the energy as a sum of translational , rotational and vibrational entropy terms calculated via the harmonic approximation ( 42 ) for the temperature 300 k. vibrational analysis was performed on the solute molecules extracted out of the solvent box . the solute structure was minimized using the conjugate - gradient method to an rms gradient of 10 kcal/(mol ) with a distance - dependent dielectric of 4r applied to approximate the screening of electrostatic interactions by solvent , where r is the distance between two atoms , as performed previously ( 22 ) . to better understand the contributions of the various energetic terms to complex formation this typically involved calculation of the difference between a selected term for the complex and those of the individual molecules comprising the complex . in the case of the interaction energies their contribution was simply the sum of the contribution between the individual molecules in each complex ( i.e. for the cbfrd dimer the eie between cbf and rd was included ) . however , in the case of the trimer formation via dimer + monomer complexation the eie contribution includes those between the monomer being added and the two molecules in the original dimer along with the change in the interaction energy between the molecules in the original dimer upon going to a trimer . for example , eie , cbf-rd + dna = eie , cbf-dna + eie , rd - dna eie , cbf-rd(dimer to trimer ) . this term accounts for gains or losses in the interaction energy of the molecules in a dimer when the third molecule binds to form a trimer . interaction energies , einter , and strain energies , estrain , were calculated for each individual species using an infinite cutoff in order to mimic the long - range electrostatic interactions treated via pme in the md simulations . energy calculations were performed on the 900 structures from each simulation system with statistical analysis performed by dividing each 4.5 ns simulation into nine 500 ps time blocks . statistical analysis was performed by obtaining the averages from each time block , with those averages treated as independent data points , allowing calculation of the overall averages , and standard errors as described previously ( 43 ) . the entropy calculations were performed on 30 structures extracted from the simulation trajectories at 150 ps time intervals , with the statistical analysis derived by dividing the 30 observations into ten 450 ps independent time blocks from which the averages and the standard errors were obtained . overall rms differences of all simulated structures versus the crystallographic structure of the trimer are shown in supplementary table s3 . md simulation studies were undertaken on the rd to understand the nature of interactions with the dna and with the binding partner , cbf. average rms differences for all non - hydrogen atoms with respect to the crystallographic ternary complex structures were 1.62 0.04 , 3.56 0.10 , 2.15 0.04 , 3.23 0.16 , 3.63 0.11 and 2.81 0.05 for the dna , rd , cbf , rd dna , rd cbf and cbfrd dna structures , respectively , indicating that the overall structures were maintained in the simulations . the structure of the trimer after a 4.5 ns simulation is illustrated in figure 1 . analysis focused on the overall binding as calculated using equation 1 followed by investigation of the contributions of different components to the overall binding ( equations 2 and 4 ) . this was followed by identifying contributions of different regions of the proteins and dna to binding , ultimately analyzing the role of individual amino acids . emphasis was placed on using the present results to interpret the body of experimental data available on rd . free energies of complex formation for the dimers and the trimer were calculated using equation 1 based on the data presented in supplementary table s4 . the resulting free energies of binding for the various complexes are presented in table 1 and are included in the thermodynamic cycle shown in figure 2 . two free energies of binding are reported , gbind and gbind , ts , that exclude and include the configuration entropy , tsmm , respectively . for gbind the calculated energies are all favorable , although the interactions involving addition of cbf are extremely exothermic . in contrast , upon inclusion of the entropy the events that involve dna binding become unfavorable . in addition , the calculations predict that rd cbf binding is more favorable than rd dna binding , in contrast to experimental observations ( 16 ) . the presence of these discrepancies is not surprising due to a variety of assumptions in the calculations , including the omission of energetic contributions , such as the solvation and entropic contributions of the added counterions ( 23 ) , limitations in the potential energy function and the force field ( 44 ) along with possible limitations in the sampling of conformational space . dna interaction energies in the force field by < 4% would account for the prediction that binding of rd to cbf is more favorable than rd binding to dna . with respect to the tsmm , the use of gas phase minimized structures may be problematic , especially in the case of the polyanionic dna where the electrostatic screening due to the 4r dielectric is expected to be insufficient when compared with the proteins . however , when the two types of binding free energy are compared , the trends are similar . moreover , in both scenarios , the binding of dna to the rd cbf dimer to form the trimer is more favorable than the binding of rd and dna to form the rd thus , it appears that the present results , although not in quantitative agreement with experiment due to assumptions and limitations in the theoretical models , are representative of the experimental regimen , allowing for atomic details of events driving binding to be elucidated . in addition , the emphasis in this work on differences in energies and structures between the systems rather than absolute energies should allow for increased confidence in the observations and the conclusions based on those observations . concerning the convergence of the present results , the energies calculated from the two independent dna simulations are within the estimated errors ( supplementary table s4 ) , suggesting that the present calculations have adequately converged . analysis of the thermodynamic cycle in figure 2 predicts that binding of both cbf and dna are cooperative . these results are consistent with previous studies showing the binding of dna to rd to be enhanced by the binding of cbf as well as the binding of cbf to rd to be enhanced by dna based on electrophoretic mobility shift assay ( 10,16,18 ) . as discussed , such cooperativity may be important for maintaining binding of rd to dna via stabilization of the ternary complex to insure adequate transcriptional activation . tighter protein protein interactions due to dna binding have been observed in other types of transcription factors ( 45 ) . contributions from solvation , strain and interaction energies to the calculated binding energies are shown in table 1 . as expected , the solvation energies are all unfavorable upon binding , with the unfavorable contributions being significantly larger for the interactions involving the highly solvated polyanionic dna . the strain energies are all also unfavorable with the exception of formation of the cbfrd dimer . typically , it would be expected that the protein protein or protein dna interactions would lead to an unfavorable strain contribution upon binding ( 23 ) . ongoing studies in our laboratory are investigating the unusual behavior in the cbfrd dimer interaction . countering the unfavorable solvation and clearly , the free energies of binding are driven by the large , opposing forces of solvation and , typically , strain energies versus interaction energies between the macromolecules in the complexes . data at the bottom of table 1 addresses the contributions to the calculated gain in binding energy associated with cooperativity upon going from the dimers to the trimer . contributing to the cooperativity is a decrease in the unfavorable solvation that occurs upon binding , evidenced by the trimer the more favorable change in interaction energy occurred with both the rd dna and the rd cbf interactions in the trimer ( table 1 ) ; details of the contributions of different regions of the proteins to this will be discussed below . countering these contributions are unfavorable increases in the strain energy , consistent with gains in interaction energies , as discussed above . thus , the cooperativity of dna and cbf binding to rd is predicted to be dominated by gains in the favorable interactions between the macromolecules and with lowering of the solvation penalty to complex formation . a more detailed understanding of the atomic determinants of cooperativity associated with rd binding to cbf and dna was obtained via interaction energy partitioning analysis between different regions of the rd and both cbf and dna . this was facilitated by partitioning rd into a variety of segments as specified in table 2 and shown in figure 1 . several regions of the protein have been previously indicated to contribute to either rd dna or rd cbf interactions ( 13 ) . residues adjacent to the respective binding partner but not included in a defined segment are referred to as other - dna and other - cbf and all residues not previously defined are referred to as non - interacting - cbf , allowing for the quantification of their contributions to binding . of note are the wing residues , which are primarily important for dna binding , while residues in wing1 are proximal to the wing residues , contacting both cbf and the wing residues . for all these segments of rd , interaction energies were calculated between the respective monomers in the dimers as well as in the trimer . as discussed above , cooperativity exists between both dna and cbf with respect to their binding to rd . dna interaction energies in both the trimer and the dimer complexes along with the change in interaction energy upon going from the rd dna dimer to the trimer , with those contributions broken down into the segments presented in table 2 . upon trimer formation the total rd dna interaction energy becomes more favorable , with three of the segments contributing significantly to the more favorable interaction energy in the trimer . these include wing , a loop and other - dna segments . the largest contribution is from the wing segment , consistent with its location in the vicinity of both the dna and the cbf. this location allows for interactions of cbf to be communicated to wing via wing1 ( see below for additional details ) , as discussed previously ( 13 ) . interestingly , the other - dna region makes a significant contribution to cooperativity by its unfavorable contribution in the rd such a change indicates that the binding of cbf leads to a conformational change in the core region of rd that favors interactions with the dna and , due to the other - dna region not being in direct contact with the dna , suggests that long - range interactions contribute to binding cooperativity . to better understand the role of the wing segment in facilitation of dna binding upon trimer formation , the contribution of this region was broken down into a per residue basis ( table 4 ) . overall , the majority of the favorable interactions are made by arg139 , arg142 and lys144 , as expected due to their positive charge and the polyanionic nature of dna . with all three residues in addition , more favorable interactions occur with ser140 , gly143 and ser145 , with the contribution of gly143 being relatively large . thus , specific residues in the wing contribute to more favorable interactions with the dna upon formation of the ternary complex . details of the role of cbf and the wing1 segment to these contributions will be discussed below . similar to rd dna binding , the rd to cbf interaction energy is increased upon trimer formation . the overall cooperativity gain of 60 kcal / mol is due to the wing1 and n - terminal segments . the contribution of the wing segment to cbf binding becomes less favorable whereas the contribution of this segment becomes more favorable to dna binding upon trimerization ( table 3 ) . such a scenario suggests a subtle balance between interactions of the wing and wing1 segments that contribute to the cooperative binding . interestingly , as with the other - dna segment discussed above for rd dna binding , the n - terminal segment makes a favorable contribution to the cooperativity of rd cbf binding although it is spatially remote from the dna in the ternary complex . this indicates that binding of dna to form the trimer leads to global conformational changes in rd that favor rd cbf interactions , further indicating the contribution of long - range interactions to cooperativity . residue breakdown of the interaction energy contributions of the wing1 and n - terminal regions to cbfrd interactions is presented in table 6 . the energetic contribution of a number of the residues changes significantly upon going from the cbfrd dimer to the trimer , with both favorable and unfavorable contributions occurring . the most significant contributions , both favorable , occur with glu116 in wing1 and asp66 in the n - terminal segment . the importance of glu116 for the binding of cbf has not previously been described , although perturbed chemical shifts of glu116 have been observed in nmr studies in the presence of either cbf or dna ( 13,17,46 ) and a significant decrease in the order parameter of glu116 has been observed upon going from the rd the interaction energies between rd glu116 and cbf in the binary and ternary complexes are 20 8 and 67 9 kcal / mol , respectively . in the present study , it was found that rd glu116 interacts with cbf residue arg33 . interestingly , the experimental change in kd was not accounted for in terms of any residue(s ) in the rd . to understand this omission , distances between the two residues in the binary and ternary complexes as a function of time were obtained ( supplementary figure s1 ) . it is observed that in the ternary complex during the md simulation , the sidechains of rd glu116 and cbf arg33 move to within 23 , in contrast to the oe2-nh2 distance being 9.4 in the crystallographic structure of the ternary complex ( 13 ) . in contrast , in the binary complex crystal structure the oe2-nh2 distance is 12 ; this distance initially decreases to shorter values during the md simulation , but upon further simulation time assumes a stable conformation with a distance of 12 between the oe2-nh2 atoms . to better understand the significant changes in the rd glu116 and cbf arg33 distances for the different systems the experimental structure along with final time frames from the 4.5 ns rd cbf binary and ternary simulations were obtained ( figure 3 ) . in the rd cbf dimer , rd glu116 and cbf arg33 are relatively far apart ( figure 3a ) . upon going to the ternary complex in the md simulation it is clear that a salt bridge forms between glu116 and arg33 ( figure 3b ) . the formation of this salt bridge is associated with a shift in the position of the arg33 sidechain towards the rd , which is suggested to be associated with the electrostatic field due to the presence of dna in the ternary complex as evidenced by the favorable interactions between the cbf arg33 and the dna ( 252.2 6.1 kcal / mol ) . in addition , energy partitioning analysis shows that arg33 of cbf overall disfavors rd cbf binding in the trimer with this effect significantly increased in the rd cbf dimer by 40 kcal / mol [ arg33-rd(trimer ) = 11.0 4.9 and arg33-rd(dimer ) = 50.3 3.3 ] , further disfavoring the rd glu116 to cbf arg33 interaction in the dimer . however , the question remains why is the rd glu116 and cbf arg33 interaction not observed in the ternary complex crystal structure ( figure 3c ) . although speculative , it may be due to the presence of higher salt concentrations in the experimental study ( 200 mm nacl ) ( 13 ) , which may partially screen the arg33dna interactions . this screening combined with competing interactions involving other residues , such as the rd tyr113 to cbf arg33 interaction that occurs in the crystal and rd cbf dimer md structures , may lead to the subtle difference between the two systems . supporting this possibility is the nmr observed decrease in the glu116 order parameter in the ternary complex ( 20 ) , suggesting that in solution and at lower salt concentration the proposed salt bridge may be present . other experimental results indicate a 5-fold increase in the dissociation constant upon formation of the ternary complex from the rd dna dimer ( 18 ) , which may be related to tyr113 properly orienting arg33 for the actual binding event . regardless , the rd glu116 to cbf arg33 salt bridge along with the additional interactions leads to the more favorable interaction energy between cbf and rd in the ternary versus the binary complex ( table 3 ) . thus , it is predicted that glu116 of rd has a significant impact on the binding of cbf to rd as well as on the cooperativity of rd binding to dna and cbf. notably , binding of cbf to the rd dna dimer has been shown via nmr to decrease the conformational variability of glu116 ( 20 ) , supporting the impact of rd interactions with its binding partners on this residue . the interaction energies for met106 , ala107 , asn109 , asn112 , tyr113 and ser114 ( table 6 ) are consistent with the experimental mutation data . mutation of these residues decreases rd cbf binding in the experimental study ( 17,18 ) , consistent with the favorable interaction energies of these residues with cbf. however , mutation of asn109 to alanine decreased the rd cbf binding by 60-fold while the calculated interaction energy is relatively small . this discrepancy may be due to mutation induced structural perturbations of the rd ( i.e. partial unfolding ) , leading to the experimentally observed decrease in binding ( 18 ) . recently , it has been shown that polar residues at protein protein interfaces provide structural rigidity so as to maintain specificity and to reduce the entropic cost upon binding ( 47 ) . thus , asn109 may play an important role in maintenance of the structural integrity of rd with a modest contribution to affinity associated with direct interactions with cbf. the n - terminal segment makes a significant , favorable gain in interaction energy with cbf upon going from the rd cbf binary to the ternary complex ( table 5 ) . as shown in table 6 , the contribution was dominated by asp66 . however , experimental mutation of asp66 to alanine only moderately affected the rd cbf binding strength ( 19 ) . visual inspection of the binary md , ternary md and crystal structures ( figure 4 ) , along with energy partitioning analysis shows asp66 to interact favorably , via its backbone carbonyl , with lys11 of cbf , with this interaction becoming a salt bridge involving the sidechains in the trimer . cbf dimer md and crystal structures and the trimer md structure appears to be due to a competitive interaction between asp66 with his163 in rd ( figure 4a and c , respectively ) . consistent with the results discussed above , this suggests that the electrostatic field imposed by the polyanionic dna in the ternary simulations disrupts the asp66-his163 interaction seen in the dimer and helps in orienting asp66 toward lys11 ( figure 4c ) , with the presence of 200 mm nacl in the experimental structure potentially damping this interaction . his163 was treated as neutral in the md simulations ; hypothetically , a different result might be obtained if his163 was treated as being protonated . presumably this would create a stronger interaction between rd asp66 and his163 , such that this interaction would dominate in both the ternary and the binary md simulations , with the asp66 backbone carbonyl oxygen interacting with the lys11 sidechain amino group in both cases . such a scenario where the interaction of asp66 with lys11 involves the formers backbone atoms could explain why a significant contribution of this residue to binding was not observed experimentally . area2 residues make favorable contributions to the interaction energy between rd and cbf ( table 5 ) . although this region does not contribute to the cooperativity , as evidenced by the similar contribution in the binary and the ternary complexes , the large favorable interaction energy contribution makes it of interest to investigate the role of individual residues in this region to binding ( table 6 ) . the importance of thr161 is thought to be due to its interaction with asn104 of cbf. indeed , visual inspection of the rd cbf binary md , ternary md and crystal structures shows two hydrogen bonds between these residues [ asn(nh2)-thr(og1 ) and asn(od1-thr(hn ) ] ( figure 5 ) . however , calculation of the residue residue interaction energy between rd thr161 and cbf asn104 yields a value of 4.9 0.4 kcal / mol , including the contribution from the thr hn atom , which does not correlate well with the change in dissociation constant in the t161a mutation . alternatively , calculation of the contribution of thr161 to the strain energy of rd yields values of 21.0 1.8 and 26.5 0.2 kcal / mol in the rd monomer and rd cbf dimer , respectively . interestingly , it has been reported that the mutation of thr161 to alanine leads to decreased exchange broadening in rd alone in nmr experiments , indicating a decrease in the conformational flexibility of residue 161 and surrounding residues ( 18 ) . this observation combined with the present energetic results suggests that thr161 may play a role in maintaining the conformation of the local region of rd , thereby impacting binding , rather than contributing directly to protein protein interactions . such a role of thr161 is consistent with the idea that conserved polar residues at protein protein binding sites provide structural rigidity ( 47 ) . the role of such conserved residues is primarily structural rather than functional . as discussed previously ( 13 ) and as emphasized above , the region of rd that includes the segments wing and wing1 appears to be of central importance for binding cooperativity . presented in table 7 are the interaction energies between wing and wing1 , including a breakdown of the interaction of wing residues with all wing1 residues and vice versa . upon going from rd to the rd wing1 total interaction energy becomes significantly more favorable , even though favorable intermolecular interactions occur between wing1 and cbf in the rd this suggests that the binding of cbf to rd , while forming a favorable interaction of cbf with wing1 also induces a conformational change in wing1 that favors the wing wing1 interaction . indeed , figure 6 shows that two salt bridges , arg142-glu111 and lys144-asp110 , are present in the rd cbf dimer ( figure 6b ) ; this represents a gain in interaction energy over the hydrogen bonds between lys144 and asp110 and glu111 in rd alone ( figure 6a ) . indeed , these favorable interactions contribute to the gain in favorable strain energy upon formation of the rd cbf dimer discussed above ( table 1 ) . moreover , nmr experiments indicate all four of these residues to undergo changes in mobility upon going from the rd thus , these intramolecular interactions make a significant contribution to the binding of rd to cbf. in contrast to the rd cbf interaction , upon going from rd alone to the rd this is consistent with the significantly favorable interaction energy between rd and dna in the binary complex , such that when the wing residues interact with the dna , the wing wing1 interactions are diminished ( table 7 ) . similarly , the wing wing1 interaction energy in the trimer is not significantly different as that in rd alone . visual analysis of the rd structure in the monomer ( figure 6a ) , the rd dna complex ( figure 6c ) and the ternary complex ( figure 6d ) , reveals the spatial relationships of residues asp110 , glu111 , arg142 and lys144 to be similar , consistent with the interaction energy contributions in these systems ( table 7 ) . in the rd dna binary and ternary complexes , it is evident that the negatively charged dna attracts arg142 away from the wing1 residues , contributing to the less favorable interactions . in the rd monomer , the lack of salt bridges between arg142 and asp110 or glu111 may be related to repulsion between the two positively charged residues , arg142 and lys144 , such that the system gains more stabilization by orienting arg142 toward the solvent . the balance of attraction and repulsion taking place between closely situated positively and negatively charged residues suggests a subtle balance of interactions , such that their relative orientations are sensitive to the influence of solvent and the presence of dna and cbf. as stated above , formation of the ternary complex leads to a wing energy partitioning analysis ( table 7 ) shows significantly less favorable interaction energies involving arg142 , asp110 and glu111 as compared with the cbfrd binary complex . the less favorable interactions of asp110 and glu111 are consistent with the loss of salt bridges with arg142 , although more favorable interactions with lys144 occur ( figure 6d ) . thus , a subtle balance of intramolecular and intermolecular interactions appear to contribute to the observed cooperativity . interaction of rd with dna leads to favorable intermolecular interactions ( table 3 ) with a simultaneous loss of wing however , upon formation of the ternary complex , the binding of cbf leads to the favorable wing interestingly , the majority of residues predicted to make the largest contributions to cooperativity are charged ( i.e. rd residues asp66 , asp110 , glu111 , glu116 , arg139 , arg142 and lys144 ) . consequently , the long - range character of electrostatic interactions may be an important factor in the observed cooperativity . for example , asp to glu mutations , or vice versa , of the acidic residues suggested to be important for cooperativity may alter the subtle balance of intermolecular and intramolecular interactions contributing to the cooperativity . in addition , mutation of any of the charged residues discussed above to neutral species would be expected to impact cooperativity . asp110 and glu111 may be the most interesting as their impact on cbf binding would be expected to be minimal while their impact on cooperativity should be large . md simulations of rd along with its binding partners cbf and dna , as well as the cbfrd and rd dna binary complexes and the cbfrd dna ternary complex have been performed to better understand the cooperative nature of the binding of these residues . in accordance with the experiment , the simulation data show cooperative binding of dna in the presence of cbf as well as predict that binding of dna to rd facilitates rd cbf binding , indicating cooperativity for this interaction . these observations are consistent with the experimental data ( 16,18 ) , supporting that ability of the applied simulation models to represent the experimental regimen . energetic and structural analysis allow for contributions to the binding and cooperativity to be understood in terms of different regions of the rd protein as well as the contribution of individual residues . of note these interactions are , to a large extent , dominated by charged residues suggesting that long - range interactions may contribute to cooperativity . from these results a number of residues of interest for mutational analysis are suggested . ribbon image of the cbfrd dna after 4.5 ns dynamics simulation including coloring of the rd segments used for analysis ( table 2 ) . dna ( green ) , cbf ( tan ) and residues surrounding omitted residues 7179 in cbf ( red ) . overall binding pathways rd dna , rd cbf and rd cbfdna . free energies before and after the / represent gbind and gbind , ts , as reported in table 1 . cbf interactions involving rd residues tyr113 , glu116 , arg118 and arg139 , and cbf residues arg33 and arg35 . images from snapshots at 4.5 ns from the ( a ) rd cbf binary and ( b ) ternary md simulations and ( c ) from the x - ray crystal structure . images from snapshots at 4.5 ns from the ( a ) rd cbf binary and ( b ) ternary md simulations and ( c ) from the x - ray crystal structure . rd cbf interactions involving rd residue thr161 and cbf residue asn104 . images from snapshots at 4.5 ns from the ( a ) rd cbf binary and ( b ) ternary md simulations and ( c ) from the x - ray crystal structure . wing1 interaction in the md simulation structures at 4.5 ns for ( a ) rd , ( b ) rd cbf , ( c ) rd dna and calculated binding energies and component contributions energies in kcal / mol calculated using equations 14 from the data reported in supplementary table s4 . free energy of binding based on the differences in the free energy of each monomer or complex , g , in supplementary table s4 . it should be noted that the sum of the terms gsolvation , estrain and eie are not exactly equivalent to gbind due to rounding errors . based on the sums , free energies of binding of 1.3 , 81.4 , 86.5 , 6.4 , 87.8 and 5.2 kcal / mol were obtained for the rd + dna , cbf + rd , rd dna + cbf , rd cbf + dna , trimer total and trimer definitions of regions of the rd interacting with dna or cbf color as shown in figure 1 . dna interaction energy energies in kcal / mol as mean se , ie = cbfrd dna rd dna . interaction energy of rd wing residues with dna energies in kcal / mol as mean se , ie = cbfrd dna rd dna . cbf interaction energy energies in kcal / mol as mean se , ie = cbfrd dna cbfrd . interaction energy of selected rd residues with cbf energies in kcal / mol as mean se , ie = cbfrd dna cbfrd . interaction energies between individual wing and wing1 residues in rd alone and in the three complexes energies in kcal / mol .

the runt domain ( rd ) is the dna - binding region of the runx genes . a related protein , known as core binding factor ( cbf ) also binds to the rd to enhance rd dna interaction by 6- to 10-fold . here , we report results from molecular dynamics ( md ) simulations of rd alone , as a dimer in complexes with dna and cbf and in a ternary complex with dna and cbf. consistent with the experimental findings , in the presence of cbf the estimated free energy of binding of rd to the dna is more favorable , which is shown to be due to more favorable intermolecular interactions and desolvation contributions . also contributing to the enhanced binding are favorable intramolecular interactions between the wing residues ( rd residues 139145 ) and the wing1 residues ( rd residues 104116 ) . the simulation studies also indicate that the rd cbf binding is more favorable in the presence of dna due to a more favorable rd cbf interaction energy . in addition , it is predicted that long - range interactions involving ionic residues contribute to binding cooperativity . results from the md calculations are used to interpret a variety of experimental mutagenesis data . a novel role for rd glu116 to the rd cbf interaction is predicted .

INTRODUCTION METHODS RESULTS AND DISCUSSION CONCLUSION SUPPLEMENTARY MATERIAL Supplementary Material Figures and Tables

the runt domain ( rd ) is the dna - binding domain of p53 type transcription factors known as core binding factor ( cbf ) subunits . a related protein , known as cbf , binds to the rd of the cbfs and enhances the dna - binding affinity of the rd . in addition , the involvement of runx genes with angiogenesis also makes the rd a potential target in the development and treatment of many types of cancers . the 3d structures of the rd in the free state , bound to dna , cbf and both dna and cbf have been determined using nuclear magnetic resonance ( nmr ) spectroscopy and/or x - ray crystallography ( 1014 ) . biochemical and biophysical studies have been performed characterizing the binding of rd to dna in the presence and absence of cbf ( 15,16 ) . in the presence of cbf , the dissociation constant ( kd ) for rd dna decreases 6- to 10-fold ( 16 ) . in addition , a recent nmr - based study has identified a variety of residues involved in the cooperativity associated with cbf binding to rd ( 20 ) . thus , computational analysis can fill gaps in understanding the rd dna , rd cbf and cbfrd dna ternary complex interactions . in the present work , the rd was subjected to molecular dynamic ( md ) simulations in the free state and in several complexes . from the md simulations , in combination with free energy component analysis ( 2224 ) based on the generalized - born model ( 25,26 ) , overall binding energies , desolvation effects and detailed interaction energies these results , along with previous structural analysis and observations , were used to interpret a variety of experimental data involving rd dna and rd cbf interactions ( 1619,27 ) . the only exception was the second dna simulation , which was initiated with the dna in the canonical b form ( 34 ) , as obtained from the program quanta ( accelrys inc . ) omission of residues 7179 of cbf , which are spatially removed from the rd in the experimental structure ( figure 1 ) , was performed by creating a peptide bond between residues 70 and 80 and relaxing the structure during the minimization and md simulation protocols described below . for complexes , emm , can be separated into interaction energy , eie and strain , estrain components : 4emm , complex = eie+estrain , where eie is the sum of the vdw and electrostatic interaction energy between individual molecules in a complex and estrain is the sum of emm terms for each molecule in the complex ( i.e. however , in the case of the trimer formation via dimer + monomer complexation the eie contribution includes those between the monomer being added and the two molecules in the original dimer along with the change in the interaction energy between the molecules in the original dimer upon going to a trimer . this term accounts for gains or losses in the interaction energy of the molecules in a dimer when the third molecule binds to form a trimer . md simulation studies were undertaken on the rd to understand the nature of interactions with the dna and with the binding partner , cbf. average rms differences for all non - hydrogen atoms with respect to the crystallographic ternary complex structures were 1.62 0.04 , 3.56 0.10 , 2.15 0.04 , 3.23 0.16 , 3.63 0.11 and 2.81 0.05 for the dna , rd , cbf , rd dna , rd cbf and cbfrd dna structures , respectively , indicating that the overall structures were maintained in the simulations . in addition , the calculations predict that rd cbf binding is more favorable than rd dna binding , in contrast to experimental observations ( 16 ) . the presence of these discrepancies is not surprising due to a variety of assumptions in the calculations , including the omission of energetic contributions , such as the solvation and entropic contributions of the added counterions ( 23 ) , limitations in the potential energy function and the force field ( 44 ) along with possible limitations in the sampling of conformational space . dna interaction energies in the force field by < 4% would account for the prediction that binding of rd to cbf is more favorable than rd binding to dna . with respect to the tsmm , the use of gas phase minimized structures may be problematic , especially in the case of the polyanionic dna where the electrostatic screening due to the 4r dielectric is expected to be insufficient when compared with the proteins . moreover , in both scenarios , the binding of dna to the rd cbf dimer to form the trimer is more favorable than the binding of rd and dna to form the rd thus , it appears that the present results , although not in quantitative agreement with experiment due to assumptions and limitations in the theoretical models , are representative of the experimental regimen , allowing for atomic details of events driving binding to be elucidated . in addition , the emphasis in this work on differences in energies and structures between the systems rather than absolute energies should allow for increased confidence in the observations and the conclusions based on those observations . these results are consistent with previous studies showing the binding of dna to rd to be enhanced by the binding of cbf as well as the binding of cbf to rd to be enhanced by dna based on electrophoretic mobility shift assay ( 10,16,18 ) . as discussed , such cooperativity may be important for maintaining binding of rd to dna via stabilization of the ternary complex to insure adequate transcriptional activation . contributing to the cooperativity is a decrease in the unfavorable solvation that occurs upon binding , evidenced by the trimer the more favorable change in interaction energy occurred with both the rd dna and the rd cbf interactions in the trimer ( table 1 ) ; details of the contributions of different regions of the proteins to this will be discussed below . thus , the cooperativity of dna and cbf binding to rd is predicted to be dominated by gains in the favorable interactions between the macromolecules and with lowering of the solvation penalty to complex formation . a more detailed understanding of the atomic determinants of cooperativity associated with rd binding to cbf and dna was obtained via interaction energy partitioning analysis between different regions of the rd and both cbf and dna . residues adjacent to the respective binding partner but not included in a defined segment are referred to as other - dna and other - cbf and all residues not previously defined are referred to as non - interacting - cbf , allowing for the quantification of their contributions to binding . of note are the wing residues , which are primarily important for dna binding , while residues in wing1 are proximal to the wing residues , contacting both cbf and the wing residues . dna interaction energies in both the trimer and the dimer complexes along with the change in interaction energy upon going from the rd dna dimer to the trimer , with those contributions broken down into the segments presented in table 2 . upon trimer formation the total rd dna interaction energy becomes more favorable , with three of the segments contributing significantly to the more favorable interaction energy in the trimer . the largest contribution is from the wing segment , consistent with its location in the vicinity of both the dna and the cbf. interestingly , the other - dna region makes a significant contribution to cooperativity by its unfavorable contribution in the rd such a change indicates that the binding of cbf leads to a conformational change in the core region of rd that favors interactions with the dna and , due to the other - dna region not being in direct contact with the dna , suggests that long - range interactions contribute to binding cooperativity . overall , the majority of the favorable interactions are made by arg139 , arg142 and lys144 , as expected due to their positive charge and the polyanionic nature of dna . thus , specific residues in the wing contribute to more favorable interactions with the dna upon formation of the ternary complex . details of the role of cbf and the wing1 segment to these contributions will be discussed below . similar to rd dna binding , the rd to cbf interaction energy is increased upon trimer formation . interestingly , as with the other - dna segment discussed above for rd dna binding , the n - terminal segment makes a favorable contribution to the cooperativity of rd cbf binding although it is spatially remote from the dna in the ternary complex . this indicates that binding of dna to form the trimer leads to global conformational changes in rd that favor rd cbf interactions , further indicating the contribution of long - range interactions to cooperativity . the importance of glu116 for the binding of cbf has not previously been described , although perturbed chemical shifts of glu116 have been observed in nmr studies in the presence of either cbf or dna ( 13,17,46 ) and a significant decrease in the order parameter of glu116 has been observed upon going from the rd the interaction energies between rd glu116 and cbf in the binary and ternary complexes are 20 8 and 67 9 kcal / mol , respectively . it is observed that in the ternary complex during the md simulation , the sidechains of rd glu116 and cbf arg33 move to within 23 , in contrast to the oe2-nh2 distance being 9.4 in the crystallographic structure of the ternary complex ( 13 ) . to better understand the significant changes in the rd glu116 and cbf arg33 distances for the different systems the experimental structure along with final time frames from the 4.5 ns rd cbf binary and ternary simulations were obtained ( figure 3 ) . in the rd cbf dimer , rd glu116 and cbf arg33 are relatively far apart ( figure 3a ) . upon going to the ternary complex in the md simulation it is clear that a salt bridge forms between glu116 and arg33 ( figure 3b ) . the formation of this salt bridge is associated with a shift in the position of the arg33 sidechain towards the rd , which is suggested to be associated with the electrostatic field due to the presence of dna in the ternary complex as evidenced by the favorable interactions between the cbf arg33 and the dna ( 252.2 6.1 kcal / mol ) . in addition , energy partitioning analysis shows that arg33 of cbf overall disfavors rd cbf binding in the trimer with this effect significantly increased in the rd cbf dimer by 40 kcal / mol [ arg33-rd(trimer ) = 11.0 4.9 and arg33-rd(dimer ) = 50.3 3.3 ] , further disfavoring the rd glu116 to cbf arg33 interaction in the dimer . however , the question remains why is the rd glu116 and cbf arg33 interaction not observed in the ternary complex crystal structure ( figure 3c ) . although speculative , it may be due to the presence of higher salt concentrations in the experimental study ( 200 mm nacl ) ( 13 ) , which may partially screen the arg33dna interactions . this screening combined with competing interactions involving other residues , such as the rd tyr113 to cbf arg33 interaction that occurs in the crystal and rd cbf dimer md structures , may lead to the subtle difference between the two systems . other experimental results indicate a 5-fold increase in the dissociation constant upon formation of the ternary complex from the rd dna dimer ( 18 ) , which may be related to tyr113 properly orienting arg33 for the actual binding event . regardless , the rd glu116 to cbf arg33 salt bridge along with the additional interactions leads to the more favorable interaction energy between cbf and rd in the ternary versus the binary complex ( table 3 ) . thus , it is predicted that glu116 of rd has a significant impact on the binding of cbf to rd as well as on the cooperativity of rd binding to dna and cbf. notably , binding of cbf to the rd dna dimer has been shown via nmr to decrease the conformational variability of glu116 ( 20 ) , supporting the impact of rd interactions with its binding partners on this residue . mutation of these residues decreases rd cbf binding in the experimental study ( 17,18 ) , consistent with the favorable interaction energies of these residues with cbf. the n - terminal segment makes a significant , favorable gain in interaction energy with cbf upon going from the rd cbf binary to the ternary complex ( table 5 ) . cbf dimer md and crystal structures and the trimer md structure appears to be due to a competitive interaction between asp66 with his163 in rd ( figure 4a and c , respectively ) . consistent with the results discussed above , this suggests that the electrostatic field imposed by the polyanionic dna in the ternary simulations disrupts the asp66-his163 interaction seen in the dimer and helps in orienting asp66 toward lys11 ( figure 4c ) , with the presence of 200 mm nacl in the experimental structure potentially damping this interaction . although this region does not contribute to the cooperativity , as evidenced by the similar contribution in the binary and the ternary complexes , the large favorable interaction energy contribution makes it of interest to investigate the role of individual residues in this region to binding ( table 6 ) . however , calculation of the residue residue interaction energy between rd thr161 and cbf asn104 yields a value of 4.9 0.4 kcal / mol , including the contribution from the thr hn atom , which does not correlate well with the change in dissociation constant in the t161a mutation . alternatively , calculation of the contribution of thr161 to the strain energy of rd yields values of 21.0 1.8 and 26.5 0.2 kcal / mol in the rd monomer and rd cbf dimer , respectively . interestingly , it has been reported that the mutation of thr161 to alanine leads to decreased exchange broadening in rd alone in nmr experiments , indicating a decrease in the conformational flexibility of residue 161 and surrounding residues ( 18 ) . as discussed previously ( 13 ) and as emphasized above , the region of rd that includes the segments wing and wing1 appears to be of central importance for binding cooperativity . upon going from rd to the rd wing1 total interaction energy becomes significantly more favorable , even though favorable intermolecular interactions occur between wing1 and cbf in the rd this suggests that the binding of cbf to rd , while forming a favorable interaction of cbf with wing1 also induces a conformational change in wing1 that favors the wing wing1 interaction . indeed , figure 6 shows that two salt bridges , arg142-glu111 and lys144-asp110 , are present in the rd cbf dimer ( figure 6b ) ; this represents a gain in interaction energy over the hydrogen bonds between lys144 and asp110 and glu111 in rd alone ( figure 6a ) . indeed , these favorable interactions contribute to the gain in favorable strain energy upon formation of the rd cbf dimer discussed above ( table 1 ) . moreover , nmr experiments indicate all four of these residues to undergo changes in mobility upon going from the rd thus , these intramolecular interactions make a significant contribution to the binding of rd to cbf. in contrast to the rd cbf interaction , upon going from rd alone to the rd this is consistent with the significantly favorable interaction energy between rd and dna in the binary complex , such that when the wing residues interact with the dna , the wing wing1 interactions are diminished ( table 7 ) . similarly , the wing wing1 interaction energy in the trimer is not significantly different as that in rd alone . visual analysis of the rd structure in the monomer ( figure 6a ) , the rd dna complex ( figure 6c ) and the ternary complex ( figure 6d ) , reveals the spatial relationships of residues asp110 , glu111 , arg142 and lys144 to be similar , consistent with the interaction energy contributions in these systems ( table 7 ) . in the rd dna binary and ternary complexes , it is evident that the negatively charged dna attracts arg142 away from the wing1 residues , contributing to the less favorable interactions . the balance of attraction and repulsion taking place between closely situated positively and negatively charged residues suggests a subtle balance of interactions , such that their relative orientations are sensitive to the influence of solvent and the presence of dna and cbf. interaction of rd with dna leads to favorable intermolecular interactions ( table 3 ) with a simultaneous loss of wing however , upon formation of the ternary complex , the binding of cbf leads to the favorable wing interestingly , the majority of residues predicted to make the largest contributions to cooperativity are charged ( i.e. for example , asp to glu mutations , or vice versa , of the acidic residues suggested to be important for cooperativity may alter the subtle balance of intermolecular and intramolecular interactions contributing to the cooperativity . md simulations of rd along with its binding partners cbf and dna , as well as the cbfrd and rd dna binary complexes and the cbfrd dna ternary complex have been performed to better understand the cooperative nature of the binding of these residues . in accordance with the experiment , the simulation data show cooperative binding of dna in the presence of cbf as well as predict that binding of dna to rd facilitates rd cbf binding , indicating cooperativity for this interaction . of note these interactions are , to a large extent , dominated by charged residues suggesting that long - range interactions may contribute to cooperativity . wing1 interaction in the md simulation structures at 4.5 ns for ( a ) rd , ( b ) rd cbf , ( c ) rd dna and calculated binding energies and component contributions energies in kcal / mol calculated using equations 14 from the data reported in supplementary table s4 . free energy of binding based on the differences in the free energy of each monomer or complex , g , in supplementary table s4 . based on the sums , free energies of binding of 1.3 , 81.4 , 86.5 , 6.4 , 87.8 and 5.2 kcal / mol were obtained for the rd + dna , cbf + rd , rd dna + cbf , rd cbf + dna , trimer total and trimer definitions of regions of the rd interacting with dna or cbf color as shown in figure 1 . interaction energy of rd wing residues with dna energies in kcal / mol as mean se , ie = cbfrd dna rd dna .

staphylococcus aureus and coagulase - negative staphylococci ( cons ) are well known to most clinicians as a cause of nosocomial and community - associated infections worldwide ( 1 , 2 ) . staphylococci are able to develop resistance to many routinely used antibiotics to which they were susceptible first ( 1 ) . moreover , this condition has become more complicated because of various factors such as horizontal acquisition of foreign genetic elements and ability to establish persistent infections ( e.g. biofilm formation ) in the host . nowadays , due to wide spread use of indwelling and prosthetic medical devices , cons have gained much attention as considerable agents involved in nosocomial infections ( 3 ) . a major part of these infections are caused by the transmission of the bacteria from hospital employees as reservoir to patients . in particular , this situation can lead to serious infections in immunocompromised patients who are hospitalized with underlying diseases ( 4 - 8 ) . macrolides including erythromycin are the antibiotics used against gram - positive and some gram - negative bacteria . three mechanisms in gram - positive bacteria that result in resistance to erythromycin are as follows : ( i ) modification in the ribosomal target site , mediated by the methyltransferases encoded by erm ( erythromycin resistance methylase ) genes , ( ii ) efflux pump encoded by msra / b ( macrolide specific resistance genes ) and ( iii ) erea / b ( erythromycin esterases ) genes ( 9 - 12 ) . among these mechanisms , many reported and sequenced erm genes , three major genes of erma , ermb and ermc are present in staphylococci ( 12 , 13 ) . due to the development of resistant strains , epidemiological data on antibiotic susceptibility of staphylococcus strains isolated from nasal carriers in each region can be helpful to select appropriate drugs to eradicate carriage states , control the nosocomial infections and also treat the patients ( 14 , 15 ) . the current study aimed to investigate the antibiotic susceptibility profile and the prevalence of the erma , ermb , ermc and msra genes among staphylococcal strains isolated from the nares of hospital personnel in khorramabad , iran . during this cross - sectional study , a total of 340 volunteers from hospital staff ( males and females ) , employed in various wards of four referral and state hospitals ( shohaday - e - ashayer , asalian , shahid madani and taemin ejtemai ) in khorramabad city ( west of iran ) were included and screened for nasal carriage of s. aureus from july 2011 to january 2012.the subjects with a history of any antibiotic consumption for at least ten days at the time of sampling were excluded . the sample size was estimated via formula to calculate the ratio with an accuracy of 0.05 ( d = 0.05 ) . to collect samples , three hospitals under study were general ( 400 , 350 and 150 beds ) and one hospital was specialized ( 100 beds ) . finally 100 adults of both genders ( 40 males and 60 females ) were enrolled . information about variables including gender , work experience and the ward which hospital staff worked was collected by a questionnaire . the samples were collected from both anterior nares of the subjects by rotating a sterile cotton wool swab . the swabs were placed and transported in 3 ml tryptic soy broth ( merck , germany ) and incubated at 37c . after overnight incubation , a loop full of inoculated broth medium was subcultured on 10% sheep blood agar , mannitol salt agar and nutrient agar ( merck , germany ) ( 16 ) . presumptive staphylococcal colonies ( growth on mannitol salt agar , gram - positive and catalase - positive cocci ) were tested further for the production of coagulase and dnase enzymes according to the standard bacteriological procedures ( 17 ) . isolates with positive reactions ( coagulase - positive and dnase - positive ) were identified as s. aureus ; other staphylococcal species with negative test results designated as cons . susceptibility of staphylococcus isolates to antibiotics ( mast , merseyside , united kingdom ) including erythromycin ( 15 g ) , clarithromycin ( 15 g ) , azithromycin ( 15 g ) , cefoxitin ( 30 g ) , penicillin g ( 10 u ) , vancomycin ( 30 g ) , tetracycline ( 30 g ) , clindamycin ( 2 g ) , trimethoprim / sulfamethoxazole ( 1.25/23.75 g ) and rifampin ( 5 g ) was determined by the kirby - bauer disk diffusion method on muller - hinton agar , according to the procedures described by the clinical and laboratory standards institute ( clsi ) ( 18 ) . total dna ( consisting of the chromosomal and the plasmid ) of all the isolates was extracted and collected using accuprep genomic dna extraction kit ( bioneer , korea ) with some modifications . briefly , a 1 ml of luria - bertani broth ( lb ) bacterial colony of each strain was suspended in 1 ml of lysogeny broth ( lb ) medium , and incubated at 37c for 18 hours . the bacterial cells were harvested by centrifugation at 7,500 g for 10 minutes and resuspended in 200 l of phosphate - buffered saline ( pbs ) and mixed well . to ensure adequate cell breakage , 10 l lysostaphin ( 100 g / ml in sterile deionized water ; sigma ) was added to the bacterial mixture and incubated at 37c for 30 minutes ( 19 ) . then , 10 l of proteinase k ( 20 mg / ml ) was added to the sample and after incubating at 56c for 30 minutes , the process was continued according to the manufacturer 's instructions . the four primer pairs used to amplify the erma , ermb , ermc and msra genes are listed in table 1 ( 12 ) . in the current study , multiplex polymerase chain reaction ( pcr ) amplification was performed for erma , ermc and msra genes as previously described by zmantar et al . ( 12 ) in a 25 l reaction mixture consisting of : 1x pcr reaction buffer , 2.5 mm mgcl2 , 0.2 mm dntp , 25 pmol of each of the three primer pairs , 1.2 u of taq dna polymerase ( fermentas , lithuania ) and 200 ng dna template . for ermb gene amplification , the ermb primers ( 25 pmol ) were added in a separate pcr reaction under the same above - mentioned conditions . amplification was carried out using a gradient thermal cycler ( bio - rad , my cycler , us ) as the following program : five minutes at 94c , 30 cycles of amplification , consisting one minute at 95c , 0.5 minute at 55c and two minutes at 72c , ending with 10 minutes at 72c ( final extension ) . to verify amplification , 5 l of pcr products were subjected to agarose gel electrophoresis ( 1.5% agarose , 1x tae , 100v ) . ethidium bromide - stained dna fragments were then visualized on a uv transilluminator at 300 nm in comparison with a 50-bp molecular size standard ladder ( fermentas , lithuania ) . to verify the authenticity of the amplicons , pcr products of three positive samples containing one of the genes of ermb , ermc or msra as representative the resulting sequences were aligned with corresponding sequences in the genbank database using at the national center for biotechnology information ( ncbi ) blast program ( http://blast.ncbi.nlm.nih.gov/blast.cgi ? program = blastn and page - type = blast search and link - loc = blasthome ) . to minimize the random errors , chi - square test and t - test were used to determine any significant differences between prevalence of the tested genes among s. aureus and cons strains . the study was approved by ethical committee of lorestan university of medical sciences , khorramabad , iran ( no . during this cross - sectional study , a total of 340 volunteers from hospital staff ( males and females ) , employed in various wards of four referral and state hospitals ( shohaday - e - ashayer , asalian , shahid madani and taemin ejtemai ) in khorramabad city ( west of iran ) were included and screened for nasal carriage of s. aureus from july 2011 to january 2012.the subjects with a history of any antibiotic consumption for at least ten days at the time of sampling were excluded . the sample size was estimated via formula to calculate the ratio with an accuracy of 0.05 ( d = 0.05 ) . to collect samples , three hospitals under study were general ( 400 , 350 and 150 beds ) and one hospital was specialized ( 100 beds ) . finally 100 adults of both genders ( 40 males and 60 females ) were enrolled . information about variables including gender , work experience and the ward which hospital staff worked was collected by a questionnaire . the samples were collected from both anterior nares of the subjects by rotating a sterile cotton wool swab . the swabs were placed and transported in 3 ml tryptic soy broth ( merck , germany ) and incubated at 37c . after overnight incubation , a loop full of inoculated broth medium was subcultured on 10% sheep blood agar , mannitol salt agar and nutrient agar ( merck , germany ) ( 16 ) . presumptive staphylococcal colonies ( growth on mannitol salt agar , gram - positive and catalase - positive cocci ) were tested further for the production of coagulase and dnase enzymes according to the standard bacteriological procedures ( 17 ) . isolates with positive reactions ( coagulase - positive and dnase - positive ) were identified as s. aureus ; other staphylococcal species with negative test results designated as cons . susceptibility of staphylococcus isolates to antibiotics ( mast , merseyside , united kingdom ) including erythromycin ( 15 g ) , clarithromycin ( 15 g ) , azithromycin ( 15 g ) , cefoxitin ( 30 g ) , penicillin g ( 10 u ) , vancomycin ( 30 g ) , tetracycline ( 30 g ) , clindamycin ( 2 g ) , trimethoprim / sulfamethoxazole ( 1.25/23.75 g ) and rifampin ( 5 g ) was determined by the kirby - bauer disk diffusion method on muller - hinton agar , according to the procedures described by the clinical and laboratory standards institute ( clsi ) ( 18 ) . total dna ( consisting of the chromosomal and the plasmid ) of all the isolates was extracted and collected using accuprep genomic dna extraction kit ( bioneer , korea ) with some modifications . briefly , a 1 ml of luria - bertani broth ( lb ) bacterial colony of each strain was suspended in 1 ml of lysogeny broth ( lb ) medium , and incubated at 37c for 18 hours . the bacterial cells were harvested by centrifugation at 7,500 g for 10 minutes and resuspended in 200 l of phosphate - buffered saline ( pbs ) and mixed well . to ensure adequate cell breakage , 10 l lysostaphin ( 100 g / ml in sterile deionized water ; sigma ) was added to the bacterial mixture and incubated at 37c for 30 minutes ( 19 ) . then , 10 l of proteinase k ( 20 mg / ml ) was added to the sample and after incubating at 56c for 30 minutes , the process was continued according to the manufacturer 's instructions . the four primer pairs used to amplify the erma , ermb , ermc and msra genes are listed in table 1 ( 12 ) . in the current study , multiplex polymerase chain reaction ( pcr ) amplification was performed for erma , ermc and msra genes as previously described by zmantar et al . ( 12 ) in a 25 l reaction mixture consisting of : 1x pcr reaction buffer , 2.5 mm mgcl2 , 0.2 mm dntp , 25 pmol of each of the three primer pairs , 1.2 u of taq dna polymerase ( fermentas , lithuania ) and 200 ng dna template . for ermb gene amplification , the ermb primers ( 25 pmol ) were added in a separate pcr reaction under the same above - mentioned conditions . amplification was carried out using a gradient thermal cycler ( bio - rad , my cycler , us ) as the following program : five minutes at 94c , 30 cycles of amplification , consisting one minute at 95c , 0.5 minute at 55c and two minutes at 72c , ending with 10 minutes at 72c ( final extension ) . to verify amplification , 5 l of pcr products were subjected to agarose gel electrophoresis ( 1.5% agarose , 1x tae , 100v ) . ethidium bromide - stained dna fragments were then visualized on a uv transilluminator at 300 nm in comparison with a 50-bp molecular size standard ladder ( fermentas , lithuania ) . to verify the authenticity of the amplicons , pcr products of three positive samples containing one of the genes of ermb , ermc or msra as representative the resulting sequences were aligned with corresponding sequences in the genbank database using at the national center for biotechnology information ( ncbi ) blast program ( http://blast.ncbi.nlm.nih.gov/blast.cgi ? program = blastn and page - type = blast search and link - loc = blasthome ) . to minimize the random errors , chi - square test and t - test were used to determine any significant differences between prevalence of the tested genes among s. aureus and cons strains . the study was approved by ethical committee of lorestan university of medical sciences , khorramabad , iran ( no . a total of 51 s. aureus strains were isolated from anterior nares of the 340 screened subjects ( i.e. the nasal carriage rate for s. aureus was 15% ) . of the 51 s. aureus - carriers , 22 ( 43.1% ) were males and 29 ( 56.9% ) were females . on the other hand , 49 randomly selected cons strains , isolated from nares of non - s . aureus carriers [ 18 males ( 36.7% ) , 31 females ( 63.3% ) ] were also included and tested in the study . there was no statistical difference between the two groups ( s. aureus nasal carriers and cons ) in terms of gender ( p = 0.72 ) . average work experience for cons obtained subjects was 6.4 0.89 years , and within s. aureus - nasal carriers were 4.8 0.68 years . based on the t - test result , difference in the mean of work experience between the two groups was not statistically significant ( p = 0.22 ) . the resistance profile for all isolates to macrolides as well as other tested antibiotics is listed in table 2 . the majority of isolates expressed resistance to penicillin ( 96% ) . no resistance to vancomycin was noted . twenty - nine ( 29% ) isolates were resistant to cefoxitin ( eight s. aureus and twenty - one cons ) . fifty - three ( 53% ) isolates were simultaneously resistant to erythromycin , azithromycin and clarithromycin ( cross - resistance ) ; while 8 ( 8% ) isolates had various macrolide susceptibility patterns . for example , two isolates were intermediate to erythromycin , while they were resistant to azithromycin and clarithromycin . in addition , two isolates were found susceptible to erythromycin , while they were resistant to clarithromycin . the frequency of resistance to all tested antibiotics between cons and s. aureus isolates were statistically significant ( p < 0.001 ) . distribution of susceptibility phenotypes to erythromycin among the isolates of s. aureus and cons according to the gender is shown in table 3 . statistical analyses showed that among the s. aureus isolates difference in prevalence of resistance to erythromycin was significant between males and females ( p = 0.011 ) ; while such difference was not significant within cons isolates ( p = 0.771 ) . abbreviation : cons , coagulase - negative staphylococci . comparison between isolates performed by using chi - square test , p value < 0.001 . all of the isolates were screened for the presence of the four genes erma , ermb , ermc and msra as the main causative agents of resistance to macrolides . as predicted , the positive isolates for the erma , ermb , ermc and msra genes showed 139 , 142 , 190 and 163 bp bands , respectively ( figure 1 ) . the frequency rate of erma , ermb , ermc and msra genes were 3% , 5% , 33% and 20% , respectively ; separately illustrated for s. aureus and cons isolates in figure 2 . it was found that among the 53 isolates resistant to erythromycin , 44 ( 83% ) isolates ( eight s. aureus and thirty - six cons ) carried at least one of the four tested genes . of these erythromycin - resistant isolates , nine ( approximately 17% ) isolates ( one s. aureus and eight cons ) harbored genes of ermc and msra , and one ( % 1.8 ) cons isolate also harbored erma and ermc genes simultaneously . however , combination of three or four genes was not found among the tested isolates ( table 4 ) . eight ( 8% ) isolates had intermediate phenotype to erythromycin , in which 4 ( 50% ) isolates carried ermb or ermc genes ( table 4 ) . in addition , it was found that out of 39 erythromycin - susceptible isolates , 36 ( 92.3% ) isolates did not harbor any of the four antibiotic resistant genes , but the remaining isolates ( three isolates ) were positive for ermb or ermc genes , while they were still susceptible to erythromycin ( table 4 ) . interestingly , of the 29 methicillin - resistant staphylococci , 22 ( 75.9% ) isolates were resistant to erythromycin and 19 ( 65.5% ) isolates harbored at least one of the tested genes . a , multiplex pcr amplification of erma , ermc and msra genes ; source of dna for lanes : 1 , negative control strain of erythromycin - sensitive staphylococcus aureus atcc 25923 ; lanes 2 , 4 and 7 , three different s. aureus isolates ( ermc ) ; lane 3 , a coagulase - negative staphylococci ( cons ) isolate ( erma ) ; lanes 5 and 6 , two different cons isolates contain ermc and msra genes simultaneously ; lane 8 , a cons isolate contains msra gene . b , pcr products resulted from amplification of ermb gene ; lanes 1 and 2 , two different s. aureus isolates contain ermb gene ; lane 3 , s. aureus atcc 29213 ( negative control ) ; lanes m , dna molecular size marker ( 50-bp ladder ; fermentas , lithonia ) ; the electrophoresis was run on 1.5% agarose gel , stained with ethidium bromide . nucleotide sequences were submitted to genbank under the accession numbers ab937975 , ab937976 and ab937977 for the ermb , ermc and msra genes , respectively . a total of 51 s. aureus strains were isolated from anterior nares of the 340 screened subjects ( i.e. the nasal carriage rate for s. aureus was 15% ) . of the 51 s. aureus - carriers , 22 ( 43.1% ) were males and 29 ( 56.9% ) were females . on the other hand , 49 randomly selected cons strains , isolated from nares of non - s . aureus carriers [ 18 males ( 36.7% ) , 31 females ( 63.3% ) ] were also included and tested in the study . there was no statistical difference between the two groups ( s. aureus nasal carriers and cons ) in terms of gender ( p = 0.72 ) . average work experience for cons obtained subjects was 6.4 0.89 years , and within s. aureus - nasal carriers were 4.8 0.68 years . based on the t - test result , difference in the mean of work experience between the two groups was not statistically significant ( p = 0.22 ) . the resistance profile for all isolates to macrolides as well as other tested antibiotics is listed in table 2 . the majority of isolates expressed resistance to penicillin ( 96% ) . no resistance to vancomycin was noted . twenty - nine ( 29% ) isolates were resistant to cefoxitin ( eight s. aureus and twenty - one cons ) . fifty - three ( 53% ) isolates were simultaneously resistant to erythromycin , azithromycin and clarithromycin ( cross - resistance ) ; while 8 ( 8% ) isolates had various macrolide susceptibility patterns . for example , two isolates were intermediate to erythromycin , while they were resistant to azithromycin and clarithromycin . in addition , two isolates were found susceptible to erythromycin , while they were resistant to clarithromycin . the frequency of resistance to all tested antibiotics between cons and s. aureus isolates were statistically significant ( p < 0.001 ) . distribution of susceptibility phenotypes to erythromycin among the isolates of s. aureus and cons according to the gender is shown in table 3 . statistical analyses showed that among the s. aureus isolates difference in prevalence of resistance to erythromycin was significant between males and females ( p = 0.011 ) ; while such difference was not significant within cons isolates ( p = 0.771 ) . abbreviation : cons , coagulase - negative staphylococci . comparison between isolates performed by using chi - square test , p value < 0.001 . all of the isolates were screened for the presence of the four genes erma , ermb , ermc and msra as the main causative agents of resistance to macrolides . as predicted , the positive isolates for the erma , ermb , ermc and msra genes showed 139 , 142 , 190 and 163 bp bands , respectively ( figure 1 ) . the frequency rate of erma , ermb , ermc and msra genes were 3% , 5% , 33% and 20% , respectively ; separately illustrated for s. aureus and cons isolates in figure 2 . it was found that among the 53 isolates resistant to erythromycin , 44 ( 83% ) isolates ( eight s. aureus and thirty - six cons ) carried at least one of the four tested genes . of these erythromycin - resistant isolates , nine ( approximately 17% ) isolates ( one s. aureus and eight cons ) harbored genes of ermc and msra , and one ( % 1.8 ) cons isolate also harbored erma and ermc genes simultaneously . however , combination of three or four genes was not found among the tested isolates ( table 4 ) . eight ( 8% ) isolates had intermediate phenotype to erythromycin , in which 4 ( 50% ) isolates carried ermb or ermc genes ( table 4 ) . in addition , it was found that out of 39 erythromycin - susceptible isolates , 36 ( 92.3% ) isolates did not harbor any of the four antibiotic resistant genes , but the remaining isolates ( three isolates ) were positive for ermb or ermc genes , while they were still susceptible to erythromycin ( table 4 ) . interestingly , of the 29 methicillin - resistant staphylococci , 22 ( 75.9% ) isolates were resistant to erythromycin and 19 ( 65.5% ) isolates harbored at least one of the tested genes . a , multiplex pcr amplification of erma , ermc and msra genes ; source of dna for lanes : 1 , negative control strain of erythromycin - sensitive staphylococcus aureus atcc 25923 ; lanes 2 , 4 and 7 , three different s. aureus isolates ( ermc ) ; lane 3 , a coagulase - negative staphylococci ( cons ) isolate ( erma ) ; lanes 5 and 6 , two different cons isolates contain ermc and msra genes simultaneously ; lane 8 , a cons isolate contains msra gene . b , pcr products resulted from amplification of ermb gene ; lanes 1 and 2 , two different s. aureus isolates contain ermb gene ; lane 3 , s. aureus atcc 29213 ( negative control ) ; lanes m , dna molecular size marker ( 50-bp ladder ; fermentas , lithonia ) ; the electrophoresis was run on 1.5% agarose gel , stained with ethidium bromide . nucleotide sequences were submitted to genbank under the accession numbers ab937975 , ab937976 and ab937977 for the ermb , ermc and msra genes , respectively . staphylococcus aureus carrier status can lead to nosocomial infections in hospitalized patients ( 6 , 8) . on the other hand , cons are leading cause agents of opportunistic and device - related infections ( 3 ) . in the current study , in several studies performed in various regions of the world , this carriage status in healthcare workers have been reported ranging ranging from 18.6% to 50% ( 20 ) . moreover , in iran , the frequency of nasal carriage among hospital employees is reported approximately 12.7% - 45% ( 21 ) . unfortunately , the results of disk diffusion testing showed that all the evaluated isolates were multidrug - resistant . it is a serious threat when a methicillin - resistant stain is transferred and spread among hospitalized patients ( therapeutic failure ) ( 22 ) . it was observed that the frequency of resistance among cons isolates was significantly higher than those among s. aureus isolates ; and this finding was also attributable to resistance to erythromycin and cefoxitin ( table 2 ) . while the majority of the erythromycin - resistant strains had cross - resistance to other macrolides ; however eight strains exhibited heterogeneous susceptibility pattern toward macrolide antibiotics ( table 2 ) . it is presumed that this discrepancy is due to alterations in the chemical structure of various generations of macrolides . the association between resistance to methicillin and the presence of tested genes was significant ( p = 0.031 ) . the simple explanation for this phenomenon is carrying and transmission of the various adjacent resistance genes on genetic elements such as plasmids . several studies concerning the distribution of erm genes and efflux pumps are conducted using southern hybridization and pcr techniques ( 13 , 23 , 24 ) . despite previous studies in which have primarily focused on clinical strains of staphylococci ( 1 , 11 , 23 - 28 ) , the current study presents the first report on the occurrence of main genes implicated in macrolides- resistance in staphylococcus strains obtained from nares of hospital employees . the obtained pcr results demonstrated that the most prevalent gene in both s. aureus and cons strains was ermc ( 33% ) . also , the frequency of this gene ( ermc ) in erythromycin - resistant s. aureus and cons were 13.7% and 53% , respectively . ( 25 ) performed with cons in turkey in which a frequency of 30% for ermc was yielded . martineau et al . ( 24 ) reported an occurrence of 66% for s. epidermidis strains . ( 9 ) found that the ermc gene is located on a small plasmid ; therefore , horizontal transmission of this gene from resistant strains to susceptible strains is unavoidable . regarding erma , some studies showed that this gene , as the most prevalent gene among clinical strains of s. aureus , has a frequency of 21% to 67% ( 1 , 24 - 27 ) . similar findings were also published in denmark , where erma was responsible for 80.6% resistance to erythromycin among 428 tested clinical s. aureus strains from 1959 to 1988 ( 23 ) . in contrast , the current study pcr results demonstrated that erma was not observed in the tested s. aureus strains . it was also found that ermb gene was carried in a minority of s. aureus ( 7.8% ) and cons ( 2% ) strains ; while previous studies revealed that this gene was primarily originated from animal strains and generally its frequency was low ( 24 - 27 ) . however , zmantar et al . in two different studies ( 12 , 28 ) reported that ermb was the most common gene when they evaluated 81 s. aureus strains isolated from human auricular . finally , msra gene , encoding an atp - dependent efflux pump ( 24 ) , was more prevalent in cons ( 41.8% ) than s. aureus strains ( 20% ) . ( 26 ) who reported the prevalence of msra gene as 14.6% and 2.1% among cons and s. aureus strains , respectively . similar data were also obtained ( 41% ) from a study conducted by zmantar et al . some staphylococcal strains in the current study showed discrepancies between the genotype and phenotype . so that , three strains ( two s. aureus and one cons ) harbored ermb or ermc gene , while they were susceptible to erythromycin . ( 1 ) also hypothesized that this phenomenon may be caused by down - regulation or mutation in promoter region of erm gens . on the other hand , martineau et al . ( 24 ) demonstrated that such strains carrying the resistant genes would exhibit phonotypical resistance , upon subculturing in media with increasing gradients of the corresponding antibiotic . moreover , nine staphylococcus isolates were resistant to erythromycin , but did not carry any of the tested erythromycin - resistant genes . authors presume that other variants of erm genes or efflux pump ( msrb ) involve in this subject , which were not evaluated in the study ( 1 , 24 , 29 , 30 ) . although the current study is the first report on the occurrence of genetic determinants responsible for macrolide resistance among staphylococcus strains originated from hospital employees ; however , lack of further in vitro investigations , such as determining the minimum inhibitory concentration ( mic ) for erythromycin , detection of other genes involved in macrolide resistance and limited sample size , were the weakness of the current study . in conclusion , no entire association was found between genotype and phenotype methods to detect macrolides resistance . in addition , distribution of genetically erythromycin - resistant isolates is geographically different among staphylococci . it is recommend controlling s. aureus in nasal carriers by proved approaches such as local or systemic administration of effective antibiotics or bacterial interference . in addition , to prevent cons - associated nosocomial and indwelling medical device infections , it is recommended that healthcare workers wash hands and avoid touching their noses during the patient examination , use sterile masks and gloves and finally , insert prosthetic and catheter devices aseptically .

backgroundepidemiological data on antibiotic susceptibility of staphylococcus strains isolated from nasal carriers in each region can be helpful to select appropriate drugs to eradicate carriage states , control nosocomial infections and also treat patients.objectivesthe current study aimed to investigate the antibiotic resistance profile and the molecular prevalence of the erma , ermb , ermc and msra genes among staphylococcus strains isolated from the anterior nares of hospital employees.patients and methodsin this cross - sectional study , a total of 100 staphylococcus isolates , 51 staphylococcus aureus , 49 coagulase - negative staphylococci ( cons ) were isolated from the anterior nares of hospital employees in khorramabad , iran . susceptibility pattern to macrolide antibiotics were determined using the disk diffusion method . the polymerase chain reaction ( pcr ) assay was applied to determine the major erythromycin - resistant genes ( erma , ermb , ermc and msra).resultsfifty - three ( 53% ) isolates were simultaneously resistant to erythromycin , azithromycin and clarithromycin ( cross - resistance ) ; while 8 ( 8% ) isolates had variable macrolide susceptibility pattern . among the s. aureus isolates , the difference in prevalence of resistance to erythromycin between males and females was significant ( p = 0.011 ) . the frequency of erma , ermb , ermc , and msra genes were 3% , 5% , 33% and 20% , respectively . it was also found that out of 53 isolates resistant to erythromycin , 44 ( 83% ) isolates ( eight s. aureus and thirty - six cons strains ) carried at least one of the four tested genes . eight ( 8% ) isolates had intermediate phenotype to erythromycin , in which 4 ( 50% ) isolates carried ermb or ermc genes . in addition , out of 39 erythromycin - susceptible isolates , 3 ( 7.7% ) isolates were positive for ermb or ermc genes.conclusionsno entire association was found between genotype and phenotype methods to detect macrolides - resistant isolates . in addition , distribution of genetically erythromycin - resistant isolates is geographically different among staphylococci . it is recommend removing s. aureus from nasal carriers by proved approaches such as local or systemic administration of effective antibiotics or bacterial interference .

1. Background 2. Objectives 3. Patients and Methods 3.1. Isolation and Identification of Nasal Staphylococci 3.2. Bacterial Susceptibility Testing 3.3. DNA Extraction 3.4. Primers and PCR 3.5. Statistical Analysis 3.6. Ethical Issues 4. Results 4.1. Staphylococcal Isolates and its Susceptibility Patterns 4.2. Distribution of Macrolides Resistance Genes Using PCR 4.3. Nucleotide Sequence Accession Numbers 5. Discussion

staphylococcus aureus and coagulase - negative staphylococci ( cons ) are well known to most clinicians as a cause of nosocomial and community - associated infections worldwide ( 1 , 2 ) . among these mechanisms , many reported and sequenced erm genes , three major genes of erma , ermb and ermc are present in staphylococci ( 12 , 13 ) . due to the development of resistant strains , epidemiological data on antibiotic susceptibility of staphylococcus strains isolated from nasal carriers in each region can be helpful to select appropriate drugs to eradicate carriage states , control the nosocomial infections and also treat the patients ( 14 , 15 ) . the current study aimed to investigate the antibiotic susceptibility profile and the prevalence of the erma , ermb , ermc and msra genes among staphylococcal strains isolated from the nares of hospital personnel in khorramabad , iran . during this cross - sectional study , a total of 340 volunteers from hospital staff ( males and females ) , employed in various wards of four referral and state hospitals ( shohaday - e - ashayer , asalian , shahid madani and taemin ejtemai ) in khorramabad city ( west of iran ) were included and screened for nasal carriage of s. aureus from july 2011 to january 2012.the subjects with a history of any antibiotic consumption for at least ten days at the time of sampling were excluded . susceptibility of staphylococcus isolates to antibiotics ( mast , merseyside , united kingdom ) including erythromycin ( 15 g ) , clarithromycin ( 15 g ) , azithromycin ( 15 g ) , cefoxitin ( 30 g ) , penicillin g ( 10 u ) , vancomycin ( 30 g ) , tetracycline ( 30 g ) , clindamycin ( 2 g ) , trimethoprim / sulfamethoxazole ( 1.25/23.75 g ) and rifampin ( 5 g ) was determined by the kirby - bauer disk diffusion method on muller - hinton agar , according to the procedures described by the clinical and laboratory standards institute ( clsi ) ( 18 ) . the four primer pairs used to amplify the erma , ermb , ermc and msra genes are listed in table 1 ( 12 ) . in the current study , multiplex polymerase chain reaction ( pcr ) amplification was performed for erma , ermc and msra genes as previously described by zmantar et al . for ermb gene amplification , the ermb primers ( 25 pmol ) were added in a separate pcr reaction under the same above - mentioned conditions . to verify the authenticity of the amplicons , pcr products of three positive samples containing one of the genes of ermb , ermc or msra as representative the resulting sequences were aligned with corresponding sequences in the genbank database using at the national center for biotechnology information ( ncbi ) blast program ( http://blast.ncbi.nlm.nih.gov/blast.cgi ? to minimize the random errors , chi - square test and t - test were used to determine any significant differences between prevalence of the tested genes among s. aureus and cons strains . during this cross - sectional study , a total of 340 volunteers from hospital staff ( males and females ) , employed in various wards of four referral and state hospitals ( shohaday - e - ashayer , asalian , shahid madani and taemin ejtemai ) in khorramabad city ( west of iran ) were included and screened for nasal carriage of s. aureus from july 2011 to january 2012.the subjects with a history of any antibiotic consumption for at least ten days at the time of sampling were excluded . isolates with positive reactions ( coagulase - positive and dnase - positive ) were identified as s. aureus ; other staphylococcal species with negative test results designated as cons . susceptibility of staphylococcus isolates to antibiotics ( mast , merseyside , united kingdom ) including erythromycin ( 15 g ) , clarithromycin ( 15 g ) , azithromycin ( 15 g ) , cefoxitin ( 30 g ) , penicillin g ( 10 u ) , vancomycin ( 30 g ) , tetracycline ( 30 g ) , clindamycin ( 2 g ) , trimethoprim / sulfamethoxazole ( 1.25/23.75 g ) and rifampin ( 5 g ) was determined by the kirby - bauer disk diffusion method on muller - hinton agar , according to the procedures described by the clinical and laboratory standards institute ( clsi ) ( 18 ) . the four primer pairs used to amplify the erma , ermb , ermc and msra genes are listed in table 1 ( 12 ) . in the current study , multiplex polymerase chain reaction ( pcr ) amplification was performed for erma , ermc and msra genes as previously described by zmantar et al . to verify the authenticity of the amplicons , pcr products of three positive samples containing one of the genes of ermb , ermc or msra as representative the resulting sequences were aligned with corresponding sequences in the genbank database using at the national center for biotechnology information ( ncbi ) blast program ( http://blast.ncbi.nlm.nih.gov/blast.cgi ? to minimize the random errors , chi - square test and t - test were used to determine any significant differences between prevalence of the tested genes among s. aureus and cons strains . a total of 51 s. aureus strains were isolated from anterior nares of the 340 screened subjects ( i.e. of the 51 s. aureus - carriers , 22 ( 43.1% ) were males and 29 ( 56.9% ) were females . on the other hand , 49 randomly selected cons strains , isolated from nares of non - s . there was no statistical difference between the two groups ( s. aureus nasal carriers and cons ) in terms of gender ( p = 0.72 ) . based on the t - test result , difference in the mean of work experience between the two groups was not statistically significant ( p = 0.22 ) . twenty - nine ( 29% ) isolates were resistant to cefoxitin ( eight s. aureus and twenty - one cons ) . fifty - three ( 53% ) isolates were simultaneously resistant to erythromycin , azithromycin and clarithromycin ( cross - resistance ) ; while 8 ( 8% ) isolates had various macrolide susceptibility patterns . for example , two isolates were intermediate to erythromycin , while they were resistant to azithromycin and clarithromycin . in addition , two isolates were found susceptible to erythromycin , while they were resistant to clarithromycin . the frequency of resistance to all tested antibiotics between cons and s. aureus isolates were statistically significant ( p < 0.001 ) . distribution of susceptibility phenotypes to erythromycin among the isolates of s. aureus and cons according to the gender is shown in table 3 . statistical analyses showed that among the s. aureus isolates difference in prevalence of resistance to erythromycin was significant between males and females ( p = 0.011 ) ; while such difference was not significant within cons isolates ( p = 0.771 ) . abbreviation : cons , coagulase - negative staphylococci . all of the isolates were screened for the presence of the four genes erma , ermb , ermc and msra as the main causative agents of resistance to macrolides . as predicted , the positive isolates for the erma , ermb , ermc and msra genes showed 139 , 142 , 190 and 163 bp bands , respectively ( figure 1 ) . the frequency rate of erma , ermb , ermc and msra genes were 3% , 5% , 33% and 20% , respectively ; separately illustrated for s. aureus and cons isolates in figure 2 . it was found that among the 53 isolates resistant to erythromycin , 44 ( 83% ) isolates ( eight s. aureus and thirty - six cons ) carried at least one of the four tested genes . of these erythromycin - resistant isolates , nine ( approximately 17% ) isolates ( one s. aureus and eight cons ) harbored genes of ermc and msra , and one ( % 1.8 ) cons isolate also harbored erma and ermc genes simultaneously . eight ( 8% ) isolates had intermediate phenotype to erythromycin , in which 4 ( 50% ) isolates carried ermb or ermc genes ( table 4 ) . in addition , it was found that out of 39 erythromycin - susceptible isolates , 36 ( 92.3% ) isolates did not harbor any of the four antibiotic resistant genes , but the remaining isolates ( three isolates ) were positive for ermb or ermc genes , while they were still susceptible to erythromycin ( table 4 ) . interestingly , of the 29 methicillin - resistant staphylococci , 22 ( 75.9% ) isolates were resistant to erythromycin and 19 ( 65.5% ) isolates harbored at least one of the tested genes . a , multiplex pcr amplification of erma , ermc and msra genes ; source of dna for lanes : 1 , negative control strain of erythromycin - sensitive staphylococcus aureus atcc 25923 ; lanes 2 , 4 and 7 , three different s. aureus isolates ( ermc ) ; lane 3 , a coagulase - negative staphylococci ( cons ) isolate ( erma ) ; lanes 5 and 6 , two different cons isolates contain ermc and msra genes simultaneously ; lane 8 , a cons isolate contains msra gene . b , pcr products resulted from amplification of ermb gene ; lanes 1 and 2 , two different s. aureus isolates contain ermb gene ; lane 3 , s. aureus atcc 29213 ( negative control ) ; lanes m , dna molecular size marker ( 50-bp ladder ; fermentas , lithonia ) ; the electrophoresis was run on 1.5% agarose gel , stained with ethidium bromide . nucleotide sequences were submitted to genbank under the accession numbers ab937975 , ab937976 and ab937977 for the ermb , ermc and msra genes , respectively . a total of 51 s. aureus strains were isolated from anterior nares of the 340 screened subjects ( i.e. of the 51 s. aureus - carriers , 22 ( 43.1% ) were males and 29 ( 56.9% ) were females . on the other hand , 49 randomly selected cons strains , isolated from nares of non - s . there was no statistical difference between the two groups ( s. aureus nasal carriers and cons ) in terms of gender ( p = 0.72 ) . average work experience for cons obtained subjects was 6.4 0.89 years , and within s. aureus - nasal carriers were 4.8 0.68 years . based on the t - test result , difference in the mean of work experience between the two groups was not statistically significant ( p = 0.22 ) . twenty - nine ( 29% ) isolates were resistant to cefoxitin ( eight s. aureus and twenty - one cons ) . fifty - three ( 53% ) isolates were simultaneously resistant to erythromycin , azithromycin and clarithromycin ( cross - resistance ) ; while 8 ( 8% ) isolates had various macrolide susceptibility patterns . for example , two isolates were intermediate to erythromycin , while they were resistant to azithromycin and clarithromycin . in addition , two isolates were found susceptible to erythromycin , while they were resistant to clarithromycin . the frequency of resistance to all tested antibiotics between cons and s. aureus isolates were statistically significant ( p < 0.001 ) . distribution of susceptibility phenotypes to erythromycin among the isolates of s. aureus and cons according to the gender is shown in table 3 . statistical analyses showed that among the s. aureus isolates difference in prevalence of resistance to erythromycin was significant between males and females ( p = 0.011 ) ; while such difference was not significant within cons isolates ( p = 0.771 ) . abbreviation : cons , coagulase - negative staphylococci . all of the isolates were screened for the presence of the four genes erma , ermb , ermc and msra as the main causative agents of resistance to macrolides . as predicted , the positive isolates for the erma , ermb , ermc and msra genes showed 139 , 142 , 190 and 163 bp bands , respectively ( figure 1 ) . the frequency rate of erma , ermb , ermc and msra genes were 3% , 5% , 33% and 20% , respectively ; separately illustrated for s. aureus and cons isolates in figure 2 . it was found that among the 53 isolates resistant to erythromycin , 44 ( 83% ) isolates ( eight s. aureus and thirty - six cons ) carried at least one of the four tested genes . of these erythromycin - resistant isolates , nine ( approximately 17% ) isolates ( one s. aureus and eight cons ) harbored genes of ermc and msra , and one ( % 1.8 ) cons isolate also harbored erma and ermc genes simultaneously . eight ( 8% ) isolates had intermediate phenotype to erythromycin , in which 4 ( 50% ) isolates carried ermb or ermc genes ( table 4 ) . in addition , it was found that out of 39 erythromycin - susceptible isolates , 36 ( 92.3% ) isolates did not harbor any of the four antibiotic resistant genes , but the remaining isolates ( three isolates ) were positive for ermb or ermc genes , while they were still susceptible to erythromycin ( table 4 ) . interestingly , of the 29 methicillin - resistant staphylococci , 22 ( 75.9% ) isolates were resistant to erythromycin and 19 ( 65.5% ) isolates harbored at least one of the tested genes . a , multiplex pcr amplification of erma , ermc and msra genes ; source of dna for lanes : 1 , negative control strain of erythromycin - sensitive staphylococcus aureus atcc 25923 ; lanes 2 , 4 and 7 , three different s. aureus isolates ( ermc ) ; lane 3 , a coagulase - negative staphylococci ( cons ) isolate ( erma ) ; lanes 5 and 6 , two different cons isolates contain ermc and msra genes simultaneously ; lane 8 , a cons isolate contains msra gene . nucleotide sequences were submitted to genbank under the accession numbers ab937975 , ab937976 and ab937977 for the ermb , ermc and msra genes , respectively . in the current study , in several studies performed in various regions of the world , this carriage status in healthcare workers have been reported ranging ranging from 18.6% to 50% ( 20 ) . moreover , in iran , the frequency of nasal carriage among hospital employees is reported approximately 12.7% - 45% ( 21 ) . unfortunately , the results of disk diffusion testing showed that all the evaluated isolates were multidrug - resistant . it was observed that the frequency of resistance among cons isolates was significantly higher than those among s. aureus isolates ; and this finding was also attributable to resistance to erythromycin and cefoxitin ( table 2 ) . while the majority of the erythromycin - resistant strains had cross - resistance to other macrolides ; however eight strains exhibited heterogeneous susceptibility pattern toward macrolide antibiotics ( table 2 ) . the association between resistance to methicillin and the presence of tested genes was significant ( p = 0.031 ) . despite previous studies in which have primarily focused on clinical strains of staphylococci ( 1 , 11 , 23 - 28 ) , the current study presents the first report on the occurrence of main genes implicated in macrolides- resistance in staphylococcus strains obtained from nares of hospital employees . also , the frequency of this gene ( ermc ) in erythromycin - resistant s. aureus and cons were 13.7% and 53% , respectively . regarding erma , some studies showed that this gene , as the most prevalent gene among clinical strains of s. aureus , has a frequency of 21% to 67% ( 1 , 24 - 27 ) . similar findings were also published in denmark , where erma was responsible for 80.6% resistance to erythromycin among 428 tested clinical s. aureus strains from 1959 to 1988 ( 23 ) . it was also found that ermb gene was carried in a minority of s. aureus ( 7.8% ) and cons ( 2% ) strains ; while previous studies revealed that this gene was primarily originated from animal strains and generally its frequency was low ( 24 - 27 ) . in two different studies ( 12 , 28 ) reported that ermb was the most common gene when they evaluated 81 s. aureus strains isolated from human auricular . so that , three strains ( two s. aureus and one cons ) harbored ermb or ermc gene , while they were susceptible to erythromycin . moreover , nine staphylococcus isolates were resistant to erythromycin , but did not carry any of the tested erythromycin - resistant genes . although the current study is the first report on the occurrence of genetic determinants responsible for macrolide resistance among staphylococcus strains originated from hospital employees ; however , lack of further in vitro investigations , such as determining the minimum inhibitory concentration ( mic ) for erythromycin , detection of other genes involved in macrolide resistance and limited sample size , were the weakness of the current study . in conclusion , no entire association was found between genotype and phenotype methods to detect macrolides resistance . in addition , distribution of genetically erythromycin - resistant isolates is geographically different among staphylococci . it is recommend controlling s. aureus in nasal carriers by proved approaches such as local or systemic administration of effective antibiotics or bacterial interference . in addition , to prevent cons - associated nosocomial and indwelling medical device infections , it is recommended that healthcare workers wash hands and avoid touching their noses during the patient examination , use sterile masks and gloves and finally , insert prosthetic and catheter devices aseptically .

male sprague dawley rats ( 4 weeks old ; 7580 g ) , purchased from slc , inc . , were maintained at a temperature of 2326c and a relative humidity of 5065 % for 2 weeks after arrival . rats were divided into five groups ( hfd - control , hfd-5 % ele , hfd-003 % asp , hfd-01 % asp and hfd-03 % asp ; n 6 for each ) based on body weight . under hfd ( 26 mj/100 g ) conditions , all animals were fed a commercial diet containing 297 % lard ( oriental yeast co. ltd . ) , water ( 77 % ) , protein ( 236 % ) , lipid ( 350 % ) , ash ( 61 % ) , dietary fibre ( 29 % ) and nitrogen - free extract ( 247 % ) . test foods were prepared by adding 5 % ele and 00303 % asp or by adding 5 % casein as a control to determine the effects of high - dose administration in rats . the test foods were provided ad libitum for 3 months to examine the potential anti - obesity and anti - metabolic syndrome effects of ele and asp . the doses given to animals fed a hfd were determined based on the findings of fujikawa et al . . after chronic administration of ele and asp , body weight was measured , and the rats were killed by decapitation without stress . wat ( perirenal wat and epididymal wat ) , bat and skeletal muscle ( the sol . the institutional animal care and use committees at mie university faculty of medicine and the suzuka university of medical science approved the animal facilities and study protocols . all procedures were performed in accordance with the national institutes of health guidelines for animal care ( 1996 ) . ele was prepared as follows : fresh eucommia leaves were treated with steam at 100110c and then dried and roasted . a quantity of 2 tonnes of roasted eucommia leaves was steeped in 10 tonnes of hot water at 90c for 1 h , and the extract was filtered and concentrated . the concentrate was left standing for 1 d. the concentrate was then filtered and concentrated , vacuum - dried and powdered ( yield : 18 % ) . we measured each of the major components ( i.e. gea , asp and cha ) in each sample using hplc with an ods column . the experimentally used ele was composed of gea ( 630 mg / g ) , asp ( 452 mg / g ) and cha ( 440 mg / g ) . were extracted with hot water for 10 h at 60c , vacuum - dried and powdered to obtain the ele . the extract was subjected to diaion hp-20p column chromatography with a gradient of water methanol ( 1:0 to 0:1 ) to yield water fractions , 30 % methanol fractions , 50 % methanol fractions , 80 % methanol fractions and 100 % methanol fractions in the order of elution . the 30 % methanol fractions were chromatographed over ymc s-15/30 120a ods with a gradient of water methanol ( 1:1 to 6:1 ) and then purified by daisogel sp-120 - 40/60-ods - b ( 100 1000 mm ) , eluted with 80 % methanol , vacuum - dried and freeze - dried , and washed with acetone to yield asp ( purity : 995 % ) . body weight was measured weekly throughout the experimental period . after killing , the perirenal wat , epididymal wat , bat and sol . m. of the hind leg were removed , and the wet weights were measured . the blood was centrifuged ( 3000 rpm , 30 min ) , and the separated plasma was stored at 80c until measurement . plasma glucose and insulin were measured as biomarkers of carbohydrate metabolism ; total cholesterol , tga and nefa in the plasma were measured as biomarkers of lipid metabolism . insulin levels were measured by elisa using an ultra - high - sensitivity rat insulin assay kit ( morinaga milk industry co. ltd . ) , adiponectin levels were measured using a mouse / rat adiponectin elisa kit ( otsuka pharmaceutical co. ltd ) , and tnf- levels were measured using a rat tnf- assay kit ( immuno - biological laboratories co. ltd . ) . frozen rat liver was thawed and homogenised on ice with six volumes of a 025 m - sucrose solution containing 1 mm - edta and 3 mm - tris - hcl ( ph 72 ) and centrifuged at 500 g for 10 min ( 4c ) . the supernatant was centrifuged at 9000 g for 10 min , followed by centrifugation of the resultant supernatant at 105 000 g for 1 h. the supernatant containing the cytosol was used for the measurement of fatty acid synthase ( fas ) activity . after spinning at 500 g , the pellet containing the mitochondria was suspended in a small volume of a 025 m - sucrose solution . after centrifugation at 9000 g for 10 min , the supernatant was used to measure carnitine palmitoyltransferase 1 ( cpt1 ) activity . fas activity was determined using a method with extensive modification based on that described by kelley et al . . the cytosolic fraction ( 20100 l ) was mixed with 500 l of 02 m - potassium phosphate buffer ( ph 70 ) , 20 l of 25 mm - acetyl - coa , 30 l of 10 mm - nadph and 20 l of 10 mm - malonyl - coa and then measured at 340 nm ( 30c ) for 23 min in a spectrophotometer . cpt1 activity was determined by a method with extensive modification based on that described by markwell et al . . the mitochondrial fraction ( 515 l ) was mixed with 500 l of 116 mm - tris - hcl ( ph 80 ) buffer , 25 mm - edta , 5 m-5,5-dithiobis-2-nitrobenzoate ( dtnb ) , 02 % triton x-100 , 2 mm - palmitoyl - coa and 125 mm - l - carnitine . the reaction was initiated by the addition of enzyme at 25c , and the rate of coa release from palmitoyl - coa was measured at 412 nm using spectrophotometry . total rna was extracted using a tripure isolation kit ( roche ) , and the rna concentrations were determined spectrophotometrically at 260 nm . the quality of the rna was determined spectrophotometrically using a 260/280 nm absorbance ratio ; the values of all the samples were 1820 . after total rna extraction , the samples were treated with a turbo dna - free kit ( ambion ) for 30 min at 37c to remove any genomic dna . the dnase - treated rna ( 035 g ) was utilised to synthesise first - strand cdna using the revertraace qpcr rt kit ( toyobo ) . the cdna products ( 50 ng/l ) were analysed by rt - pcr using power sybr green pcr master mix ( applied biosystems ) with an abi prism 7300 ( applied biosystems ) . the amplification program included an initial denaturation step at 95c for 10 min , forty cycles of denaturation at 95c for 15 s and annealing / extension at 5960c for 1 min . the glyceraldehyde-3-phosphate dehydrogenase mrna level was determined and used as an internal control . each gene was normalised to glyceraldehyde-3-phosphate dehydrogenase by subtracting the cycle threshold ( ct ) value of glyceraldehyde-3-phosphate dehydrogenase from the ct value of the gene target ( ct[target ] ) . the relative expression of the target gene was calculated using sds v1.2 with the relative quantification ( respiratory quotient ) software ( applied biosystems ) , with ct[target ] compared with the ct values of the reference ; i.e. , ct = ct[target ] ct[reference ] . the degree of difference ( expressed in fold difference ) between the target and the reference was calculated as 2 . the melting point analysis detected no non - specific amplification in the cdna samples . the slopes of the amplification curves did not differ between groups during the mrna analysis , and no differences in the amplification efficiency were observed . the sequences of the specific primers used in the power sybr green pcr master mix protocol are listed in supplementary table s1 ( available online at http://journals.cambridge.org/jns ) . each pcr primer was designed using dinasis pro v2.7 software ( hitachi software engineering co. ltd ) , and the oligonucleotides were purchased from greiner bio - one co. ltd . the values are expressed as means with their standard errors derived from measurements of six rats ( n 6 ) . one - way anova was used for inter - group comparisons and for linear trend and quadratic trend tests . when the anova results revealed significant differences , contrast tests were used to identify significant differences from hfd-5 % ele or hfd - control group . in the statistics for each contrast test , we preplanned to leave out the cubic response but compared 5 % ele or control to the average of the three asp treatments . this is assuming that we do not have a specific asp treatment that we expect to compare to 5 % ele a priori : coefficients for linear ( 04597 , 03314 , 00321 08233 ) and quadratic ( 05334 , 000171 , 08025 , 02673 ) responses with control ( 0 % ) , 003 % asp , 01 % asp and 03 % asp ; coefficients for control v. 5 % ele ( 1 , 0 , 0 , 0 , 1 ) and asp v. 5 % ele ( 0 1 , 1 , 1 , 3 ) with control , 003 % asp , 01 % asp , 03 % asp and 5 % ele . compared with the hfd - control group , final body weight , body weight gain and food intake decreased significantly in 3 months in the hfd-5 % ele group ( each p < 005 ) , and the dose - dependent decrease was observed in the hfd-00303 % asp group compared with the hfd - control group ( each p < 005 , table 1 ) . no difference in the final body weight , body weight gain and food intake was observed between the hfd-5 % ele and the average of all hfd-0103 % asp groups ( table 1 ) . table 1.effects of asperuloside ( asp ) on body weight , white adipose tissue ( wat ) weight , brown adipose tissue ( bat ) weight , soleus muscle ( sol . m ) weight and plasma indices in high - fat diet ( hfd)-fed rats(mean values with their standard errors)hfd - asphfd - elehfd - cont . ( n 6)003 % ( n 6)01 % ( n 6)03 % ( n 6)5 % ( n 6)mean se mean se mean se mean se mean se initial body weight ( g per rat)7101071215725057100671707food intake ( g / d per rat)*2782221332177271492015616final body weight ( g per rat)*5649516194658461746217body weight gain ( g per rat)*493104451839383 90739017relative wat weight ( % ) perirenal*27031502140113011501epididymal26022502220120012002relative bat weight ( % ) * 024002031001033002037002039004relative sol . m. weight ( % ) 007001007001007001007001007001glucose ( mg / l)*162171150137139442133855141363insulin ( ng / ml)*77065211390833063407tag ( mg / l)*22603781605301119923510712431143237nefa ( eq / l)*63913374490560402721639732094372303total cholesterol ( mg / l)*8803472125708246642659128adiponectin ( g / l)*295396484533546tnf- ( pg / ml)*198318213651319879270689689101ele , eucommia leaf extract ; cont . , control . * linear contrast with control ( 0 % ) , 003 % , 01 % and 03 % asp ( p < 005). contrast for control v. 5 % ele ( p < 005). contrast for the average of all asp treatments v. 5 % ele ( p < 005 ) . effects of asperuloside ( asp ) on body weight , white adipose tissue ( wat ) weight , brown adipose tissue ( bat ) weight , soleus muscle ( sol . m ) weight and plasma indices in high - fat diet ( hfd)-fed rats ( mean values with their standard errors ) ele , eucommia leaf extract ; cont . , * linear contrast with control ( 0 % ) , 003 % , 01 % and 03 % asp ( p < 005 ) . contrast for control v. 5 % ele ( p < 005 ) . contrast for the average of all asp treatments v. 5 % ele ( p < 005 ) . compared with the hfd - control group , the relative weight of the perirenal wat significantly decreased in 3 months in the hfd-5 % ele group ( p < 005 ) , and a significant and dose dependent decrease was observed in the hfd-00303 % asp group when compared with the hfd - control group ( p < 005 , table 1 ) . these changes in the asp groups were dose - dependent ( p < 005 , table 1 ) . no difference in the relative weight of the perirenal wat was observed between the hfd-5 % ele and the average of the hfd-0103 % asp groups ( table 1 ) . the relative bat weight increased in the hfd-5 % ele group under chronic administration ( p < 005 , table 1 ) . the asp groups exhibited a significant increase with linearity when compared with the hfd - control group ( p < 005 , table 1 ) . a significant difference in relative bat weight was observed between the hfd-5 % ele and the average of the three hfd - asp groups ( p < 005 , table 1 ) . the control group exhibited primary signs of progressive metabolic syndrome with plasma parameters in agreement with a previous report . in comparison with the hfd - control group , plasma nefa levels significantly decreased in the hfd-5 % ele group during 3 months of hfd feeding ( p < 005 , table 1 ) . the asp groups exhibited a significant decrease with linearity when compared with the hfd - control group ( p < 005 , table 1 ) . administration of asp dose dependently reduced the plasma tag level in rats fed a hfd when compared with the hfd - control rats ( p < 005 ) , and a significant decrease was seen in the hfd-5 % ele group ( p < 005 , table 1 ) . no difference in the plasma level of nefa and tag was observed between the hfd-5 % ele and the average of all hfd-0103 % asp groups ( table 1 ) . a significantly decreased total cholesterol level was observed in 5 % ele - administered rats under hfd conditions ( p < 005 , table 1 ) . the asp groups exhibited a significant decrease of the plasma total cholesterol level in a dose - dependent fashion when compared with the hfd - control group ( p < 005 ) , and a significant difference was observed between the hfd-5 % ele and the average of the hfd-0103 % asp groups ( p < 005 , table 1 ) . a dose - dependently reduced plasma glucose level was observed in the hfd - asp groups compared with the hfd - control group after 3 months of hfd feeding ( p < 005 ) ; however , no difference in plasma glucose level was observed between the hfd-5 % ele and the average of the three hfd - asp groups ( table 1 ) . administration of 5 % ele yielded markedly decreased plasma insulin levels under hfd conditions ( p < 005 ) , and linearly decreased plasma insulin levels were also observed in rats administered 0103 % asp contrasted with the hfd - control rats ( p < 005 , table 1 ) . no difference in the plasma insulin level was observed between the hfd-5 % ele and the average of the hfd - asp groups ( table 1 ) . in the hfd-5 % ele group , significantly increased levels of plasma adiponectin were observed ; a dose - dependent increase was also observed in the hfd - asp groups compared with the hfd - control group ( each p < 005 , table 1 ) . no difference in the plasma level of adiponectin was observed between the hfd-5 % ele and the average of the three hfd - asp groups ( table 1 ) . in contrast , administration of 5 % ele yielded a considerably decreased plasma tnf- level under hfd conditions ; a dose - dependently decreased tnf- level was also observed in the asp - administered rats under hfd conditions compared with the hfd - control rats ( each p < 005 , table 1 ) . no difference in the plasma level of tnf- was observed between the hfd-5 % ele and the average of the hfd-0103 % asp groups ( table 1 ) . under hfd conditions , chronic administration of 5 % ele significantly induced the expression of glucokinase , citrate synthase ( cs ) , dihydrolipoamide succinyl transferase ( ogdh ) , nadh dehydrogenase flavoprotein 1 ( comp i ) and cytochrome c oxidase , subunit 4a ( comp iv ) in the rat liver ( each p < 005 , table 2 ) . the asp groups exhibited a significant and dose - dependent increase of glucokinase , cs , ogdh , comp i and comp iv mrna when compared with the hfd - control group ( p < 005 ) , and the significantly increased mrna level of cs , ogdh , comp i and comp iv in the average of all the hfd-00303 % asp groups was lower than that of the hfd-5 % ele group ( p < 005 , table 2 ) . a significant difference in glucokinase mrna level was not observed between the hfd-5 % ele and the average of the hfd-00303 % asp groups ( table 2 ) . in the perirenal wat of rats fed a hfd , the levels of isocitrate dehydrogenase 3 ( idh3 ) and comp i mrna were markedly decreased by chronic administration of 5 % ele ( each p < 005 , table 3 ) . the asp groups exhibited a significant decrease of glut4 , idh3 and comp i mrna with linearity when compared with the hfd - control group ( each p < 005 , table 3 ) . no difference was observed between the hfd-5 % ele and the average of the three hfd - asp groups ( table 3 ) . in the sol . m. of rats under hfd conditions , the 5 % ele group had significantly increased levels of glut4 , cs , idh3 , ogdh , succinate dehydrogenase complex , subunit a , flavoprotein , comp i , comp iv and atp synthase , h transporting , mitochondrial f1 complex , delta subunit ( comp v ) mrna ( each p < 005 , table 4 ) . similarly , the asp groups had a markedly increased mrna level of these genes with linearity when compared with the hfd - control group ( each p < 005 , table 4 ) . a difference except for idh3 mrna was observed between the hfd-5 % ele and the average of the hfd - asp groups ( each p < 005 , table 4 ) . table 2.gene expression analysis by real - time pcr in liver after administration of asperuloside ( asp ) under high - fat diet ( hfd ) conditions(mean values with their standard errors)hfd - asp ( fold of cont.)hfd - ele ( fold of cont.)hfd - cont . ( n 6)003 % ( n 6)01 % ( n 6)03 % ( n 6)5 % ( n 6)gene namemean se mean se mean se mean se mean se glycolytic systemgck*100011147011182015219025260042tca cyclecs*100011096004190007197008215008ogdh*100012098008149014178008289005electron transfer systemcomp i*100010096006142010206010239008comp iv*100012105006084011061008062007fatty acid synthesisfas*100010124013167017176003205022fatty acid transporterfatp*100010124013167017176003205022fatty acid -oxidationcptl*100010123015157010176016235007acadvi*100010122005137006205012184013fatty acid -oxidation ( catalytic)hadh ii*100015135007177017232020227016hadh sc*100012095007`149012180010172009cont . , control ; ele , eucommia leaf extract ; gck , glucokinase ; cs , citrate synthase ; ogdh , dihydrolipoamide succinyl transferase ; comp i , nadh dehydrogenase flavoprotein 1 ; comp iv , cytochrome c oxidase , subunit 4a ; fas , fatty acid synthase ; fatp , fatty acid transport protein ; cpt1 , carnitine palmitoyltransferase 1 ; acadvl , acyl - coa dehydrogenase , very long chain ; hadh ii , hydroxyacyl - coa dehydrogenase type ii ; hadh sc , l-3-hydroxyacyl - coa dehydrogenase , short chain . * linear contrast with control ( 0 % ) , 003 % , 01 % and 03 % asp ( p < 005). contrast for control v. 5 % ele ( p < 005). contrast for the average of all asp treatments v. 5 % ele ( p < 005 ) . table 3.gene expression analysis by real - time pcr in perirenal white adipose tissue after administration of asperuloside ( asp ) under high - fat diet ( hfd ) conditions(mean values with their standard errors)hfd - asp ( fold of cont.)hfd - ele ( fold of cont.)hfd - cont . ( n 6)003 % ( n 6)01 % ( n 6)03 % ( n 6)5 % ( n 6)gene namemean se mean se mean se mean se mean se glutglut4 * 100008080007066004057009079010glycolytic systemhk2100007095018078006066004106020tca cyclecs100004110014081005067004098014idh3*100006102014089009057009060007ogdh100012111018069004093033093025electron transfer systemcomp i*100005103011066002058003064007comp iv100008113009100006064027099026lipolysishsl*100017173043209048273021176049fatty acid synthesisfas*100005067003048004042004069005fatty acid transporterfatp100006088005085005052005063006fatty acid - oxidationcpt2100011125019144013171032161018acadvl*100010143007177026270028193009fatty acid receptor and adipocytokineppar*100009118002180012211008198012adiponectin*100011101002238022303014247011resistin*100005097007086004066005068008cont . , control ; ele , eucommia leaf extract ; hk2 , hexokinase 2 ; cs , citrate synthase ; idh3 , isocitrate dehydrogenase 3 ; ogdh , dihydrolipoamide succinyl transferase ; comp i , nadh dehydrogenase flavoprotein 1 ; comp iv , cytochrome c oxidase , subunit 4a ; hsl , hormone - sensitive lipase ; fas , fatty acid synthase ; fatp , fatty acid transporter protein ; cpt2 , carnitine palmitoyltransferase 2 ; acadvl , acyl - coa dehydrogenase , very long chain . * linear contrast with control ( 0 % ) , 003 % , 01 % and 03 % asp ( p < 005). contrast for control v. 5 % ele ( p < 0.05). contrast for the average of all asp treatments v. 5 % ele ( p < 0.05 ) . table 4.gene expression analysis by real - time pcr in soleus muscle after administration of asperuloside ( asp ) under high - fat diet ( hfd ) conditions(mean values with their standard errors)hfd - asp ( fold of cont.)hfd - ele ( fold of cont.)hfd - cont . ( n 6)003 % ( n 6)01 % ( n 6)03 % ( n 6)5 % ( n 6)gene namemean se mean se mean se mean se mean se glutglut4*100019129006191021220021229020glycolytic systemhk2100007091008101014095009083008tca cyclecs*100005116007132012209008190005idh3*100009114007129008171009157010ogdh*100010101007171009223012242016sdha ( comp ii)*100012142007213019254013264016electron transfer systemcomp i*100017155029211016218018256020comp iv*100010104002144010158005152002comp iv*100009110002123003189011107004fatty acid synthesisfas100016069007081008079015073009fatty acid transporterfas100014137017115012101012104008ketone bodies utilisationscos*100008123004213009227009197014p3kcot*100012109012250012261015227017ketone body 's utilisation ( acetoacetic acid acetyl coa)ogdh*1000101010071 71009223012242016sdha ( comp ii)*100012142007213019254013264016cont . , control ; ele , eucommia leaf extract ; hk2 , hexokinase 2 ; cs , citrate synthase ; idh3 , isocitrate dehydrogenase 3 ; ogdh , dihydrolipoamide succinyl transferase ; sdha ( comp ii ) , succinate dehydrogenase complex , subunit a , flavoprotein ; comp i , nadh dehydrogenase flavoprotein 1 ; comp iv , cytochrome c oxidase , subunit 4a ; comp v , atp synthase , h transporting , mitochondrial f1 complex , delta subunit ; fas , fatty acid synthase ; fatp , fatty acid transporter protein ; scos , succinyl coa synthase ; p3kcot , peroxisomal 3-ketoacyl - coa thiolase . * linear contrast with control ( 0 % ) , 003 % , 01 % and 03 % asp ( p < 005). contrast for control v. 5 % ele ( p < 0.05). contrast for the average of all asp treatments v. 5 % ele ( p < 0.05 ) . gene expression analysis by real - time pcr in liver after administration of asperuloside ( asp ) under high - fat diet ( hfd ) conditions ( mean values with their standard errors ) cont . , control ; ele , eucommia leaf extract ; gck , glucokinase ; cs , citrate synthase ; ogdh , dihydrolipoamide succinyl transferase ; comp i , nadh dehydrogenase flavoprotein 1 ; comp iv , cytochrome c oxidase , subunit 4a ; fas , fatty acid synthase ; fatp , fatty acid transport protein ; cpt1 , carnitine palmitoyltransferase 1 ; acadvl , acyl - coa dehydrogenase , very long chain ; hadh ii , hydroxyacyl - coa dehydrogenase type ii ; hadh sc , l-3-hydroxyacyl - coa dehydrogenase , short chain . * linear contrast with control ( 0 % ) , 003 % , 01 % and 03 % asp ( p < 005 ) . contrast for control v. 5 % ele ( p < 005 ) . contrast for the average of all asp treatments v. 5 % ele ( p < 005 ) . gene expression analysis by real - time pcr in perirenal white adipose tissue after administration of asperuloside ( asp ) under high - fat diet ( hfd ) conditions ( mean values with their standard errors ) cont . , control ; ele , eucommia leaf extract ; hk2 , hexokinase 2 ; cs , citrate synthase ; idh3 , isocitrate dehydrogenase 3 ; ogdh , dihydrolipoamide succinyl transferase ; comp i , nadh dehydrogenase flavoprotein 1 ; comp iv , cytochrome c oxidase , subunit 4a ; hsl , hormone - sensitive lipase ; fas , fatty acid synthase ; fatp , fatty acid transporter protein ; cpt2 , carnitine palmitoyltransferase 2 ; acadvl , acyl - coa dehydrogenase , very long chain . * linear contrast with control ( 0 % ) , 003 % , 01 % and 03 % asp ( p < 005 ) . contrast for control v. 5 % ele ( p < 0.05 ) . contrast for the average of all asp treatments v. 5 % ele ( p < 0.05 ) . gene expression analysis by real - time pcr in soleus muscle after administration of asperuloside ( asp ) under high - fat diet ( hfd ) conditions ( mean values with their standard errors ) cont . , control ; ele , eucommia leaf extract ; hk2 , hexokinase 2 ; cs , citrate synthase ; idh3 , isocitrate dehydrogenase 3 ; ogdh , dihydrolipoamide succinyl transferase ; sdha ( comp ii ) , succinate dehydrogenase complex , subunit a , flavoprotein ; comp i , nadh dehydrogenase flavoprotein 1 ; comp iv , cytochrome c oxidase , subunit 4a ; comp v , atp synthase , h transporting , mitochondrial f1 complex , delta subunit ; fas , fatty acid synthase ; fatp , fatty acid transporter protein ; scos , succinyl coa synthase ; p3kcot , peroxisomal 3-ketoacyl - coa thiolase . * linear contrast with control ( 0 % ) , 003 % , 01 % and 03 % asp ( p < 005 ) . contrast for control v. 5 % ele ( p < 0.05 ) . contrast for the average of all asp treatments v. 5 % ele ( p < 0.05 ) . in the rat liver under hfd conditions , chronic administration of 5 % ele significantly decreased the level of expression of fas mrna and a marked increased the mrna levels of fa transport protein , cpt1 , acyl - coa dehydrogenase , very long chain ( acadvl ) , hydroxyacyl - coa dehydrogenase type ii and l-3-hydroxyacyl - coa dehydrogenase , short chain ( each p < 005 , table 2 ) . the hfd - asp groups also exhibited a significant decrease in fas mrna and markedly increase in these mrna in the liver in a dose - dependent fashion when compared with the hfd - control group ( each p < 005 , table 2 ) . significant differences in acadvl , hydroxyacyl - coa dehydrogenase type ii and l-3-hydroxyacyl - coa dehydrogenase , short chain mrna levels were observed between the hfd-5 % ele and the average of all the hfd-00303 % asp groups ( p < 005 ) , but not in fas , fa transport protein and cpt1 mrna levels ( table 2 ) . the hfd-5 % ele condition did not affect the hormone - sensitive lipase mrna level in perirenal wat ( table 3 ) . although the hfd - asp groups had a clearly increased hormone - sensitive lipase mrna level with linearity when compared with the hfd - control group ( p < 005 ) , no difference was observed between the hfd-5 % ele and the average of the hfd-00303 % asp groups ( table 3 ) . the hfd-5 % ele group exhibited marked suppression of mrna expression of fas in perirenal wat ( p < 005 , table 3 ) . the hfd - asp groups exhibited significantly decreased levels of fas mrna with linearity when compared with the hfd - control group ( p < 005 ) and these significant down - regulations in the average of the hfd-00303 % asp groups were clearly lower than that of the hfd-5 % ele group ( p < 005 , table 3 ) . the 5 % ele group had significantly increased perirenal wat acadvl mrna levels under hfd conditions ( p < 005 , table 3 ) . although the hfd - asp groups exhibited significantly increased levels of acadvl mrna with linearity when compared with the hfd - control group ( p < 005 ) , no difference was also observed between the hfd-5 % ele and the average of the three hfd - asp groups ( p < 005 , table 3 ) . there were no significant differences among any of the groups in the level of mrna expression of fas or fa transport protein in the sol . chronic administration of 5 % ele led to significantly increased mrna levels of succinyl coa synthase , peroxisomal 3-ketoacyl - coa thiolase , ogdh and succinate dehydrogenase complex , subunit a , flavoprotein in the sol . m. of rats under a hfd ( each p < 005 , table 4 ) . the hfd - asp groups exhibited a significant up - regulation of these genes with linearity when compared with the hfd - control group ( each p < 005 , table 4 ) . no difference was not observed between the hfd-5 % ele and the average of all the hfd-00303 % asp groups ( table 4 ) . in the perirenal wat of rats fed a hfd , chronic administration of 5 % ele led to a significant increase in ppar and adiponectin mrna levels ( each p < 005 , table 3 ) . the asp groups exhibited dose - dependent increases in the expression of both genes when compared with the hfd - control rats ( p < 005 , table 3 ) . no difference in the increased ppar and adiponectin mrna levels was observed between the hfd-5 % ele and the average of all hfd - asp groups ( table 3 ) . while 5 % ele yielded significantly decreased resistin mrna levels ( p < 005 , table 3 ) , no difference in the mrna level was observed between the hfd-5 % ele and the hfd - asp groups ( table 3 ) . the hfd - asp groups exhibited significant down - regulation of the gene in a dose - dependent fashion when compared with the hfd - control group ( p < 005 , table 3 ) . in the bat of hfd - fed rats , chronic administration of 5 % ele led to significantly increased uncoupling protein 2 ( ucp2 ) mrna levels ( p < 005 ) , but the 5 % ele administration did not affect the expression of other ucp mrna ( table 5 ) . % asp administration clearly increased the mrna levels of ucp1 and ucp2 in a dose - dependent fashion when compared with the hfd - control group ( each p < 005 , table 5 ) . no difference in the increased ucp1 and ucp2 mrna levels was observed between the hfd-5 % ele and the average of the three hfd - asp groups ( table 5 ) . table 5.gene expression analysis by real - time pcr in brown adipose tissue after administration of asperuloside ( asp ) under high - fat diet ( hfd ) conditions(mean values with their standard errors)hfd - asp ( fold of cont.)hfd - ele ( fold of cont.)hfd - cont . ( n 6)003 % ( n 6)01 % ( n 6)03 % ( n 6)5 % ( n 6)gene namemean se mean se mean se mean se mean se uncoupling atp synthesis from oxidative metabolismucp1 * 100007098004138010198007101005ucp2*100008110007142013164007226014ucp3100007096006092006089004109002cont . , control ; ele , eucommia leaf extract ; ucp , uncoupling protein . * linear contrast with control ( 0 % ) , 003 % , 01 % and 03 % asp ( p < 005). contrast for control v. 5 % ele ( p < 0.05 ) . gene expression analysis by real - time pcr in brown adipose tissue after administration of asperuloside ( asp ) under high - fat diet ( hfd ) conditions ( mean values with their standard errors ) cont . , control ; ele , eucommia leaf extract ; ucp , uncoupling protein . * linear contrast with control ( 0 % ) , 003 % , 01 % and 03 % asp ( p < 005 ) . contrast for control v. 5 % ele ( p < 0.05 ) . in rats under hfd conditions , chronic administration of 5 % ele led to a significant increase in mitochondrial cpt1 activity in the liver compared with the hfd - control group ( p < 005 , fig . moreover , chronic administration of asp dose - dependently increased the mitochondrial cpt1 activity in the liver of rats under hfd conditions when compared with the hfd - control rats ( p < 005 , fig . 1(a ) ) . in contrast , significant changes in cytosolic fas activity could not be observed during rt - pcr analysis in ele and asp groups ( fig . 1.effect of asperuloside ( asp ) on the activity of hepatic carnitine palmitoyltransferase 1 ( cpt1 ) ( a ) and hepatic fatty acid synthase ( fas ) ( b ) in high - fat diet ( hfd)-fed rats . values are means ( n 6 ) with standard errors represented by vertical bars . quadratic contrast with control ( 0 % ) , 003 , 01 and 03 % asp ( p < 005 ) . contrast for control v. 5 % ele ( p < 005 ) . effect of asperuloside ( asp ) on the activity of hepatic carnitine palmitoyltransferase 1 ( cpt1 ) ( a ) and hepatic fatty acid synthase ( fas ) ( b ) in high - fat diet ( hfd)-fed rats . values are means ( n 6 ) with standard errors represented by vertical bars . quadratic contrast with control ( 0 % ) , 003 , 01 and 03 % asp ( p < 005 ) . contrast for control v. 5 % ele ( p < 005 ) . similarly to our previous study , 3-month administration of asp controlled the body weight gain , food intake and relative wat weight and increased the relative bat weight with prominently decreased plasma levels of tag , nefa and total cholesterol in hfd rats . the administration of asp at a dose of 01 % exhibited effects comparable to those observed with the administration of ele at a dose of 5 % . the effects were maintained at a dose of 03 % . moreover , the results agreed with previous findings that showed that asp but not gea and cha decreases the body weight and wat weight via significantly reduced circulating nefa levels in hfd - fed mice . for carbohydrate metabolism , no difference in plasma glucose level was observed in rats administered ele under hfd conditions . on the other hand , some studies reported that ele controls the plasma glucose level in type 1 diabetes model rats and type 2 diabetes model mice ( c57bl / ksj - db / db mice ) . the hfd model animals , which were employed in this study , showed a characteristic increase in plasma insulin levels , and the magnitude of this increase was considerably decreased by ele administration over 3 months . our study showed that 5 % ele administration decreased the plasma level of insulin under hfd conditions , and a significant and dose - dependent decrease was observed in rats fed 00303 % asp , resulting in a significant decrease in plasma glucose level in a dose - dependent fashion . this suggests that asp may improve insulin resistance in rat models of hfd - induced obesity . rt - pcr analysis showed that chronic administration of asp to rats under a hfd significantly increased the mrna levels of cs , idh3 , ogdh , sdha , comp i , comp iv and comp v in skeletal muscles . therefore , there is a possible increase in the abundance of enzymes in these pathways that could increase the capacity of the glycolytic system , tricarboxylic acid cycle and electron transport system in skeletal muscles , increasing the mrna levels of glucokinase , cs , ogdh , comp i and comp iv in the liver and markedly decreasing the mrna levels of idh3 and comp i in the wat . glut4 mrna expression was increased in skeletal muscle and decreased in wat under the same dietary conditions . in the hfd model rats , the effect of asp on carbohydrate metabolism resembled that of ele , supporting the results of a previous study . thus , asp may be an important component of ele responsible for the control of carbohydrate metabolism . similar to ele - fed rats , the asp - fed animals might no longer be able to maintain the shape of the adipose cells that store tag because of the dose - dependently decreased atp production in the wat under hfd conditions . a diminished perirenal wat cell size was observed in rats fed ele and asp under hfd conditions ( preliminary histological results of perirenal wat cells showed that the range of most adipose cell areas ( m ) and the occupancy ( % ) in the cells within view ( n 4 ) are as follows : hfd - control , 200142 000 m/946 % ; hfd-5 % ele , 200124 000 m/922 % ; hfd-03 % asp , 18000 m/915 % ) . the asp ( ele , p < 005 ) groups might increase the use of glucose in skeletal muscles , decreasing the circulating glucose level under hfd conditions . one study showed that increases in plasma levels of tag , nefa and total cholesterol are directly related to higher incidences of obesity and other lipid - associated diseases . in a recent study , 3-month administration of 9 % ele suppressed plasma tag levels in rats under hfd conditions . a hypolipidaemic effect of ele has been reported in which ele significantly attenuates the increased tag level in plasma following a single administration of lipids . eucommia leaves stimulated liver lipid metabolism and decreased the plasma level of tag and total cholesterol in hamsters . in addition , a recent study showed that ele might affect the control of absorption of tag derived from foods into the bloodstream as well as the activation of lipid metabolism in the liver , in particular fatty acid ( fa ) -oxidation . in this study , chronic administration of 5 % ele led to pronounced decreases in plasma levels of nefa , tag and total cholesterol under hfd conditions , and significant and dose - dependent decreases were observed in rats administered asp , suggesting that chronic administration of ele and asp may improve hyperlipidaemia in rats with hfd - induced obesity . furthermore , rt - pcr analysis showed that chronic administration of asp dose - dependently increased the level of fa transport protein , cpt1 and acadvl mrna and suppressed the level of fas mrna in the liver of rats on a hfd , as well as ele groups . the results show that there is a possible increase in the abundance of enzymes in pathways that could increase the capacity for fa -oxidation . in hfd - fed rats , uptake of fa into the liver was increased by the administration of either asp or ele . the uptake was followed by an increased fa -oxidation and atp production , which decreased plasma nefa levels . in contrast , the lipid metabolic enzyme activity assay revealed that the administration of asp or ele stimulated mitochondrial cpt1 activity in the liver but not fas activity in the cytosol . although we can not conclude that asp and ele directly promote fa -oxidation , the fa transport system , which reaches the mitochondrion before -oxidation , is activated . these results , in addition to the results of the rt - pcr analysis and the determination of lipid plasma levels , show that asp and ele have lipid metabolic - stimulating effects that may be influenced by the activation of fa -oxidation . concomitantly , significant activation of lipolysis was observed in the wat of rats supplemented with asp ; however , a similar effect was not observed in animals fed hfd-5 % ele . similar to chronic administration of ele ( in this study and a previous study ) , chronic administration of asp significantly induced mrna expression of ppar and adiponectin , which may depend on the accumulation of visceral fat to improve insulin resistance or hyperlipaemia . the increased expression of these mrna may activate fa -oxidation in the wat of rats under hfd conditions . in this study , chronic administration of asp and ele was found to significantly decrease the plasma levels of nefa and tnf- and significantly increase the plasma level of adiponectin . one study reported that ele suppressed hepatic activities of fas and 3-hydroxy-3-methylglutaryl ( hmg)-coa reductase and increased hepatic -oxidation activity in hfd - fed hamsters , resulting in decreased plasma levels of fa and cholesterol as well as hepatic levels of cholesterol . moreover , our results showed that chronic administration of any tested dose of asp or ele caused simultaneous activation of ketone bodies use in skeletal muscle and hepatic fa -oxidation in rats under hfd conditions . ucp , of which at least three closely related homologues have been cloned , are fa anion transporters in addition to proton transporters . ucp1 ( thermogenin ) mediates non - shivering thermogenesis in the bat , uncoupling the respiratory chain and allowing fast substrate oxidation with a low rate of atp generation . interestingly , chronic administration of asp at a dose from 003 to 03 % increased the bat ucp1 mrna level in a dose - dependent fashion under hfd conditions ; in contrast , such an effect was not observed when administering 5 % ele . thus , based on the rt - pcr analysis of gene expression , the up - regulation of ucp1 mrna expression by asp was attenuated by other ingredients in the ele . chronic administration of asp and ele increased the ucp2 mrna level in the bat of rats under hfd conditions . one study showed that the skeletal muscle ucp3 mrna level was unchanged , but the ucp3 protein level was significantly increased in conjugated linoleic acid - fed mice . our recent results showed that administration of 01 % asp for 1 month significantly enhanced the bmr and decreased the respiratory quotient , which might stimulate lipid metabolisms in rats fed a hfd . in conclusion , asp administration resulted in decreased atp production in the wat , accelerated fa -oxidation in the liver and increased use of ketone bodies and glucose in skeletal muscle and may have increased non - shivering thermogenesis due to ucp1 expression in the bat . this study showed that asp may act as a major ingredient in ele , relating nearly every metabolic function across several organs , except for the activation of the bat ucp1-induced thermogenesis .

eucommia leaves ( eucommia ulmoides oliver ) contain chlorogenic acid ( a caffeic acid derivative ) and geniposidic acid and asperuloside ( asp ) , iridoid glucosides used in beverages . we used a metabolic syndrome rat model , produced by feeding a 35 % high - fat diet ( hfd ) , to examine potential anti - obesity and anti - metabolic syndrome effects and mechanisms of chronic administration of asp . these effects were compared with eucommia leaf extract ( ele ) , the positive control , which exhibits anti - obesity effects . a total of six rats were studied for 3 months in five groups . asp suppressed body weight , visceral fat weight , food intake and circulating levels of glucose , insulin and lipids , and increased the plasma adiponectin level in rats on a hfd . these effects are similar to those of ele , except for the influence on the plasma glucose level . rt pcr studies showed that asp ( like ele with known anti - obesity effects ) diminished isocitrate dehydrogenase 3 , nadh dehydrogenase flavoprotein 1 ( comp i ) mrna and fatty acid synthase levels ( white adipose tissue ) , increased carnitine palmitoyltransferase 1 and acyl - coa dehydrogenase , very - long - chain mrna levels ( liver ) , and increased glut4 , citrate synthase , isocitrate dehydrogenase 3 , succinyl coa synthase , peroxisomal 3-ketoacyl - coa thiolase , dihydrolipoamide succinyl transferase and succinate dehydrogenase mrna levels ( skeletal muscle ) under hfd conditions . interestingly , asp administration resulted in significantly increased mrna levels of uncoupling protein 1 ( ucp1 ) in the brown adipose tissue of hfd - fed rats ; ele did not affect the expression of ucp1 . the increased expression of ucp1 may be negated by many ingredients other than asp in the ele . these findings suggest that chronic administration of asp stimulates anti - obesity and anti - metabolic syndrome activity in hfd - fed rats across several organs , similar to ele administration ; thus , asp may be an important ingredient of ele .

Materials and methods Results Discussion

rats were divided into five groups ( hfd - control , hfd-5 % ele , hfd-003 % asp , hfd-01 % asp and hfd-03 % asp ; n 6 for each ) based on body weight . the test foods were provided ad libitum for 3 months to examine the potential anti - obesity and anti - metabolic syndrome effects of ele and asp . compared with the hfd - control group , final body weight , body weight gain and food intake decreased significantly in 3 months in the hfd-5 % ele group ( each p < 005 ) , and the dose - dependent decrease was observed in the hfd-00303 % asp group compared with the hfd - control group ( each p < 005 , table 1 ) . table 1.effects of asperuloside ( asp ) on body weight , white adipose tissue ( wat ) weight , brown adipose tissue ( bat ) weight , soleus muscle ( sol . effects of asperuloside ( asp ) on body weight , white adipose tissue ( wat ) weight , brown adipose tissue ( bat ) weight , soleus muscle ( sol . m ) weight and plasma indices in high - fat diet ( hfd)-fed rats ( mean values with their standard errors ) ele , eucommia leaf extract ; cont . compared with the hfd - control group , the relative weight of the perirenal wat significantly decreased in 3 months in the hfd-5 % ele group ( p < 005 ) , and a significant and dose dependent decrease was observed in the hfd-00303 % asp group when compared with the hfd - control group ( p < 005 , table 1 ) . administration of asp dose dependently reduced the plasma tag level in rats fed a hfd when compared with the hfd - control rats ( p < 005 ) , and a significant decrease was seen in the hfd-5 % ele group ( p < 005 , table 1 ) . a dose - dependently reduced plasma glucose level was observed in the hfd - asp groups compared with the hfd - control group after 3 months of hfd feeding ( p < 005 ) ; however , no difference in plasma glucose level was observed between the hfd-5 % ele and the average of the three hfd - asp groups ( table 1 ) . administration of 5 % ele yielded markedly decreased plasma insulin levels under hfd conditions ( p < 005 ) , and linearly decreased plasma insulin levels were also observed in rats administered 0103 % asp contrasted with the hfd - control rats ( p < 005 , table 1 ) . under hfd conditions , chronic administration of 5 % ele significantly induced the expression of glucokinase , citrate synthase ( cs ) , dihydrolipoamide succinyl transferase ( ogdh ) , nadh dehydrogenase flavoprotein 1 ( comp i ) and cytochrome c oxidase , subunit 4a ( comp iv ) in the rat liver ( each p < 005 , table 2 ) . the asp groups exhibited a significant and dose - dependent increase of glucokinase , cs , ogdh , comp i and comp iv mrna when compared with the hfd - control group ( p < 005 ) , and the significantly increased mrna level of cs , ogdh , comp i and comp iv in the average of all the hfd-00303 % asp groups was lower than that of the hfd-5 % ele group ( p < 005 , table 2 ) . in the perirenal wat of rats fed a hfd , the levels of isocitrate dehydrogenase 3 ( idh3 ) and comp i mrna were markedly decreased by chronic administration of 5 % ele ( each p < 005 , table 3 ) . m. of rats under hfd conditions , the 5 % ele group had significantly increased levels of glut4 , cs , idh3 , ogdh , succinate dehydrogenase complex , subunit a , flavoprotein , comp i , comp iv and atp synthase , h transporting , mitochondrial f1 complex , delta subunit ( comp v ) mrna ( each p < 005 , table 4 ) . table 2.gene expression analysis by real - time pcr in liver after administration of asperuloside ( asp ) under high - fat diet ( hfd ) conditions(mean values with their standard errors)hfd - asp ( fold of cont. , control ; ele , eucommia leaf extract ; gck , glucokinase ; cs , citrate synthase ; ogdh , dihydrolipoamide succinyl transferase ; comp i , nadh dehydrogenase flavoprotein 1 ; comp iv , cytochrome c oxidase , subunit 4a ; fas , fatty acid synthase ; fatp , fatty acid transport protein ; cpt1 , carnitine palmitoyltransferase 1 ; acadvl , acyl - coa dehydrogenase , very long chain ; hadh ii , hydroxyacyl - coa dehydrogenase type ii ; hadh sc , l-3-hydroxyacyl - coa dehydrogenase , short chain . table 3.gene expression analysis by real - time pcr in perirenal white adipose tissue after administration of asperuloside ( asp ) under high - fat diet ( hfd ) conditions(mean values with their standard errors)hfd - asp ( fold of cont. , control ; ele , eucommia leaf extract ; hk2 , hexokinase 2 ; cs , citrate synthase ; idh3 , isocitrate dehydrogenase 3 ; ogdh , dihydrolipoamide succinyl transferase ; comp i , nadh dehydrogenase flavoprotein 1 ; comp iv , cytochrome c oxidase , subunit 4a ; hsl , hormone - sensitive lipase ; fas , fatty acid synthase ; fatp , fatty acid transporter protein ; cpt2 , carnitine palmitoyltransferase 2 ; acadvl , acyl - coa dehydrogenase , very long chain . table 4.gene expression analysis by real - time pcr in soleus muscle after administration of asperuloside ( asp ) under high - fat diet ( hfd ) conditions(mean values with their standard errors)hfd - asp ( fold of cont. , control ; ele , eucommia leaf extract ; hk2 , hexokinase 2 ; cs , citrate synthase ; idh3 , isocitrate dehydrogenase 3 ; ogdh , dihydrolipoamide succinyl transferase ; sdha ( comp ii ) , succinate dehydrogenase complex , subunit a , flavoprotein ; comp i , nadh dehydrogenase flavoprotein 1 ; comp iv , cytochrome c oxidase , subunit 4a ; comp v , atp synthase , h transporting , mitochondrial f1 complex , delta subunit ; fas , fatty acid synthase ; fatp , fatty acid transporter protein ; scos , succinyl coa synthase ; p3kcot , peroxisomal 3-ketoacyl - coa thiolase . gene expression analysis by real - time pcr in liver after administration of asperuloside ( asp ) under high - fat diet ( hfd ) conditions ( mean values with their standard errors ) cont . , control ; ele , eucommia leaf extract ; gck , glucokinase ; cs , citrate synthase ; ogdh , dihydrolipoamide succinyl transferase ; comp i , nadh dehydrogenase flavoprotein 1 ; comp iv , cytochrome c oxidase , subunit 4a ; fas , fatty acid synthase ; fatp , fatty acid transport protein ; cpt1 , carnitine palmitoyltransferase 1 ; acadvl , acyl - coa dehydrogenase , very long chain ; hadh ii , hydroxyacyl - coa dehydrogenase type ii ; hadh sc , l-3-hydroxyacyl - coa dehydrogenase , short chain . gene expression analysis by real - time pcr in perirenal white adipose tissue after administration of asperuloside ( asp ) under high - fat diet ( hfd ) conditions ( mean values with their standard errors ) cont . , control ; ele , eucommia leaf extract ; hk2 , hexokinase 2 ; cs , citrate synthase ; idh3 , isocitrate dehydrogenase 3 ; ogdh , dihydrolipoamide succinyl transferase ; comp i , nadh dehydrogenase flavoprotein 1 ; comp iv , cytochrome c oxidase , subunit 4a ; hsl , hormone - sensitive lipase ; fas , fatty acid synthase ; fatp , fatty acid transporter protein ; cpt2 , carnitine palmitoyltransferase 2 ; acadvl , acyl - coa dehydrogenase , very long chain . gene expression analysis by real - time pcr in soleus muscle after administration of asperuloside ( asp ) under high - fat diet ( hfd ) conditions ( mean values with their standard errors ) cont . , control ; ele , eucommia leaf extract ; hk2 , hexokinase 2 ; cs , citrate synthase ; idh3 , isocitrate dehydrogenase 3 ; ogdh , dihydrolipoamide succinyl transferase ; sdha ( comp ii ) , succinate dehydrogenase complex , subunit a , flavoprotein ; comp i , nadh dehydrogenase flavoprotein 1 ; comp iv , cytochrome c oxidase , subunit 4a ; comp v , atp synthase , h transporting , mitochondrial f1 complex , delta subunit ; fas , fatty acid synthase ; fatp , fatty acid transporter protein ; scos , succinyl coa synthase ; p3kcot , peroxisomal 3-ketoacyl - coa thiolase . in the rat liver under hfd conditions , chronic administration of 5 % ele significantly decreased the level of expression of fas mrna and a marked increased the mrna levels of fa transport protein , cpt1 , acyl - coa dehydrogenase , very long chain ( acadvl ) , hydroxyacyl - coa dehydrogenase type ii and l-3-hydroxyacyl - coa dehydrogenase , short chain ( each p < 005 , table 2 ) . significant differences in acadvl , hydroxyacyl - coa dehydrogenase type ii and l-3-hydroxyacyl - coa dehydrogenase , short chain mrna levels were observed between the hfd-5 % ele and the average of all the hfd-00303 % asp groups ( p < 005 ) , but not in fas , fa transport protein and cpt1 mrna levels ( table 2 ) . chronic administration of 5 % ele led to significantly increased mrna levels of succinyl coa synthase , peroxisomal 3-ketoacyl - coa thiolase , ogdh and succinate dehydrogenase complex , subunit a , flavoprotein in the sol . in the perirenal wat of rats fed a hfd , chronic administration of 5 % ele led to a significant increase in ppar and adiponectin mrna levels ( each p < 005 , table 3 ) . in the bat of hfd - fed rats , chronic administration of 5 % ele led to significantly increased uncoupling protein 2 ( ucp2 ) mrna levels ( p < 005 ) , but the 5 % ele administration did not affect the expression of other ucp mrna ( table 5 ) . % asp administration clearly increased the mrna levels of ucp1 and ucp2 in a dose - dependent fashion when compared with the hfd - control group ( each p < 005 , table 5 ) . table 5.gene expression analysis by real - time pcr in brown adipose tissue after administration of asperuloside ( asp ) under high - fat diet ( hfd ) conditions(mean values with their standard errors)hfd - asp ( fold of cont. gene expression analysis by real - time pcr in brown adipose tissue after administration of asperuloside ( asp ) under high - fat diet ( hfd ) conditions ( mean values with their standard errors ) cont . in rats under hfd conditions , chronic administration of 5 % ele led to a significant increase in mitochondrial cpt1 activity in the liver compared with the hfd - control group ( p < 005 , fig . moreover , chronic administration of asp dose - dependently increased the mitochondrial cpt1 activity in the liver of rats under hfd conditions when compared with the hfd - control rats ( p < 005 , fig . 1.effect of asperuloside ( asp ) on the activity of hepatic carnitine palmitoyltransferase 1 ( cpt1 ) ( a ) and hepatic fatty acid synthase ( fas ) ( b ) in high - fat diet ( hfd)-fed rats . effect of asperuloside ( asp ) on the activity of hepatic carnitine palmitoyltransferase 1 ( cpt1 ) ( a ) and hepatic fatty acid synthase ( fas ) ( b ) in high - fat diet ( hfd)-fed rats . similarly to our previous study , 3-month administration of asp controlled the body weight gain , food intake and relative wat weight and increased the relative bat weight with prominently decreased plasma levels of tag , nefa and total cholesterol in hfd rats . moreover , the results agreed with previous findings that showed that asp but not gea and cha decreases the body weight and wat weight via significantly reduced circulating nefa levels in hfd - fed mice . our study showed that 5 % ele administration decreased the plasma level of insulin under hfd conditions , and a significant and dose - dependent decrease was observed in rats fed 00303 % asp , resulting in a significant decrease in plasma glucose level in a dose - dependent fashion . rt - pcr analysis showed that chronic administration of asp to rats under a hfd significantly increased the mrna levels of cs , idh3 , ogdh , sdha , comp i , comp iv and comp v in skeletal muscles . thus , asp may be an important component of ele responsible for the control of carbohydrate metabolism . similar to ele - fed rats , the asp - fed animals might no longer be able to maintain the shape of the adipose cells that store tag because of the dose - dependently decreased atp production in the wat under hfd conditions . a diminished perirenal wat cell size was observed in rats fed ele and asp under hfd conditions ( preliminary histological results of perirenal wat cells showed that the range of most adipose cell areas ( m ) and the occupancy ( % ) in the cells within view ( n 4 ) are as follows : hfd - control , 200142 000 m/946 % ; hfd-5 % ele , 200124 000 m/922 % ; hfd-03 % asp , 18000 m/915 % ) . the asp ( ele , p < 005 ) groups might increase the use of glucose in skeletal muscles , decreasing the circulating glucose level under hfd conditions . in this study , chronic administration of 5 % ele led to pronounced decreases in plasma levels of nefa , tag and total cholesterol under hfd conditions , and significant and dose - dependent decreases were observed in rats administered asp , suggesting that chronic administration of ele and asp may improve hyperlipidaemia in rats with hfd - induced obesity . furthermore , rt - pcr analysis showed that chronic administration of asp dose - dependently increased the level of fa transport protein , cpt1 and acadvl mrna and suppressed the level of fas mrna in the liver of rats on a hfd , as well as ele groups . similar to chronic administration of ele ( in this study and a previous study ) , chronic administration of asp significantly induced mrna expression of ppar and adiponectin , which may depend on the accumulation of visceral fat to improve insulin resistance or hyperlipaemia . the increased expression of these mrna may activate fa -oxidation in the wat of rats under hfd conditions . moreover , our results showed that chronic administration of any tested dose of asp or ele caused simultaneous activation of ketone bodies use in skeletal muscle and hepatic fa -oxidation in rats under hfd conditions . interestingly , chronic administration of asp at a dose from 003 to 03 % increased the bat ucp1 mrna level in a dose - dependent fashion under hfd conditions ; in contrast , such an effect was not observed when administering 5 % ele . chronic administration of asp and ele increased the ucp2 mrna level in the bat of rats under hfd conditions . in conclusion , asp administration resulted in decreased atp production in the wat , accelerated fa -oxidation in the liver and increased use of ketone bodies and glucose in skeletal muscle and may have increased non - shivering thermogenesis due to ucp1 expression in the bat . this study showed that asp may act as a major ingredient in ele , relating nearly every metabolic function across several organs , except for the activation of the bat ucp1-induced thermogenesis .

the causes of cognitive disability vary with the severity of the condition : moderate - to - severe intellectual disability ( defined as an intelligence quotient [ iq ] score less than 50 ) is much more likely to be due to a single pathological cause ( genetic or environmental ) than mild mr ( defined as an iq score between 50 and 70 ) , which is often thought to be multifactorial in origin . chromosomal and genetic disorders account for 30% to 40% of moderate - to - severe mr ; environmental insults explain a further 10% to 30% , and the cause is unknown in about 40% of cases . genetic and environmental causes explain , in roughly equal proportions , about 30% of mild intellectual disability ; an etiological diagnosis is not obtained in the remaining 70% of cases . table i summarizes data from epidemiological studies of low iq , following the convention of separating mild disability from moderate to severe . while controversy has long surrounded the extent to which genetic variation contributes to variation in intellectual function , there is now little doubt that moderate - to - severe intellectual disability is due primarily to chromosomal and genetic abnormalities . the largest individual contributors are down 's syndrome , chromosomal rearrangements , and x - linked mental retardation ( xlmr ) ( table i ) . small chromosomal rearrangements , affecting the ends ( telomeres ) of chromosomes have emerged as a common cause in cases until recently regarded as idiopathic , and it is likely that a considerable proportion of cases of unknown etiology will also be found to have a genetic origin . the importance of polygenic influences is inferred from the results of twin , family , and adoption studies for normal iq measures , and rarely from direct investigation of families with low iq ; studies evaluating biological and environmental risk factors in this group are singularly lacking , but there are indications that single - gene conditions and chromosomal abnormalities may be more frequent than previously assumed . table ii presents data on the genetic basis of conditions for which there is evidence that mutations give rise directly to intellectual disability . the table lists conditions where the genetic effects on intellectual function are thought to be relatively immediate , that is to say where no obvious developmental abnormality of the brain or progressive destruction of neuronal tissue results in cognitive impairment . when we consider the pathogenesis of intellectual disability , it is important to bear in mind that the phenotype involves multiple domains of intellectual functioning , often broadly divided into verbal and performance skills , but also encompassing capacities such as memory and attention , where performance is not traditionally seen as central to intellectual ability . unfortunately , we do not know whether the domains that psychologists recognize correspond to the way genes operate , whether , for instance , verbal and performance skills can be separated at a genetic level . information is lacking about genetic influences on the domains of both normal and abnormal intellectual functioning . studies of the heritability of intelligence , a measure of the extent to which genes contribute to the variation in intellectual functioning in the population , have mostly been carried out on overall measures of cognitive function , such as iq , although more recent work on speech and language development is beginning to indicate that genetic effects that have more specific influences can be identified . similarly , there have been few detailed psychometric investigations of people with intellectual disability due to a specific genetic lesion , so we do not know whether cognitive functioning is abnormal over all domains or whether there are discrete abnormalities . in fact , as discussed later , there is some evidence in favor of the latter hypothesis . genetic mapping techniques and molecular cloning have made it possible to investigate disorders where the relationship between intellectual disability and genetic defect might be immediate . these are conditions where there are no noticeable alterations in brain structures and the cause of cognitive impairment is difficult to find . in general , this distinction is reflected in the division of mr into syndromic and nonsyndromic conditions . in syndromic mr , the phenotype includes additional physical abnormalities ( such as facial dysmorphism or minor abnormalities of the hands and feet ) , while in nonsyndromic mr the only abnormality is cognitive impairment . it might appear that genetic lesions are directly responsible for intellectual disability more commonly in nonsyndromic than in syndromic conditions , but it should be borne in mind that , without an understanding of the pathogenesis , this is only an assumption . for example , phenotypes vary considerably and mutations in the same gene may give rise to both syndromic and nonsyndromic intellectual disability : mutations in rsk2 give rise to coffin - lowry syndrome ( cls ) and to nonspecific intellectual disability , and mutations in different parts of the atrx gene produce either syndromic or nonsyndromic mr . nevertheless , some remarkable advances in x - linked nonsyndromic intellectual disability are uncovering genes that act directly on cognition , probably through central nervous system ( cns ) development . this is because x - linked recessive disease is compatible with the occurrence of affected members in multiple generations ; it is therefore both recognizable as an inherited condition and amenable to genetic mapping . x - linked intellectual disability ( ie , xlmr ) is common : the frequency is estimated to be 1.8 in 1000 males with a carrier frequency of 2.4 in 1000 females . the number of recognized conditions continues to increase : currently 210 have been described , 126 mapped , and 32 cloned . fragile x syndrome is the commonest form of xlmr , with a prevalence of approximately 1 in 5000 males and causes intellectual disability in about 1 in 8000 females . affected individuals have a folate - sensitive fragile site in the region xq27.3 , associated with an expansion of a trinucleotide repeat ( cgg ) in the 5'-noncoding region of a gene that encodes an rna binding protein termed fmr1 . despite being one of the early triumphs of positional cloning , the function of fmr1 , and in particular how its deficiency gives rise to intellectual disability , is still not understood . in the normal brain , the fmr protein it can bind rna , including its own transcript , and it has been postulated that the fmr protein has a role in the machinery of translation and , as it shuttles between nucleus and cytoplasm , that it may be involved in mrna export . one explanation for the effect of the gene on brain function is that it plays a role in the maturation and pruning of dendritic spines during brain development . mutations in factors that regulate gene expression are emerging as an important genetic cause of intellectual disability . two syndromic conditions have been found in which the gene acts as a transcriptional regulator through its effect on chromatin . in rett 's syndrome , a progressive neurological disorder that affects females almost exclusively , mutations have been found in methyl - cpg - binding protein 2 ( mecp2 ) . mecp2 selectively binds cpg dinucleotides in the mammalian genome and mediates transcriptional repression through interaction with histone deacetylase and the corepressor sin3a . in the alpha - thalassemia x - linked mental retardation syndrome ( atrx ) , mutations in atrx give rise to characteristic developmental abnormalities including severe mr , facial dysmorphism , urogenital abnormalities , and alpha - thalassemia . the gene contains sequence motifs that indicate that it belongs to a group of proteins that to bind to chromatin . at a molecular level , the mutation has effects on the pattern of genomic methylation , consistent with the role of atrx in chromatin remodeling . the pleiotropic effects of mutations in mecp2 and atrx could result from the regulated expression of a restricted class of genes . investigation of a syndromic mr condition , cls , has led to the discovery of the involvement of another signaling pathway in cognitive impairment , namely the mapk - activated signaling pathway ( mapk for mitogen - activated protein kinase ) . cls is characterized by severe psychomotor retardation , facial and digital physical anomalies , and progressive skeletal deformation . the disorder was mapped by linkage to the region xp22.2 and mutations discovered in a positional candidate gene rsk2 ( also known as rps6ka3 ) . rsk2 mediates growth factor induction of cyclic adenosine monophosphate response element - binding protein ( creb ) phosphorylation , as part of a signaling pathway whereby ras - mapk and ras signals are transmitted to the nucleus to activate gene expression . remarkably , mutations in rsk2 give rise to nonsyndromic mr : patients in an xlmr family with neither facial , digital , nor skeletal anomalies compatible with cls , but with mild mr , have been found to have a mutation in exon 14 of the gene , resulting in a conservative amino acid change . a number of genetic conditions associated with intellectual disability have been found to be due to small chromosomal deletions or duplications ( typically less than 5 megabases ) and are known as segmental aneusomy syndromes ( sec tabic ii ) . the small size of some of the regions has enabled a search for dosage - sensitive genes . however , in order to prove that a deleted gene is indeed dosage - sensitive , it has been necessary to find families with point mutations in the gene that segregate with intellectual disability . this has been achieved with rubinstein - taybi syndrome ( characterized by abnormal craniofacial features , broad thumbs , and intellectual disability ) , which can arise from monosomy of a small region in 16pl3.3 . williams - beuren syndrome is a neurodevelopmental disorder characterized by congenital heart disease , infantile hypercalcemia , dysmorphic facial features , and cognitive disability . it is known that mutations affecting the elastin gene give rise to the supravalvular aortic stenosis , but there are still at least 15 candidate genes that could be involved in the unusual cognitive profile of the syndrome . these include a number of transcriptional regulators , such as williams ' syndrome transcription factor , which contains a plant homeodomain ( phd ) , limk1 , which contains one phd motif followed by a bromodomain , and the wbscr14/ws - bhlh gene , which encodes a basic - helix - loop - helix leucine zipper , characteristic of a subclass of transcription factors . two clinically distinct disorders , prader - willi and angelman syndromes ( pws and as ) , arise from abnormalities of a small region in 15ql l - ql3 . these syndromes have characteristic and distinct neurobehavioral profiles : in as the retardation is severe ( very few affected individuals can talk ) and there is ataxia , seizures , abnormal eeg , microcephaly , facial dysmorphism , hyperactivity and paroxysmal laughter . by contrast , in pws , the mr may be only mild ; there is a characteristic facial appearance and a specific behavioral abnormality , ie , hyperphagia resulting in severe obesity . despite the phenotypic differences , the basic defect is the same in the two disorders : a failure of parent - of - origin - specific gene expression . if both copies of chromosome 15 derive from the mother then the individual will have pws ; if both are from the father then the phenotype is as . the basic defect is not simply a dosage effect ; it turns out that about a quarter of cases of pws are not due to a deletion but to the inheritance of two maternal copies of chromosome 15 ( rather than the usual situation of one maternal and one paternal ) . the chromosomal region is said to bear a parent - of - origin imprint , of which the molecular signature is a difference in dna methylation . mutations in a ubiquitin protein ligase gene ( ube3a ) have been found in a few rare families with as . the gene product is part of a widely used ubiquitin - mediated protein degradation pathway . seven genes ( and candidate genes ) have been identified in the pws region , all of which appear to be brain specific . it is not known if the phenotype is due to an abnormality in a single gene . however , there is now some evidence to suggest that abnormal rna editing , due to misregulation of guide rnas , mediates the defect in pws . the nucleolus contains a large number of small rnas , termed small nucleolar rnas ( snornas ) ; the majority of these snornas function in the posttranscriptional modification of rrna nucleotides . however , it is now clear that the action of methylation guide snornas goes beyond the field of ribosome biogenesis . recently three brain - specific snornas , which are subject to genomic imprinting in mice and humans , have been discovered within the 15qll critical region for pws and as . unusually , they do not have appropriate antisense elements , so their function is not clear , but one has a similarity to the mrna encoded by the gene for the serotonin receptor-2c . the sequence matches a conserved region subject to both alternative splicing and adenosine - to - inosine editing . because of the known involvement of serotonin in appetite control and cognition , this finding raises the intriguing possibility that the defect in pws involves a defect in serotonin neurotransmission . similar problems beset attempts to understand how deletions in the region 22q11 give rise to cognitive disabilities . digeorge ( dgs ) , velocardiofacial ( vcfs ) , and conotruncal anomaly face ( ctaf ) syndromes are different phenotypic manifestations of deletions within 22q11 . both dgs and vcfs are associated with intellectual disability ; additionally psychosis is found in some patients with vcfs . cloning and sequencing of the entire region has not identified any obvious candidates for the cognitive defect and it now seems likely that the syndromes arise from combined monosomy of more than one gene . this is because x - linked recessive disease is compatible with the occurrence of affected members in multiple generations ; it is therefore both recognizable as an inherited condition and amenable to genetic mapping . x - linked intellectual disability ( ie , xlmr ) is common : the frequency is estimated to be 1.8 in 1000 males with a carrier frequency of 2.4 in 1000 females . the number of recognized conditions continues to increase : currently 210 have been described , 126 mapped , and 32 cloned . fragile x syndrome is the commonest form of xlmr , with a prevalence of approximately 1 in 5000 males and causes intellectual disability in about 1 in 8000 females . affected individuals have a folate - sensitive fragile site in the region xq27.3 , associated with an expansion of a trinucleotide repeat ( cgg ) in the 5'-noncoding region of a gene that encodes an rna binding protein termed fmr1 . despite being one of the early triumphs of positional cloning , the function of fmr1 , and in particular how its deficiency gives rise to intellectual disability , is still not understood . in the normal brain , the fmr protein it can bind rna , including its own transcript , and it has been postulated that the fmr protein has a role in the machinery of translation and , as it shuttles between nucleus and cytoplasm , that it may be involved in mrna export . one explanation for the effect of the gene on brain function is that it plays a role in the maturation and pruning of dendritic spines during brain development . mutations in factors that regulate gene expression are emerging as an important genetic cause of intellectual disability . two syndromic conditions have been found in which the gene acts as a transcriptional regulator through its effect on chromatin . in rett 's syndrome , a progressive neurological disorder that affects females almost exclusively , mutations have been found in methyl - cpg - binding protein 2 ( mecp2 ) . mecp2 selectively binds cpg dinucleotides in the mammalian genome and mediates transcriptional repression through interaction with histone deacetylase and the corepressor sin3a . in the alpha - thalassemia x - linked mental retardation syndrome ( atrx ) , mutations in atrx give rise to characteristic developmental abnormalities including severe mr , facial dysmorphism , urogenital abnormalities , and alpha - thalassemia . the gene contains sequence motifs that indicate that it belongs to a group of proteins that to bind to chromatin . at a molecular level , the mutation has effects on the pattern of genomic methylation , consistent with the role of atrx in chromatin remodeling . the pleiotropic effects of mutations in mecp2 and atrx could result from the regulated expression of a restricted class of genes . investigation of a syndromic mr condition , cls , has led to the discovery of the involvement of another signaling pathway in cognitive impairment , namely the mapk - activated signaling pathway ( mapk for mitogen - activated protein kinase ) . cls is characterized by severe psychomotor retardation , facial and digital physical anomalies , and progressive skeletal deformation . the disorder was mapped by linkage to the region xp22.2 and mutations discovered in a positional candidate gene rsk2 ( also known as rps6ka3 ) . rsk2 mediates growth factor induction of cyclic adenosine monophosphate response element - binding protein ( creb ) phosphorylation , as part of a signaling pathway whereby ras - mapk and ras signals are transmitted to the nucleus to activate gene expression . remarkably , mutations in rsk2 give rise to nonsyndromic mr : patients in an xlmr family with neither facial , digital , nor skeletal anomalies compatible with cls , but with mild mr , have been found to have a mutation in exon 14 of the gene , resulting in a conservative amino acid change . a number of genetic conditions associated with intellectual disability have been found to be due to small chromosomal deletions or duplications ( typically less than 5 megabases ) and are known as segmental aneusomy syndromes ( sec tabic ii ) . the small size of some of the regions has enabled a search for dosage - sensitive genes . however , in order to prove that a deleted gene is indeed dosage - sensitive , it has been necessary to find families with point mutations in the gene that segregate with intellectual disability . this has been achieved with rubinstein - taybi syndrome ( characterized by abnormal craniofacial features , broad thumbs , and intellectual disability ) , which can arise from monosomy of a small region in 16pl3.3 . williams - beuren syndrome is a neurodevelopmental disorder characterized by congenital heart disease , infantile hypercalcemia , dysmorphic facial features , and cognitive disability . it is known that mutations affecting the elastin gene give rise to the supravalvular aortic stenosis , but there are still at least 15 candidate genes that could be involved in the unusual cognitive profile of the syndrome . these include a number of transcriptional regulators , such as williams ' syndrome transcription factor , which contains a plant homeodomain ( phd ) , limk1 , which contains one phd motif followed by a bromodomain , and the wbscr14/ws - bhlh gene , which encodes a basic - helix - loop - helix leucine zipper , characteristic of a subclass of transcription factors . two clinically distinct disorders , prader - willi and angelman syndromes ( pws and as ) , arise from abnormalities of a small region in 15ql l - ql3 . these syndromes have characteristic and distinct neurobehavioral profiles : in as the retardation is severe ( very few affected individuals can talk ) and there is ataxia , seizures , abnormal eeg , microcephaly , facial dysmorphism , hyperactivity and paroxysmal laughter . by contrast , in pws , the mr may be only mild ; there is a characteristic facial appearance and a specific behavioral abnormality , ie , hyperphagia resulting in severe obesity . despite the phenotypic differences , the basic defect is the same in the two disorders : a failure of parent - of - origin - specific gene expression . if both copies of chromosome 15 derive from the mother then the individual will have pws ; if both are from the father then the phenotype is as . the basic defect is not simply a dosage effect ; it turns out that about a quarter of cases of pws are not due to a deletion but to the inheritance of two maternal copies of chromosome 15 ( rather than the usual situation of one maternal and one paternal ) . the chromosomal region is said to bear a parent - of - origin imprint , of which the molecular signature is a difference in dna methylation . mutations in a ubiquitin protein ligase gene ( ube3a ) have been found in a few rare families with as . the gene product is part of a widely used ubiquitin - mediated protein degradation pathway . seven genes ( and candidate genes ) have been identified in the pws region , all of which appear to be brain specific . it is not known if the phenotype is due to an abnormality in a single gene . however , there is now some evidence to suggest that abnormal rna editing , due to misregulation of guide rnas , mediates the defect in pws . the nucleolus contains a large number of small rnas , termed small nucleolar rnas ( snornas ) ; the majority of these snornas function in the posttranscriptional modification of rrna nucleotides . however , it is now clear that the action of methylation guide snornas goes beyond the field of ribosome biogenesis . recently three brain - specific snornas , which are subject to genomic imprinting in mice and humans , have been discovered within the 15qll critical region for pws and as . unusually , they do not have appropriate antisense elements , so their function is not clear , but one has a similarity to the mrna encoded by the gene for the serotonin receptor-2c . the sequence matches a conserved region subject to both alternative splicing and adenosine - to - inosine editing . because of the known involvement of serotonin in appetite control and cognition , this finding raises the intriguing possibility that the defect in pws involves a defect in serotonin neurotransmission . similar problems beset attempts to understand how deletions in the region 22q11 give rise to cognitive disabilities . digeorge ( dgs ) , velocardiofacial ( vcfs ) , and conotruncal anomaly face ( ctaf ) syndromes are different phenotypic manifestations of deletions within 22q11 . both dgs and vcfs are associated with intellectual disability ; additionally psychosis is found in some patients with vcfs . cloning and sequencing of the entire region has not identified any obvious candidates for the cognitive defect and it now seems likely that the syndromes arise from combined monosomy of more than one gene . given the difficulties encountered in investigating the segmental aneusomies , then trying to identify specific genes responsible for the abnormalities found in aneuplodies , where there is an abnormality in the number of a whole chromosome , might seem impossible . however comparison between individuals with partial aneuploidy of a chromosome has allowed the definition of critical regions in both down 's syndrome ( trisomy 21 ) and turner syndrome ( xo ) . candidate genes for some of the somatic features of turner syndrome have been proposed : shox / phog encodes a homeodomain protein that may explain the short stature , while rps4y encodes an isoform of a ribosomal small subunit protein . identification of genes for features other than short stature has been problematic . there are no candidates for the unusual cognitive profile . however , there is one report that turner syndrome patients with a paternally derived x chromosome have superior verbal abilities and skills involved in social interactions . in work on down 's syndrome , attention has been focused on the region 21q22.2 as a potential site for dosage - sensitive genes that affect learning and behavior . on the basis of transgenic mouse experiments , the gene encodes a tyrosine / serine kinase expressed in developing neuroblasts and a human gene lies in the down 's syndrome - critical chromosomal region 21q22 . however , as with the segmental aneusomies , there is a proliferation of down 's syndrome critical region ( dscr ) genes ; as yet no definitive evidence of their role in intellectual disability has been provided . perhaps the most striking finding to emerge from the study of nonsyndromic xlmr is the discovery of mutations in genes affecting different components of the rho - signaling pathway ( table ii ) . two genes , oligophrenin-1 ( ophn1 ) and arhgef6 , directly affect the rho - activation cycle . ophn1 encodes a rho - gap protein ( gap for gtpase [ guanosine triphosphatase]-activating protein ) that stimulates the intrinsic gtpase activity of rho , rac , and cdc42 . arhgef6 encodes a small cytoplasmic protein , homologous to proteins that activate rho - gtpases by exchanging guanosine diphosphate ( gdp ) for guanosine triphosphate ( gtp ) . a third gene found to be mutated in xlmr families is pak3 pak3 may well be a downstream effector of the rho - gtpases rac and cdc42 putting the message forward to the actin cytoskeleton and to transcriptional activation . guanosine nucleotide dissociation inhibitor-1 ( gdi1 ) inhibits gdp dissociation from rab3a by binding to gdp - bound rab proteins and appears to be crucial in maintaining the balance between the gtp- and gdp - bound forms of rab3 . rab3a is a small gtp - binding protein that functions in the recruitment of synaptic vesicles for exocytosis and is essential for long - term potentiation ( ltp ) in hippocampal neurons . all rab proteins are hydrophobic by nature and need gdi to mediate membrane attachment and retrieval . rab exists exclusively as a soluble complex with gdi in the cytoplasm , where it forms a reservoir to deliver rab to the membrane during assembly of a transport vesicle . how might the biology of the small gtp - binding proteins explain human cognitive function ? growth cones of developing axons find their way through the brain by sampling molecular signals , helped by gtpases . whereas cdc42 and rac1 are involved in the formation of lamellipodia and filopodia , inhibition of rho , rac , and cdc42 also reduces dendrite formation . cognitive dysfunction could therefore be due to a failure to establish correct neuronal connections during cns development . this view is supported by what is known about the function of rab3a in exocytosis . synaptic vesicles contain rab3a , the most abundant rab protein in the brain and , in one model , exocytosis leads to the dissociation of rab3a from the vesicle . since rab3a - deficient mice have no fundamental deficits in synaptic vesicle exocytosis , the protein is not essential to the process , but is required to maintain a normal reserve of synaptic vesicles . the gdi1 mutation , by disrupting rab3a traffic , is expected to alter neurotransmitter release , which might , in turn , account for the intellectual impairment . both the developmental and synaptic transmission account of rho - gtpase involvement must explain why only neurones involved in cognitive systems are disrupted . one likely explanation is that the mutations only partly disrupt the brain system on which they operate , but it could also be that compensatory mechanisms , effective in other cell types , fail when it comes to neuronal processes involved in cognitive processing . interestingly , there is also evidence that the cognitive defects associated with neurofibromatosis type 1 ( nf1 ) derive from an effect on the ras pathway . some 30% to 65% of the affected children have learning difficulties , but only 4% to 8% have mr . the nf1 gene , neurofibromin , has a gap - related domain linking it to signal transduction pathways . molecular investigation of a family with nf1 identified a mutation that disabled the rasgtpase - activating function . affected children had an iq range of 80 to 89 and impairment in both language and motor development , indicating that the gap of neurofibromin is critical to the development of these functions . tm4sf2 encodes a member of a group of proteins that complex with integrins , proteins that function as ap - heterodimers mediating adhesive interactions with the extracellular matrix and also acting to transduce signaling . evidence for the role of integrins in human cognition came from the isolation of a mutation in tm4sf2 in a patient with nonsyndromic xlmr . analysis of the expression pattern of tm4sf2 using mrna in situ hybridization on mouse brain sections revealed that it is ubiquitously expressed early in brain development . il1rapl ( interleukin-1 [ il-1 ] receptor accessory protein - like ) has , as its name suggests , homology to il-1 receptor accessory protein . the function of the fmr2 gene , associated with mild intellectual disability gene , is also unknown : it encodes a nuclear protein that may regulate transcription and available data indicate that it functions at the cell surface . the il1rapl gene was identified by analyzing overlapping microdeletions in xp22 . 1 - 21.3 associated with nonspecific mr . using dna sequence from this region , nonoverlapping deletions encompassing the il1rapl gene were found and a point mutation in this gene was discovered segregating with mr in an unrelated family . the nonsense mutation introduces a premature stop codon that leads to a barely detectable level of il1rapl transcript . the expression pattern of il1rapl mrna on mouse brains is also consistent with a role in learning in memory , as it is present in the granular layer of the dentate gyrus and the pyramidal layer of the hippocampus . examples of autosomal single - gene defects resulting in intellectual disability are very rare . however , there is one good example of a four - generation family with a speech and language disorder that , remarkably , segregates as an autosomal dominant condition . the speech and language difficulties are part of a broader syndrome that includes a lower than average iq ; affected members also have a pronounced impairment in articulation . the gene has been mapped to the chromosomal region 7q , a region also implicated in studies of autism , a polygenic condition , one characteristic of which is abnormal speech development . molecular characterization of this unusual mendelian disorder could well provide new insights into the biology of language development . there are a small number of rare developmental disorders that result in intellectual disability and are thought to have a polygenic basis . among these , autism ( a condition marked by abnormal language and social development , together with obessional behavior ) is known to have an extremely high heritability ( over 90% ) . the difficulties besetting attempts to identify the predisposing loci are common to all attempts to dissect the genetic basis of complex , polygenic phenotypes , with different studies reporting different findings ( table ii ) . at present , there is some replicated evidence pointing to a locus on chromosomal region 7q . mapping the loci determining quantitative variation in iq there has been more success mapping the genes that influence a specific intellectual function , namely reading . a locus at 6p21.3 is one of the few replicated findings in behavioral genetics , with a number of studies reporting that the locus is relatively specific for reading disability . assuming that the approach does work and that localizations for polygenic variation in intellectual disability i are found , we are faced with the question of whether genes that determine variation overlap with the mutations described above . conceivably , the same pathways are involved , in which case the combination of mapping ) and molecular pathology screening would be ideally placed to identify the many genes that are responsible for intellectual disability .

the importance of genetic influences on cognitive disability has been recognized for a long time , but molecular analysis has only recently begun to yield insights into the pathogenesis of this common and disabling condition . the availability of genome sequences has enabled the characterization of the chromosomal deletions and trisomies that result in cognitive disability , and mutations in rare single - gene conditions are being discovered . the molecular pathology of cognitive disability is turning out to be as heterogeneous as the condition itself , with unexpected complexities even in apparently simple gene - deletion syndromes . one remarkable finding from studies on x - linked mental retardation is that mutations in different small guanosine triphosphate ( gtp)-binding proteins result in cognitive disability without other somatic features . advances are also being made in cognitive disability with polygenic origins , such as dyslexia and autism . however , the genetic basis of mild intellectual disability has yet to be satisfactorily explained .

The extent to which genes are involved Syndromic intellectual disability Mendelian disorders Segmental aneusomy syndromes Aneuploidy Nonsyndromic intellectual disability Polygenic effects on intellectual disability

the causes of cognitive disability vary with the severity of the condition : moderate - to - severe intellectual disability ( defined as an intelligence quotient [ iq ] score less than 50 ) is much more likely to be due to a single pathological cause ( genetic or environmental ) than mild mr ( defined as an iq score between 50 and 70 ) , which is often thought to be multifactorial in origin . genetic and environmental causes explain , in roughly equal proportions , about 30% of mild intellectual disability ; an etiological diagnosis is not obtained in the remaining 70% of cases . while controversy has long surrounded the extent to which genetic variation contributes to variation in intellectual function , there is now little doubt that moderate - to - severe intellectual disability is due primarily to chromosomal and genetic abnormalities . the largest individual contributors are down 's syndrome , chromosomal rearrangements , and x - linked mental retardation ( xlmr ) ( table i ) . small chromosomal rearrangements , affecting the ends ( telomeres ) of chromosomes have emerged as a common cause in cases until recently regarded as idiopathic , and it is likely that a considerable proportion of cases of unknown etiology will also be found to have a genetic origin . the importance of polygenic influences is inferred from the results of twin , family , and adoption studies for normal iq measures , and rarely from direct investigation of families with low iq ; studies evaluating biological and environmental risk factors in this group are singularly lacking , but there are indications that single - gene conditions and chromosomal abnormalities may be more frequent than previously assumed . table ii presents data on the genetic basis of conditions for which there is evidence that mutations give rise directly to intellectual disability . the table lists conditions where the genetic effects on intellectual function are thought to be relatively immediate , that is to say where no obvious developmental abnormality of the brain or progressive destruction of neuronal tissue results in cognitive impairment . when we consider the pathogenesis of intellectual disability , it is important to bear in mind that the phenotype involves multiple domains of intellectual functioning , often broadly divided into verbal and performance skills , but also encompassing capacities such as memory and attention , where performance is not traditionally seen as central to intellectual ability . information is lacking about genetic influences on the domains of both normal and abnormal intellectual functioning . studies of the heritability of intelligence , a measure of the extent to which genes contribute to the variation in intellectual functioning in the population , have mostly been carried out on overall measures of cognitive function , such as iq , although more recent work on speech and language development is beginning to indicate that genetic effects that have more specific influences can be identified . similarly , there have been few detailed psychometric investigations of people with intellectual disability due to a specific genetic lesion , so we do not know whether cognitive functioning is abnormal over all domains or whether there are discrete abnormalities . genetic mapping techniques and molecular cloning have made it possible to investigate disorders where the relationship between intellectual disability and genetic defect might be immediate . these are conditions where there are no noticeable alterations in brain structures and the cause of cognitive impairment is difficult to find . in syndromic mr , the phenotype includes additional physical abnormalities ( such as facial dysmorphism or minor abnormalities of the hands and feet ) , while in nonsyndromic mr the only abnormality is cognitive impairment . it might appear that genetic lesions are directly responsible for intellectual disability more commonly in nonsyndromic than in syndromic conditions , but it should be borne in mind that , without an understanding of the pathogenesis , this is only an assumption . for example , phenotypes vary considerably and mutations in the same gene may give rise to both syndromic and nonsyndromic intellectual disability : mutations in rsk2 give rise to coffin - lowry syndrome ( cls ) and to nonspecific intellectual disability , and mutations in different parts of the atrx gene produce either syndromic or nonsyndromic mr . nevertheless , some remarkable advances in x - linked nonsyndromic intellectual disability are uncovering genes that act directly on cognition , probably through central nervous system ( cns ) development . this is because x - linked recessive disease is compatible with the occurrence of affected members in multiple generations ; it is therefore both recognizable as an inherited condition and amenable to genetic mapping . x - linked intellectual disability ( ie , xlmr ) is common : the frequency is estimated to be 1.8 in 1000 males with a carrier frequency of 2.4 in 1000 females . the number of recognized conditions continues to increase : currently 210 have been described , 126 mapped , and 32 cloned . fragile x syndrome is the commonest form of xlmr , with a prevalence of approximately 1 in 5000 males and causes intellectual disability in about 1 in 8000 females . despite being one of the early triumphs of positional cloning , the function of fmr1 , and in particular how its deficiency gives rise to intellectual disability , is still not understood . in the normal brain , the fmr protein it can bind rna , including its own transcript , and it has been postulated that the fmr protein has a role in the machinery of translation and , as it shuttles between nucleus and cytoplasm , that it may be involved in mrna export . one explanation for the effect of the gene on brain function is that it plays a role in the maturation and pruning of dendritic spines during brain development . mutations in factors that regulate gene expression are emerging as an important genetic cause of intellectual disability . in the alpha - thalassemia x - linked mental retardation syndrome ( atrx ) , mutations in atrx give rise to characteristic developmental abnormalities including severe mr , facial dysmorphism , urogenital abnormalities , and alpha - thalassemia . investigation of a syndromic mr condition , cls , has led to the discovery of the involvement of another signaling pathway in cognitive impairment , namely the mapk - activated signaling pathway ( mapk for mitogen - activated protein kinase ) . cls is characterized by severe psychomotor retardation , facial and digital physical anomalies , and progressive skeletal deformation . remarkably , mutations in rsk2 give rise to nonsyndromic mr : patients in an xlmr family with neither facial , digital , nor skeletal anomalies compatible with cls , but with mild mr , have been found to have a mutation in exon 14 of the gene , resulting in a conservative amino acid change . a number of genetic conditions associated with intellectual disability have been found to be due to small chromosomal deletions or duplications ( typically less than 5 megabases ) and are known as segmental aneusomy syndromes ( sec tabic ii ) . the small size of some of the regions has enabled a search for dosage - sensitive genes . however , in order to prove that a deleted gene is indeed dosage - sensitive , it has been necessary to find families with point mutations in the gene that segregate with intellectual disability . this has been achieved with rubinstein - taybi syndrome ( characterized by abnormal craniofacial features , broad thumbs , and intellectual disability ) , which can arise from monosomy of a small region in 16pl3.3 . williams - beuren syndrome is a neurodevelopmental disorder characterized by congenital heart disease , infantile hypercalcemia , dysmorphic facial features , and cognitive disability . it is known that mutations affecting the elastin gene give rise to the supravalvular aortic stenosis , but there are still at least 15 candidate genes that could be involved in the unusual cognitive profile of the syndrome . these include a number of transcriptional regulators , such as williams ' syndrome transcription factor , which contains a plant homeodomain ( phd ) , limk1 , which contains one phd motif followed by a bromodomain , and the wbscr14/ws - bhlh gene , which encodes a basic - helix - loop - helix leucine zipper , characteristic of a subclass of transcription factors . these syndromes have characteristic and distinct neurobehavioral profiles : in as the retardation is severe ( very few affected individuals can talk ) and there is ataxia , seizures , abnormal eeg , microcephaly , facial dysmorphism , hyperactivity and paroxysmal laughter . despite the phenotypic differences , the basic defect is the same in the two disorders : a failure of parent - of - origin - specific gene expression . the chromosomal region is said to bear a parent - of - origin imprint , of which the molecular signature is a difference in dna methylation . however , there is now some evidence to suggest that abnormal rna editing , due to misregulation of guide rnas , mediates the defect in pws . however , it is now clear that the action of methylation guide snornas goes beyond the field of ribosome biogenesis . both dgs and vcfs are associated with intellectual disability ; additionally psychosis is found in some patients with vcfs . this is because x - linked recessive disease is compatible with the occurrence of affected members in multiple generations ; it is therefore both recognizable as an inherited condition and amenable to genetic mapping . x - linked intellectual disability ( ie , xlmr ) is common : the frequency is estimated to be 1.8 in 1000 males with a carrier frequency of 2.4 in 1000 females . fragile x syndrome is the commonest form of xlmr , with a prevalence of approximately 1 in 5000 males and causes intellectual disability in about 1 in 8000 females . despite being one of the early triumphs of positional cloning , the function of fmr1 , and in particular how its deficiency gives rise to intellectual disability , is still not understood . in the normal brain , the fmr protein it can bind rna , including its own transcript , and it has been postulated that the fmr protein has a role in the machinery of translation and , as it shuttles between nucleus and cytoplasm , that it may be involved in mrna export . one explanation for the effect of the gene on brain function is that it plays a role in the maturation and pruning of dendritic spines during brain development . mutations in factors that regulate gene expression are emerging as an important genetic cause of intellectual disability . in the alpha - thalassemia x - linked mental retardation syndrome ( atrx ) , mutations in atrx give rise to characteristic developmental abnormalities including severe mr , facial dysmorphism , urogenital abnormalities , and alpha - thalassemia . at a molecular level , the mutation has effects on the pattern of genomic methylation , consistent with the role of atrx in chromatin remodeling . the pleiotropic effects of mutations in mecp2 and atrx could result from the regulated expression of a restricted class of genes . investigation of a syndromic mr condition , cls , has led to the discovery of the involvement of another signaling pathway in cognitive impairment , namely the mapk - activated signaling pathway ( mapk for mitogen - activated protein kinase ) . the disorder was mapped by linkage to the region xp22.2 and mutations discovered in a positional candidate gene rsk2 ( also known as rps6ka3 ) . remarkably , mutations in rsk2 give rise to nonsyndromic mr : patients in an xlmr family with neither facial , digital , nor skeletal anomalies compatible with cls , but with mild mr , have been found to have a mutation in exon 14 of the gene , resulting in a conservative amino acid change . a number of genetic conditions associated with intellectual disability have been found to be due to small chromosomal deletions or duplications ( typically less than 5 megabases ) and are known as segmental aneusomy syndromes ( sec tabic ii ) . the small size of some of the regions has enabled a search for dosage - sensitive genes . however , in order to prove that a deleted gene is indeed dosage - sensitive , it has been necessary to find families with point mutations in the gene that segregate with intellectual disability . this has been achieved with rubinstein - taybi syndrome ( characterized by abnormal craniofacial features , broad thumbs , and intellectual disability ) , which can arise from monosomy of a small region in 16pl3.3 . williams - beuren syndrome is a neurodevelopmental disorder characterized by congenital heart disease , infantile hypercalcemia , dysmorphic facial features , and cognitive disability . it is known that mutations affecting the elastin gene give rise to the supravalvular aortic stenosis , but there are still at least 15 candidate genes that could be involved in the unusual cognitive profile of the syndrome . these include a number of transcriptional regulators , such as williams ' syndrome transcription factor , which contains a plant homeodomain ( phd ) , limk1 , which contains one phd motif followed by a bromodomain , and the wbscr14/ws - bhlh gene , which encodes a basic - helix - loop - helix leucine zipper , characteristic of a subclass of transcription factors . these syndromes have characteristic and distinct neurobehavioral profiles : in as the retardation is severe ( very few affected individuals can talk ) and there is ataxia , seizures , abnormal eeg , microcephaly , facial dysmorphism , hyperactivity and paroxysmal laughter . despite the phenotypic differences , the basic defect is the same in the two disorders : a failure of parent - of - origin - specific gene expression . the chromosomal region is said to bear a parent - of - origin imprint , of which the molecular signature is a difference in dna methylation . mutations in a ubiquitin protein ligase gene ( ube3a ) have been found in a few rare families with as . seven genes ( and candidate genes ) have been identified in the pws region , all of which appear to be brain specific . however , there is now some evidence to suggest that abnormal rna editing , due to misregulation of guide rnas , mediates the defect in pws . however , it is now clear that the action of methylation guide snornas goes beyond the field of ribosome biogenesis . unusually , they do not have appropriate antisense elements , so their function is not clear , but one has a similarity to the mrna encoded by the gene for the serotonin receptor-2c . because of the known involvement of serotonin in appetite control and cognition , this finding raises the intriguing possibility that the defect in pws involves a defect in serotonin neurotransmission . digeorge ( dgs ) , velocardiofacial ( vcfs ) , and conotruncal anomaly face ( ctaf ) syndromes are different phenotypic manifestations of deletions within 22q11 . both dgs and vcfs are associated with intellectual disability ; additionally psychosis is found in some patients with vcfs . candidate genes for some of the somatic features of turner syndrome have been proposed : shox / phog encodes a homeodomain protein that may explain the short stature , while rps4y encodes an isoform of a ribosomal small subunit protein . identification of genes for features other than short stature has been problematic . in work on down 's syndrome , attention has been focused on the region 21q22.2 as a potential site for dosage - sensitive genes that affect learning and behavior . on the basis of transgenic mouse experiments , the gene encodes a tyrosine / serine kinase expressed in developing neuroblasts and a human gene lies in the down 's syndrome - critical chromosomal region 21q22 . however , as with the segmental aneusomies , there is a proliferation of down 's syndrome critical region ( dscr ) genes ; as yet no definitive evidence of their role in intellectual disability has been provided . perhaps the most striking finding to emerge from the study of nonsyndromic xlmr is the discovery of mutations in genes affecting different components of the rho - signaling pathway ( table ii ) . ophn1 encodes a rho - gap protein ( gap for gtpase [ guanosine triphosphatase]-activating protein ) that stimulates the intrinsic gtpase activity of rho , rac , and cdc42 . arhgef6 encodes a small cytoplasmic protein , homologous to proteins that activate rho - gtpases by exchanging guanosine diphosphate ( gdp ) for guanosine triphosphate ( gtp ) . a third gene found to be mutated in xlmr families is pak3 pak3 may well be a downstream effector of the rho - gtpases rac and cdc42 putting the message forward to the actin cytoskeleton and to transcriptional activation . guanosine nucleotide dissociation inhibitor-1 ( gdi1 ) inhibits gdp dissociation from rab3a by binding to gdp - bound rab proteins and appears to be crucial in maintaining the balance between the gtp- and gdp - bound forms of rab3 . how might the biology of the small gtp - binding proteins explain human cognitive function ? whereas cdc42 and rac1 are involved in the formation of lamellipodia and filopodia , inhibition of rho , rac , and cdc42 also reduces dendrite formation . since rab3a - deficient mice have no fundamental deficits in synaptic vesicle exocytosis , the protein is not essential to the process , but is required to maintain a normal reserve of synaptic vesicles . one likely explanation is that the mutations only partly disrupt the brain system on which they operate , but it could also be that compensatory mechanisms , effective in other cell types , fail when it comes to neuronal processes involved in cognitive processing . some 30% to 65% of the affected children have learning difficulties , but only 4% to 8% have mr . analysis of the expression pattern of tm4sf2 using mrna in situ hybridization on mouse brain sections revealed that it is ubiquitously expressed early in brain development . the function of the fmr2 gene , associated with mild intellectual disability gene , is also unknown : it encodes a nuclear protein that may regulate transcription and available data indicate that it functions at the cell surface . examples of autosomal single - gene defects resulting in intellectual disability are very rare . the gene has been mapped to the chromosomal region 7q , a region also implicated in studies of autism , a polygenic condition , one characteristic of which is abnormal speech development . molecular characterization of this unusual mendelian disorder could well provide new insights into the biology of language development . there are a small number of rare developmental disorders that result in intellectual disability and are thought to have a polygenic basis . the difficulties besetting attempts to identify the predisposing loci are common to all attempts to dissect the genetic basis of complex , polygenic phenotypes , with different studies reporting different findings ( table ii ) . mapping the loci determining quantitative variation in iq there has been more success mapping the genes that influence a specific intellectual function , namely reading . a locus at 6p21.3 is one of the few replicated findings in behavioral genetics , with a number of studies reporting that the locus is relatively specific for reading disability . assuming that the approach does work and that localizations for polygenic variation in intellectual disability i are found , we are faced with the question of whether genes that determine variation overlap with the mutations described above . conceivably , the same pathways are involved , in which case the combination of mapping ) and molecular pathology screening would be ideally placed to identify the many genes that are responsible for intellectual disability .

dental implant treatment is a successful , widespread and predictable treatment for tooth loss over the past 20 years however , an increasing number of implant failures caused by peri - implant diseases still take part in every day clinical dental practice . two forms of peri - implant inflammation have been identified in the literature : peri - implant mucositis and peri - implantitis . the american academy of periodontology ( aap ) stated that from a clinical standpoint , signs that determine the presence of peri - implant mucositis include bleeding on probing ( bop ) and/or suppuration , which are usually associated with probing depths ( pds ) 4 mm and no evidence of radiographic loss of bone beyond bone remodelling . peri - implantitis is a progressive , irreversible disease of the bone and soft tissues around osseointegrated dental implants under masticatory function that is accompanied by bone resorption , reduced osseointegration , deep pocket formation and suppuration . despite divergences in the definition of peri - implantitis and the differential diagnosis of peri - implant diseases , studies have estimated that peri - implantitis affects approximately 10% of implants and 20% of patients . according to a recent systematic review peri - implant mucositis and peri - implantitis have a prevalence ranging from 19 to 65% and from 1 to 47% , respectively . on the other hand , another systematic review reported mean prevalence for peri - implant mucositis and peri - implantitis as 43% and 22% , respectively . the combination of clinical and radiographic parameters , such as pd , bop , suppuration , mobility and marginal bone loss , are the commonly used parameters for the diagnosis of peri - implantitis . however , these diagnostic processes might not be sensitive or specific enough to distinguish disease onset , development , and activity . clinical measurements around implants as like natural teeth might be challenged by the force and direction of probing , implant geometry , prosthesis design and peri - implant soft tissue biotype . in addition , both peri - implant mucositis and peri - implantitis lesions can present with bop and/or suppuration , with pds greater than 4 mm . therefore , clinicians and researchers may often observe the early , and sometimes the late diagnosis of peri - implantitis . early detection of peri - implant destruction , as well as monitoring progression of bone loss is extremely important . currently , blunt surrogate markers are being used such as radiographs and peri - implant probing . these tests have obvious limitations as only history of disease may be detected . as main markers of peri - implantitis are bone destruction and inflammation , biomarkers and enzymes in implant sulcus fluid ( pisf ) focusing on these disease entities are of interest . active components and mechanisms involved in the destructive process may thus be important perspectives within this field . such knowledge may potentially lead to new diagnostic strategies and candidate disease markers for peri - implant conditions . a biomarker is an indicator of a biological state and can help to distinguish between normal and pathologic processes . presently , radiographs and clinical parameters such as , pd , clinical attachment level and bop generally used for peri - implant condition diagnosis . research to look at associations between certain biomarkers with health and/or disease can give more tools to clinicians for better understanding the pathogenesis of such peri - implant diseases . one of the main advantages of evaluating biomarkers is the repeatability and non - invasive nature of obtaining samples for analysis . biomarkers can be measured in secretions such as saliva and gingival crevicular fluid , or in the case of implants , peri - implant crevicular fluid ( picf ) . since then , studies have been conducted to look at a vast array of biomarkers and enzymes around dental implants as an early sign of peri - implantitis . today , there is large variation for the threshold of diagnosis for peri - implantitis , which may explain the wide range of percentages reported for its prevalence . researchers and clinicians are always looking for adjunctive measures to aid in proper disease diagnosis , and the measurement of levels of enzymes and biomarkers is possible tool , and has gathered a lot of interest . therefore , the purpose of this article was to review the current understanding of the biomarkers and enzymes associated with peri - implant diseases and how their level changes took part in the pathogenesis of the disease . protocol and registration the methods of the analysis and inclusion criteria were specified in advance and documented in a protocol . the review was registered in prospero , an international prospective register of systematic reviews . the reporting of this systematic analysis adhered to the preferred reporting items for systematic review and meta - analyses ( prisma ) statement . which biomarkers and enzymes in picf are used for distinguish between healthy implants and implants having peri - implant diseases ? do patients with peri - implant diseases ( peri - implant mucositis or peri - implantitis ) present higher levels of biomarkers and enzymes in picf ? types of publications the review included studies on humans published in the english language . letters , editorials , case reports , literature reviews , animal research , phd theses , and abstracts were excluded . a search was conducted on the national library of medicine database ( medline ) through its online site ( pubmed ) and embase databases . additionally , a hand search was conducted in the following journals : implant dentistry , clinical oral implants research ; clinical implant dentistry and related research , european journal of oral implantology , international journal of oral & maxillofacial implants , journal of oral implantology , international journal of oral and maxillofacial surgery , international journal of oral and maxillofacial surgery , journal of periodontology , journal of clinical periodontology , international journal of periodontics and restorative dentistry , journal of prosthetic dentistry , international journal of endodontics , journal of endodontics , oral surgery , oral medicine , oral pathology and oral radiology , endodontology and turkish journal of medical sciences . an organized and logical approach was used to categorize the studies dealing with the association between picf biomarkers and peri - implant diseases . the keywords and search inquiries used during the primary stage were as follows : peri implant crevicular fluid or peri - implant crevicular fluid and peri implant sulcus fluid and peri - implant sulcus fluid and peri - implantitis or peri implantitis and peri - implant inflammation or peri implant inflammation and peri - implant infection or peri - implant infection and peri - implant mucositis or peri implant mucositis and implant biomarkers and implant enzymes . the choice of keywords was intended to be broad to collect as much relevant data as possible without relying on electronic means alone to refine the search results . after advance search ; the studies dealing with peri - implantitis or peri - implant mucositis and picf biomarker or enzyme level analysis were included in the present review . based on the inclusion criteria , the authors independently screened titles and abstracts derived from the literature search ( figure 1 ) . both reviewers compared decisions and their eligibility for this review was confirmed after discussion . full articles were obtained for all the studies considered eligible for inclusion in this paper and further evaluated by both reviewers . types of publications the present review included only human studies published in the english language . letters , editorials , case reports , literature reviews , animal research , phd theses , and abstracts were excluded . the present review included all human prospective , follow - up studies , clinical trials , cohort studies , case - control studies , case series studies , published between january 1 , 1996 and march 1 , 2016 , were searched that reported on biomarkers and enzyme levels obtained by picf and/or pisf analysis . types of participants / population individuals included in the studies should have had at least one osseointegrated screw - type dental implant that presented with clinical or radiologic signs of peri - implant mucositis or peri - implantitis and subjected to picf biomarker analysis . inclusion and exclusion criteria for this systematic review , original cross - sectional and longitudinal prospective clinical studies with collection of different biomarkers and enzymes in picf from individuals with peri - implantitis ( p ) or peri - implant mucositis ( m ) were selected . letters , editorials , case reports , literature reviews , animal research , phd theses , and abstracts were excluded . other exclusion criteria were as follows : 1 ) studies with quantification of biomarkers and enzymes in tissue biopsies , serum , saliva and other biologic sources ; 2 ) assessment of only fluid volume but not biomarker and enzyme levels ; 3 ) fluid collection and analyses for determining the effect of different implant designs not inflammation ; 4 ) fluid collection during early osseointegration ; 5 ) focus on gingival distances ; 6 ) unreported implant inflammation criteria ; 7 ) not clear information about patient / implant groups whether healthy or peri - implantitis ; 8) studies assessed different biomarker genotypes . assessment of methodological quality the quality of all selected trials was assessed using the recommended approach for assessing risk of bias in studies included in cochrane reviews . evaluated parameters are : ( 1 ) random sequence generation , ( 2 ) allocation concealment , ( 3 ) blinding of participants and personnel , ( 4 ) blinding of outcome assessment , ( 5 ) incomplete outcome data , ( 6 ) selective reporting , and ( 7 ) other bias . the potential risk of bias was categorized as low , unclear or high significant heterogeneity between publications in terms of diseases definitions , assessed parameters , study designs , as well as measured outcomes , among others , prevented the quantitative synthesis of the included studies and consequently a meta - analysis could not be completed . instead , a qualitative descriptive analysis of the reported outcomes was performed and systematically reviewed in forms of tables . study selection and search results two reviewers based on the inclusion criteria independently screened titles derived from this comprehensive search . the reviewers compared decisions and resolved differences through discussion , consulting a third party when consensus could not be reached . full reports were obtained for all the studies judged eligible for inclusion in this paper . at the title and abstract stage , one reviewer accepted the citations that appeared to meet the inclusion criteria and sent them on for full - text review , with a second reviewer assessing only those citations the first reviewer believed ineligible . after title screening and abstract reading , 1267 studies were excluded because they did not fit the inclusion criteria . the participants of all studies selected were in good general health and had not received any medication ( e.g. antibiotics , and/or anti - inflammatory and/or immunosuppressive agents that could affect the peri - implantitis biologic process at the time of picf sampling . the most prevalent study design was cross - sectional ( n = 32 ) followed by interventional ( n = 5 ) . a great variability in picf collection and biomarker or enzyme assessment was observed , and many different biomarkers and enzymes have been reported across the studies . the participants of all included studies were in general health and had not received any medication that may affect the peri - implant disease process . a wide range of biomarkers and enzymes used to explore an association between studied marker and peri - implant diseases . assessed biomarkers and enzymes in picf for peri - implant diseases cs = cross - sectional ; int = interventional ; cc = case - control ; rct = randomized clinical trial ; picf = peri - implant crevicular fluid ; pisf = peri - implant sulcus fluid ; il = interleukin ; p = peri - implantitis ; m = mucositis ; h = healthy ; tnf = tumour necrosis factor ; vegf = vascular endothelial growth factor ; mmp = matrix metallo proteinase ; timp = tissue inhibitor of matrix metallo proteinase ; opg = osteoprotegerin ; pth = parathyroid hormone ; oc = osteocalcin ; opn = osteopontin ; tgf = transforming growth factor ; rankl = receptor activator of nuclear factor kappa b ligand ; pge = prostoglandine ; mip-1alpha = macrophage inflammatory protein-1alpha ; pai-2 = plasminogen activator inhibitor type 2 ; pdgf = platelet derived growth factor ; ifn = interferone ; mpo = myeloperoxidase ; no = nitricoxide ; ictp = c - telopeptide pyridinoline crosslinks of type i collagen ; ast = aspartat amino transferase ; pd = probing depth ; gi = gingival index ; pi = plaque index ; mbi = modified bleeding index ; mpi = modified plaque index . after full text reading , the 10 studies were excluded due to following reasons : 1 ) focus on the comparison of picf interleukin-1 beta ( il-1 ) and plasma tumor necrosis factor - alpha ( tnf- ) levels between systemically healthy and diabetic subjects , 2 ) directly compare the biomarkers and enzymes of picf around implants with gingival crevicular fluid ( gcf ) around natural teeth [ 11 - 15 ] , 3 ) cytokine evaluation with polymerase chain reaction instead of biomarker levels [ 16 - 18 ] , 4 ) sample composed of only failing implants instead of peri - implant diseases . the majority of the studies used the following criteria to define the peri - implantitis : at least one peri - implant site with pd > 4 mm with clinical signs of inflammation ( bop , suppuration and bone loss ) . health implants two studies included smokers and match the number of smokers in healthy and peri - implantitis groups . most articles reported the subject numbers as well as the implants evaluated in the studies however , some of the studies reported only the number of patients and some studies reported only the number of implant sites . of the included biomarkers and enzymes in picf , il-1 was the most studied parameter ( 19 studies ) followed by tnf- ( 11 studies ) , il-6 ( 11 studies ) and il-8 ( 6 studies ) . five studies reported oxidative stress parameters associated with peri - implant inflammation and 6 studies reported matrix metallo proteinase ( mmp ) levels . twenty studies compared the results between p and h sites and most studies evaluated healthy and diseased implants from different patients . results of individual studies one interventional study evaluated the levels of il-6 , opg , osteocalcin ( oc ) , leptin , osteopontin ( opn ) , parathyroid hormone ( pth ) , tnf- , adiponectin and insulin levels after surgical treatment of peri - implantitis . they reported no change according to the levels of oc , opn , pth , tnf- and insulin levels and significant reduction according to total protein , mmp-8 , il-6 , opg , leptin and adiponectin levels after surgical treatment . another interventional study also reported total amount of tnf- was significantly reduced at 3 and 12 months after therapy ( open flap debridement ) compared to baseline associated with improvements in clinical parameters . another interventional study exposed healthy implants to de novo plaque accumulation and no significant changes observed in the total amount of tnf- , il-1 and tgf-2 compared to baseline in picf . another study included three groups ( h , m , p ) of implants and reported levels of tnf- was significantly higher in p and m , tnf- levels of diseased implants decreased from baseline to three months after therapies , no differences among groups for il-4 , il-10 , il-12 and the osteoprotegerin ( opg ) and receptor activator of nfkb ligand ( rankl ) ratio was higher for healthy implants than for untreated peri - implantitis . another interventional study reported no difference between peri - implant health and disease condition according to il-1 and pge2 levels . assessment of methodological quality the results of risk of bias assessment for included studies were summarized in table 2 . risk of bias within the included studies + = low risk ; ? = unclear risk ; - = high risk . the current evidence according to the picf levels of biomarkers and enzymes that used to distinguish between healthy and inflamed implant sites and their diagnostic and prognostic potential for prediction of future peri - implantitis was assessed and results from 41 original were explored in the present review . it is obvious that a wide range of biomarkers and enzymes are reported to be involved in peri - implant inflammation ( table 1 ) . the picf levels of 13 different cytokines ( il-1 , il-2 , il-4 , il-5 , il-6 , il-7 , il-8 , il-10 , il-12 , il-17 , ifn- , pge-2 and tnf- ) have been compared in different clinical peri - implant conditions . mmps are endopeptidases capable of degrading various extracellular matrix proteins and play a role in cell proliferation , differentiation , migration , and apoptosis . peri - implantitis has been shown to demonstrate a similar pattern of destruction as periodontitis , and mmp upregulation has been associated with irreversible peri - implant connective tissue destruction . six studies assessed collagenases in picf in different peri - implantitis lesions which are important around peri - implant tissues , are mmp-1 , mmp-3 , mmp-8 , and mmp-13 and tissue inhibitors [ 21,29 - 33 ] . and according to all included studies , mmps were reported to be positively correlated with clinical inflammatory conditions around implants [ 21,29 - 33 ] . il-1 and tnf- are the two most targeted biomarkers among the majority of included studies and take part in osteoclast formation and bone resorption [ 6,34 - 36 ] . il-1 regulates the degradation of extracellular matrix components of the plasminogen system and the collagenase activity in inflammation and wound healing . it has been shown that inhibition of il-1 reduced tissue breakdown and the progression of tissue inflammation . tnf- induces fibroblast apoptosis and reduction of the repair capacity of the peri - implant tissue , but mechanical therapy seems to revert this situation . of the 19 studies assessed il-1 , 5 of them showed no statistically significant differences between healthy and diseased groups [ 20,27,38 - 40 ] . thirteen studies showed higher levels of il-1 in picf than healthy implant sites [ 7,21,22,30,35,36,41 - 47 ] . of the 10 studies assessed tnf- , 3 of them showed no relationship with this cytokine with peri - implant inflammation , while other 7 studies showed significant relationship with this cytokine [ 23,24,36,41,46 - 48 ] . these findings suggest that pro - inflammatory cytokines such as il-1 and tnf- are up to date , the most promising proteins to be used as markers in picf for differentiation between peri - implantitis and healthy implants . although ils are the most of interest in picf analyses followed by mmps , there are other biomarkers and enzymes that their levels were evaluated to reflect the local inflammatory condition of implants . myeloperoxidase ( mpo ) is an antimicrobial leukocyte - derived enzyme found in high concentrations in the primary granules of leukocytes that catalyzes the formation of a number of reactive oxidant species . three studies reported significantly higher amounts of mpo in pisf collected around implants with inflammatory lesions [ 50 - 52 ] . in the peri - implant region as well as the natural dentition , it has also been demonstrated that no metabolism is closely related to the status and degree of peri - implant inflammation . elastase is a major enzyme released from human leukocytes and contributes to tissue damage during inflammation , significantly higher amounts of alkaline phosphatase and elastase were found in pisf around implants with peri - implantitis compared with healthy controls . prostaglandin e2 ( pge2 ) is a vasodilator that increases vascular permeability at sites of inflammation and also has a role in bone resorption . one study reported that pge2 showed positive correlations with gingival index and pd , 2 studies reported no difference between peri - implant health and disease condition according to picf levels . another enzyme associated with bone resorption is cathepsin - k , which is a protease that is released during the inflammatory process after tissue injury . showed that cathepsin - k activity was positively correlated with the volume of pisf where there was also inflammatory bone loss , indicating it could be a biomarker used to predict or assess peri - implant alveolar bone loss . additional biomarkers for peri - implant bone loss have also been studied for peri - implant diseases . rankl normally binds to rank , which is originate on the surfaces of osteoclast precursors as well as mature osteoclasts , and this binding is necessary for their formation , function , and existence . opg acts as a trap receptor for rankl , and its binding prevents rankl binding to rank , thereby inhibiting the differentiation of an osteoclast precursor into a mature osteoclast . reported that srankl concentrations , opg total amounts , and opg concentrations were significantly lower in peri - implantitis group when compared to healthy group . in contrast , rankl , rank , and opg concentrations were found to be significantly higher in peri - implantitis sites compared with healthy implant sites , but the ratio of opg / rankl was not different . it was also demonstrated that the opg / rankl ratio improved with mechanical treatment of peri - implantitis sites . osteocalcin is a 5.4-kda calcium - binding protein of bone and the most abundant non - collagenous protein of the mineralized bone tissue . one study reported higher osteocalcin ( oc ) levels in picf as possible biomarker to define the inflammatory conditions around implants whereas two studies reported conflicting results for picf oc levels . first limitation of the present systematic review is the wide range of different definitions regarding peri - implant mucositis and peri - implantitis that were employed in the included investigations . another limitation is a lack of information on whether the inflammatory markers in picf were matched for the clinical parameters of the respective collection sites , such as pd , clinical attachment loss , and bop . this is a vital point due to a strong link between picf biomarker levels and severity and extent of local inflammatory disease . the majority of studies reported the mean clinical parameters of all implant sites or did not clarify whether the clinical parameters presented were related to all implant sites or to sites selected for picf sampling . due to a cyclic progression of peri - implant diseases , the immune - inflammatory event biomarkers responsible for tissue breakdown may not always be active in cross - sectional studies with a single moment of fluid collection . for better understanding of the immune inflammatory peri - implant diseases and for developing host - modulation therapies , biomarkers plays a crucial role to aid clinicians to elucidate the complex biologic process involved . based on this systematic review , it was concluded that inflammatory mediators , such as interleukin-1 beta and plasma tumor necrosis factor - alpha , in crevicular fluid collected from peri - implant pockets are the most used biomarkers to assist in the early diagnosis of peri - implantitis . it is suggested that studies should be conducted to establish a standardized method to diagnose and classify the peri - implant diseases . standardized investigations should be performed based on the criteria of subject selection , peri - implantitis diagnosis , as well as peri - implant crevicular fluid sampling method ( e.g. number and severity of sampling sites , sampling time ) , sample handling and detection sensitivity / specificity of the used assay .

abstractobjectivesto review the current understanding of the biomarkers and enzymes associated with different forms peri - implant diseases and how their level changes influence the pathogenesis of the inflammatory diseases around dental implants.material and methodsan electronic search in two different databases was performed including medline ( pubmed ) and embase between 1996 to 2016 . human studies analyse peri - implant crevicular fluid ( picf ) biomarker and enzyme levels of implants having peri - implant mucositis and peri - implantitis published in english language , were evaluated . a systematic review was performed to assess which biomarkers and enzymes in picf were used to identify the inflammatory conditions around dental implants.resultsfifty-one articles were identified of which 41 were further evaluated and included in the analysis . due to significant heterogeneity between included studies , a meta - analysis could not be performed . instead , a systematic descriptive review was performed.conclusionsbiomarkers and enzymes in peri - implant crevicular fluid have shown promising results in differentiating from peri - implant disease condition to health . however , due to inconsistent results and acquiring much evidence from cross - sectional studies , additional evidence supported by randomized - controlled trials is needed to validate the links reported .

INTRODUCTION MATERIAL AND METHODS RESULTS DISCUSSION CONCLUSIONS ACKNOWLEDGMENTS AND DISCLOSURE STATEMENTS

dental implant treatment is a successful , widespread and predictable treatment for tooth loss over the past 20 years however , an increasing number of implant failures caused by peri - implant diseases still take part in every day clinical dental practice . two forms of peri - implant inflammation have been identified in the literature : peri - implant mucositis and peri - implantitis . the american academy of periodontology ( aap ) stated that from a clinical standpoint , signs that determine the presence of peri - implant mucositis include bleeding on probing ( bop ) and/or suppuration , which are usually associated with probing depths ( pds ) 4 mm and no evidence of radiographic loss of bone beyond bone remodelling . peri - implantitis is a progressive , irreversible disease of the bone and soft tissues around osseointegrated dental implants under masticatory function that is accompanied by bone resorption , reduced osseointegration , deep pocket formation and suppuration . despite divergences in the definition of peri - implantitis and the differential diagnosis of peri - implant diseases , studies have estimated that peri - implantitis affects approximately 10% of implants and 20% of patients . according to a recent systematic review peri - implant mucositis and peri - implantitis have a prevalence ranging from 19 to 65% and from 1 to 47% , respectively . on the other hand , another systematic review reported mean prevalence for peri - implant mucositis and peri - implantitis as 43% and 22% , respectively . the combination of clinical and radiographic parameters , such as pd , bop , suppuration , mobility and marginal bone loss , are the commonly used parameters for the diagnosis of peri - implantitis . however , these diagnostic processes might not be sensitive or specific enough to distinguish disease onset , development , and activity . clinical measurements around implants as like natural teeth might be challenged by the force and direction of probing , implant geometry , prosthesis design and peri - implant soft tissue biotype . in addition , both peri - implant mucositis and peri - implantitis lesions can present with bop and/or suppuration , with pds greater than 4 mm . therefore , clinicians and researchers may often observe the early , and sometimes the late diagnosis of peri - implantitis . early detection of peri - implant destruction , as well as monitoring progression of bone loss is extremely important . currently , blunt surrogate markers are being used such as radiographs and peri - implant probing . as main markers of peri - implantitis are bone destruction and inflammation , biomarkers and enzymes in implant sulcus fluid ( pisf ) focusing on these disease entities are of interest . active components and mechanisms involved in the destructive process may thus be important perspectives within this field . such knowledge may potentially lead to new diagnostic strategies and candidate disease markers for peri - implant conditions . presently , radiographs and clinical parameters such as , pd , clinical attachment level and bop generally used for peri - implant condition diagnosis . research to look at associations between certain biomarkers with health and/or disease can give more tools to clinicians for better understanding the pathogenesis of such peri - implant diseases . biomarkers can be measured in secretions such as saliva and gingival crevicular fluid , or in the case of implants , peri - implant crevicular fluid ( picf ) . since then , studies have been conducted to look at a vast array of biomarkers and enzymes around dental implants as an early sign of peri - implantitis . today , there is large variation for the threshold of diagnosis for peri - implantitis , which may explain the wide range of percentages reported for its prevalence . therefore , the purpose of this article was to review the current understanding of the biomarkers and enzymes associated with peri - implant diseases and how their level changes took part in the pathogenesis of the disease . protocol and registration the methods of the analysis and inclusion criteria were specified in advance and documented in a protocol . the reporting of this systematic analysis adhered to the preferred reporting items for systematic review and meta - analyses ( prisma ) statement . which biomarkers and enzymes in picf are used for distinguish between healthy implants and implants having peri - implant diseases ? do patients with peri - implant diseases ( peri - implant mucositis or peri - implantitis ) present higher levels of biomarkers and enzymes in picf ? types of publications the review included studies on humans published in the english language . a search was conducted on the national library of medicine database ( medline ) through its online site ( pubmed ) and embase databases . additionally , a hand search was conducted in the following journals : implant dentistry , clinical oral implants research ; clinical implant dentistry and related research , european journal of oral implantology , international journal of oral & maxillofacial implants , journal of oral implantology , international journal of oral and maxillofacial surgery , international journal of oral and maxillofacial surgery , journal of periodontology , journal of clinical periodontology , international journal of periodontics and restorative dentistry , journal of prosthetic dentistry , international journal of endodontics , journal of endodontics , oral surgery , oral medicine , oral pathology and oral radiology , endodontology and turkish journal of medical sciences . an organized and logical approach was used to categorize the studies dealing with the association between picf biomarkers and peri - implant diseases . the keywords and search inquiries used during the primary stage were as follows : peri implant crevicular fluid or peri - implant crevicular fluid and peri implant sulcus fluid and peri - implant sulcus fluid and peri - implantitis or peri implantitis and peri - implant inflammation or peri implant inflammation and peri - implant infection or peri - implant infection and peri - implant mucositis or peri implant mucositis and implant biomarkers and implant enzymes . after advance search ; the studies dealing with peri - implantitis or peri - implant mucositis and picf biomarker or enzyme level analysis were included in the present review . full articles were obtained for all the studies considered eligible for inclusion in this paper and further evaluated by both reviewers . types of publications the present review included only human studies published in the english language . the present review included all human prospective , follow - up studies , clinical trials , cohort studies , case - control studies , case series studies , published between january 1 , 1996 and march 1 , 2016 , were searched that reported on biomarkers and enzyme levels obtained by picf and/or pisf analysis . types of participants / population individuals included in the studies should have had at least one osseointegrated screw - type dental implant that presented with clinical or radiologic signs of peri - implant mucositis or peri - implantitis and subjected to picf biomarker analysis . inclusion and exclusion criteria for this systematic review , original cross - sectional and longitudinal prospective clinical studies with collection of different biomarkers and enzymes in picf from individuals with peri - implantitis ( p ) or peri - implant mucositis ( m ) were selected . other exclusion criteria were as follows : 1 ) studies with quantification of biomarkers and enzymes in tissue biopsies , serum , saliva and other biologic sources ; 2 ) assessment of only fluid volume but not biomarker and enzyme levels ; 3 ) fluid collection and analyses for determining the effect of different implant designs not inflammation ; 4 ) fluid collection during early osseointegration ; 5 ) focus on gingival distances ; 6 ) unreported implant inflammation criteria ; 7 ) not clear information about patient / implant groups whether healthy or peri - implantitis ; 8) studies assessed different biomarker genotypes . assessment of methodological quality the quality of all selected trials was assessed using the recommended approach for assessing risk of bias in studies included in cochrane reviews . the potential risk of bias was categorized as low , unclear or high significant heterogeneity between publications in terms of diseases definitions , assessed parameters , study designs , as well as measured outcomes , among others , prevented the quantitative synthesis of the included studies and consequently a meta - analysis could not be completed . instead , a qualitative descriptive analysis of the reported outcomes was performed and systematically reviewed in forms of tables . the reviewers compared decisions and resolved differences through discussion , consulting a third party when consensus could not be reached . antibiotics , and/or anti - inflammatory and/or immunosuppressive agents that could affect the peri - implantitis biologic process at the time of picf sampling . the most prevalent study design was cross - sectional ( n = 32 ) followed by interventional ( n = 5 ) . a great variability in picf collection and biomarker or enzyme assessment was observed , and many different biomarkers and enzymes have been reported across the studies . the participants of all included studies were in general health and had not received any medication that may affect the peri - implant disease process . a wide range of biomarkers and enzymes used to explore an association between studied marker and peri - implant diseases . assessed biomarkers and enzymes in picf for peri - implant diseases cs = cross - sectional ; int = interventional ; cc = case - control ; rct = randomized clinical trial ; picf = peri - implant crevicular fluid ; pisf = peri - implant sulcus fluid ; il = interleukin ; p = peri - implantitis ; m = mucositis ; h = healthy ; tnf = tumour necrosis factor ; vegf = vascular endothelial growth factor ; mmp = matrix metallo proteinase ; timp = tissue inhibitor of matrix metallo proteinase ; opg = osteoprotegerin ; pth = parathyroid hormone ; oc = osteocalcin ; opn = osteopontin ; tgf = transforming growth factor ; rankl = receptor activator of nuclear factor kappa b ligand ; pge = prostoglandine ; mip-1alpha = macrophage inflammatory protein-1alpha ; pai-2 = plasminogen activator inhibitor type 2 ; pdgf = platelet derived growth factor ; ifn = interferone ; mpo = myeloperoxidase ; no = nitricoxide ; ictp = c - telopeptide pyridinoline crosslinks of type i collagen ; ast = aspartat amino transferase ; pd = probing depth ; gi = gingival index ; pi = plaque index ; mbi = modified bleeding index ; mpi = modified plaque index . after full text reading , the 10 studies were excluded due to following reasons : 1 ) focus on the comparison of picf interleukin-1 beta ( il-1 ) and plasma tumor necrosis factor - alpha ( tnf- ) levels between systemically healthy and diabetic subjects , 2 ) directly compare the biomarkers and enzymes of picf around implants with gingival crevicular fluid ( gcf ) around natural teeth [ 11 - 15 ] , 3 ) cytokine evaluation with polymerase chain reaction instead of biomarker levels [ 16 - 18 ] , 4 ) sample composed of only failing implants instead of peri - implant diseases . the majority of the studies used the following criteria to define the peri - implantitis : at least one peri - implant site with pd > 4 mm with clinical signs of inflammation ( bop , suppuration and bone loss ) . health implants two studies included smokers and match the number of smokers in healthy and peri - implantitis groups . most articles reported the subject numbers as well as the implants evaluated in the studies however , some of the studies reported only the number of patients and some studies reported only the number of implant sites . of the included biomarkers and enzymes in picf , il-1 was the most studied parameter ( 19 studies ) followed by tnf- ( 11 studies ) , il-6 ( 11 studies ) and il-8 ( 6 studies ) . five studies reported oxidative stress parameters associated with peri - implant inflammation and 6 studies reported matrix metallo proteinase ( mmp ) levels . results of individual studies one interventional study evaluated the levels of il-6 , opg , osteocalcin ( oc ) , leptin , osteopontin ( opn ) , parathyroid hormone ( pth ) , tnf- , adiponectin and insulin levels after surgical treatment of peri - implantitis . they reported no change according to the levels of oc , opn , pth , tnf- and insulin levels and significant reduction according to total protein , mmp-8 , il-6 , opg , leptin and adiponectin levels after surgical treatment . another interventional study exposed healthy implants to de novo plaque accumulation and no significant changes observed in the total amount of tnf- , il-1 and tgf-2 compared to baseline in picf . another study included three groups ( h , m , p ) of implants and reported levels of tnf- was significantly higher in p and m , tnf- levels of diseased implants decreased from baseline to three months after therapies , no differences among groups for il-4 , il-10 , il-12 and the osteoprotegerin ( opg ) and receptor activator of nfkb ligand ( rankl ) ratio was higher for healthy implants than for untreated peri - implantitis . another interventional study reported no difference between peri - implant health and disease condition according to il-1 and pge2 levels . risk of bias within the included studies + = low risk ; ? the current evidence according to the picf levels of biomarkers and enzymes that used to distinguish between healthy and inflamed implant sites and their diagnostic and prognostic potential for prediction of future peri - implantitis was assessed and results from 41 original were explored in the present review . it is obvious that a wide range of biomarkers and enzymes are reported to be involved in peri - implant inflammation ( table 1 ) . the picf levels of 13 different cytokines ( il-1 , il-2 , il-4 , il-5 , il-6 , il-7 , il-8 , il-10 , il-12 , il-17 , ifn- , pge-2 and tnf- ) have been compared in different clinical peri - implant conditions . peri - implantitis has been shown to demonstrate a similar pattern of destruction as periodontitis , and mmp upregulation has been associated with irreversible peri - implant connective tissue destruction . six studies assessed collagenases in picf in different peri - implantitis lesions which are important around peri - implant tissues , are mmp-1 , mmp-3 , mmp-8 , and mmp-13 and tissue inhibitors [ 21,29 - 33 ] . and according to all included studies , mmps were reported to be positively correlated with clinical inflammatory conditions around implants [ 21,29 - 33 ] . tnf- induces fibroblast apoptosis and reduction of the repair capacity of the peri - implant tissue , but mechanical therapy seems to revert this situation . thirteen studies showed higher levels of il-1 in picf than healthy implant sites [ 7,21,22,30,35,36,41 - 47 ] . of the 10 studies assessed tnf- , 3 of them showed no relationship with this cytokine with peri - implant inflammation , while other 7 studies showed significant relationship with this cytokine [ 23,24,36,41,46 - 48 ] . these findings suggest that pro - inflammatory cytokines such as il-1 and tnf- are up to date , the most promising proteins to be used as markers in picf for differentiation between peri - implantitis and healthy implants . although ils are the most of interest in picf analyses followed by mmps , there are other biomarkers and enzymes that their levels were evaluated to reflect the local inflammatory condition of implants . myeloperoxidase ( mpo ) is an antimicrobial leukocyte - derived enzyme found in high concentrations in the primary granules of leukocytes that catalyzes the formation of a number of reactive oxidant species . in the peri - implant region as well as the natural dentition , it has also been demonstrated that no metabolism is closely related to the status and degree of peri - implant inflammation . elastase is a major enzyme released from human leukocytes and contributes to tissue damage during inflammation , significantly higher amounts of alkaline phosphatase and elastase were found in pisf around implants with peri - implantitis compared with healthy controls . one study reported that pge2 showed positive correlations with gingival index and pd , 2 studies reported no difference between peri - implant health and disease condition according to picf levels . another enzyme associated with bone resorption is cathepsin - k , which is a protease that is released during the inflammatory process after tissue injury . showed that cathepsin - k activity was positively correlated with the volume of pisf where there was also inflammatory bone loss , indicating it could be a biomarker used to predict or assess peri - implant alveolar bone loss . additional biomarkers for peri - implant bone loss have also been studied for peri - implant diseases . reported that srankl concentrations , opg total amounts , and opg concentrations were significantly lower in peri - implantitis group when compared to healthy group . in contrast , rankl , rank , and opg concentrations were found to be significantly higher in peri - implantitis sites compared with healthy implant sites , but the ratio of opg / rankl was not different . it was also demonstrated that the opg / rankl ratio improved with mechanical treatment of peri - implantitis sites . one study reported higher osteocalcin ( oc ) levels in picf as possible biomarker to define the inflammatory conditions around implants whereas two studies reported conflicting results for picf oc levels . first limitation of the present systematic review is the wide range of different definitions regarding peri - implant mucositis and peri - implantitis that were employed in the included investigations . another limitation is a lack of information on whether the inflammatory markers in picf were matched for the clinical parameters of the respective collection sites , such as pd , clinical attachment loss , and bop . due to a cyclic progression of peri - implant diseases , the immune - inflammatory event biomarkers responsible for tissue breakdown may not always be active in cross - sectional studies with a single moment of fluid collection . for better understanding of the immune inflammatory peri - implant diseases and for developing host - modulation therapies , biomarkers plays a crucial role to aid clinicians to elucidate the complex biologic process involved . based on this systematic review , it was concluded that inflammatory mediators , such as interleukin-1 beta and plasma tumor necrosis factor - alpha , in crevicular fluid collected from peri - implant pockets are the most used biomarkers to assist in the early diagnosis of peri - implantitis . it is suggested that studies should be conducted to establish a standardized method to diagnose and classify the peri - implant diseases . standardized investigations should be performed based on the criteria of subject selection , peri - implantitis diagnosis , as well as peri - implant crevicular fluid sampling method ( e.g.

molecular tools such as the recently introduced method of massively parallel sequencing ( deep sequencing ) [ 1 , 2 ] greatly facilitate the study of complex bacterial communities and provide deep insights into their compositions [ 35 ] . combined with the technique of barcoded pcr amplicons , deep sequencing methods are able to process many samples at a relatively low cost per sequence [ 6 , 7 ] . deep sequencing is , therefore , a promising tool for examining the influence of nutritional and other factors on intestinal microbial communities and functionalities . however , as with any new technology , pitfalls exist . for barcoded pcr amplicon sequencing studies , nucleic acids must be extracted and the resulting dna extract should ideally represent the entire bacterial diversity in a given habitat . furthermore , barcoding requires a pcr step , which depends on primers that should ideally cover the complete bacterial diversity . finally , the evaluation of sequence reads is based on databases , most of which are not yet suited for massive sequence inputs and sequence quality is often found to be suboptimal [ 9 , 10 ] . in regard to dna extraction from complex samples , a multitude of studies have reported that any given nucleic acid extraction method is biased towards certain bacterial groups [ 1113 ] . complex samples such as environmental samples from soil , waste treatment , or the gastrointestinal tract harbour not only diverse microbial communities , but also other components including mixtures of different carbohydrates , proteins , or minerals . bacteria can adhere to these compounds and are , thus , more difficult to extract than from culture media . additionally , substances that are chemically related to nucleic acids such as polyphenolic substances ( humic acids and certain components of dietary fibre ) can be coextracted and act as powerful pcr inhibitors . gram - positive cell walls are generally more rigid than gram - negative cell walls , and the extraction of bacterial dna itself , therefore , becomes a balance between efficient cell lysis and the destruction of dna from already lysed cells . the most efficient rupturing of bacterial cell walls seems to be achieved by bead beating , although commercial kits such as the qiagen stool amp kit yield high amounts of stool dna without bead beating . the correct choice of primer binding site is naturally of primary interest for any pcr based study . for barcoded pcr amplicons , dna must be amplified in order to sequence multiple samples in a single pyrosequencing run . the hypervariable regions of the bacterial 16s rrna gene are generally the targets of choice , as the 16s rrna gene is a valuable phylogenetic marker but also has the advantage of being the most sequenced bacterial gene ; that is , sequences from pyrosequencing studies can be assigned against a large collection of reference sequences . however , it has been shown that there is no universal primer set that covers all known bacterial 16s rrna genes [ 1517 ] . the aim of this study was , therefore , to evaluate the impact of dna extraction , primer sets , and automated data evaluation on final results . the study was approved by the local state office of health and social affairs landesamt fr gesundheit und soziales , berlin ( lageso reg . a total of 12 ileal samples from 40- to 42-day - old piglets fed a standard starter diet supplemented with 200 or 3000 mg g zno ( n = 6 per group ) , respectively , were used for this study . subsequent to the euthanasia of the piglets , the gastrointestinal tracts were opened immediately , and the contents of the ileum were removed and stored at 80c . total nucleic acids were extracted from 1 g of ileal digesta by using a guanidinium thiocyanate ( 4 m ) containing lysis buffer at 90c for 2x 5 minutes , 2x 1 minute bead beating with acid washed glass beads ( 0.30.5 mm ) , subsequent phenol / chloroform ( 50 : 50 , v / v ) extraction , and isopropanol ( 98% ) precipitation . crude extracts were purified to pcr grade dna with commercial silica gel spin columns ( nucleospinkit tissue , machery - nagel , dueren , germany ) . the amount of dna was measured with fluorescence using sybr green i and calf thymus dna as reference dna . dna extraction was performed with a commercial kit ( qiagen stool kit , qiagen , hilden , germany ) and 200 mg ileal digesta in triplicate according to the instructions of the manufacturer except for an increase in temperature during the lysis step to 90c . purified dna was then pooled per sample and the dna was quantified as described above . dna samples were diluted to 100 ng l , and 1 l was used in triplicate for 25 l pcr reactions . two primer sets ( s - d - bact-0008-a - s-20/s - d - bact-0534-a - a-17 and s - d - bact-0968-a - s-18/s - d - bact-1401-a - a-17 ) at a concentration of 0.3 m were used to amplify two regions of bacterial 16s rrna genes . primers were tagged with unique hexamer nucleotides in order to sort pcr products after sequencing ( supporting information , table s1 ) . a commercial master mix kit ( hotstartaq plus master mix ; qiagen , hilden ; with added sybr green i during cycle number optimization ) was used for pcr amplification under the following cycling conditions : 1x 15 min at 95c , 32x ( for the 8f-534r set ) or 35x ( for the 968f-1401r set ) 15 sec at 95c , 30 sec at 55c , 30 sec at 72c , and 1x 1 min 20c . optimal amplification conditions were defined for each primer combination by the cycle number before the real time pcr amplification curves entered a plateau with no further increase of total fluorescence . cycling was performed on a stratagene mx3000p ( stratagene , amsterdam , the netherlands ) . pcr products were removed immediately after the last cycle and stored at 20c until further analysis . the pcr products were purified with a commercial kit ( qiaquick nucleotide removal kit , qiagen , hilden , germany ) and the amount of dna was determined as described above . equimolar dilutions of all samples were then combined into one master sample per extraction procedure . pyrosequencing was performed by agowa ( berlin , germany ) on a genome sequencer flx system using a titanium series picotiterplate , which was split in half to accommodate the two dna master samples from different extraction procedures . sequence reads were sorted according to barcodes and primer combination , resulting in 48 single data files . after removal of the sample barcodes and primer sequences , data files were uploaded to the mg - rast server [ 18 , 19 ] and processed by its seed software using silva ssu as reference databases . the phylogenetic profile of each sample was computed with the following parameters from the seed software : maximum e - value of 1e-5 , minimum percent identity of 98% , and minimum alignment length of 150 bases . sequences that were assigned as unclassified or of eukaryotic origin were not considered in the analysis process . for statistical interpretation , the next step in the analysis was the deletion of all data with four or less identical sequence reads per sample in order to increase the confidence of sequence reads and to reduce the bias through possible sequencing errors [ 21 , 22 ] . also , sequence reads that only occurred in one sample were deleted in order to focus on more common bacterial species . the remaining sequences were used to calculate the relative abundance of specific sequence reads in a sample . anova - procedures were carried out with the software spss 15.0 after using the levene test for homogeneity of variances to determine significant differences at the 0.05 level . data that failed the homogeneity of variance test was analyzed with the nonparametric mann - whitney u test to determine asymptotic significant differences . furthermore , data groups with only one data point were omitted to allow multiple comparisons for the remainder of the data groups . dna extraction , subsequent barcode pcr , and merging of 12 pcr products per extraction procedure yielded two master sample pools of 30 and 50 ng l , respectively . the length of pcr products as determined by agarose gel electrophoresis was 438532 bp for the extraction procedure i and 524608 bp for the extraction procedure ii . the 454-sequencing of two master samples yielded a total of 1.11 10 sequences with an average read length of 379 bases . after the correction for read length ( minimum 150 bases ) , 6.05 10 sequence reads were used for further analysis . there were no significant differences between dietary treatments , but high individual variation was observed . on average , 24763 ( 19867 ) and 26092 ( 18054 ) sequence reads were present in the 200 mg g zno and 3000 mg g zno experimental group , respectively . figure 1 shows sets of curves on the distribution of length of sequence reads for single samples . the primer set 8f-534r led to a more broadly distributed proportion of sequence length with a higher proportion of sequences around 300400 bases and peaks for some samples at 450 and 480 bases , respectively . in contrast , the primer set 968f-1401r displayed a sharp peak of sequence length at around 400430 bases for all samples . however , the primer set 8f-534r led to a significant shift to more gc - rich sequences , when the extraction procedure i was used . the percentage of unclassified / uncultured sequences split by extraction procedure and primer set is shown in table s2 , supplementary material available online at http://dx.doi.org/10.1155/2014/548683 . unclassified sequences in the silva database ranged from 0.2 to 6.2% of total sequences , which were mostly assigned to unclassified clostridiales ( data not shown ) . after exclusion of unclassified / eukaryotic sequences as well as filtering for minimum occurrence ( 5 sequences ) , 4.2 10 sequences remained for further analysis . table 1 shows the total number of aligned sequences for both extraction procedures and primer sets , as well as the total number of assigned bacterial genera after filtering for low sequence occurrence . a multifactorial anova analysis of the data is shown in table 2 . the extraction procedure ii proved to be superior in terms of total sequence reads and number . regarding the primer sets , the 8f-534r primer set generally led to more sequence reads and detected more bacterial genera compared to the 968f-1401r primer set with the extraction procedure ii . the removal of sequences with less than five reads per sample reduced the total amount of sequence reads only slightly , whereas a drastic reduction in assigned bacterial genera was observed ( see supporting information for data on unprocessed sequences , table s3 ) . however , the multivariate anova analysis revealed that there were highly significant interactions for extraction procedure and the choice of the primer set . significant differences were observed for most bacterial orders depending on the choice of the extraction procedure or primer set . table 3 shows the relative distribution of the 12 most prominent bacterial orders from a total of 25 orders that were detected . the lactobacillales order showed the highest amount of assigned sequence reads for all tested parameters , followed by clostridiales and enterobacteriales / actinomycetales . the extraction procedure ii showed numerically higher lactobacillales reads than the extraction procedure i. the 8f-534r primer set also had numerically higher amounts of lactobacillales reads than the 968f-1401r set , but significant differences were only found for the combination of the extraction procedure i and primer set 968f-1401r . on the contrary , the extraction procedure i generally led to a significantly higher relative abundance of clostridiales reads . very high numerical differences regarding the extraction procedure were also found for the order enterobacteriales , although no significant differences were observed due to very high individual variations . thus , actinomycetales , bacilliales , fusobacteriales , erysipelotrichales , and caulobacteriales seemed to be extracted more effectively by the extraction procedure i , while pseudomonadales , campylobacterales , and neisseriales were detected more effectively by the extraction procedure ii . similarly , fusobacteriales , burkholderiales , and campylobacterales assignments were more pronounced with the primer set 8f-534r , whereas more actinomycetales and bacilliales sequence reads were detected with the primer set 968f-1401r . on the genus level , a total of 154 bacterial genera were detected in processed sequence reads with the two extraction procedures and the two primer sets . of the total number of genera , 101 bacterial genera were detected by the silva database . table 4 shows the relative distribution of the major bacterial genera , which exceeded 0.1% of total reads in the database . regarding the extraction procedure , the combined amount of major genera for both primer sets was 26.8 ( 9.4 ) for the extraction procedure i versus 22.3 ( 2.3 ) genera for the extraction procedure ii . the most prominent differences between both extraction procedures were observed for genera of the clostridiales order , in which the extraction procedure ii led to fewer genera above 0.1% of the total sequences . according to the anova analysis , veillonella spp . , and weissella spp . regarding the primer sets , the combined amount of major genera for both extraction procedures was 22.8 ( 4.3 ) genera for primer set 8f-534r versus 26.7 ( 12.0 ) genera for primer set 968f-1401r . for instance , the 8f-534r primer set led to an average of 65.5% lactobacillus spp . sequences , while the 968f-1401r primer set only displayed an average of 25.4% . on the contrary , sequences , while the 968f-1401r primer set showed 22.1% . in detail , the primer set 8f-534r yielded significantly higher percentages for bacillus spp . , whereas the primer set 968f-1401r showed higher percentages for clostridium spp . , gemella spp . , lachnospira spp . , leuconostoc spp . , microbacterium spp contradicting results were observed for macrococcus spp . , which showed higher percentages with the 8-f-534r primers using the extraction method i , but the primer set 968f-1401r showed higher percentages with the extraction method ii . table 5 shows richness , shannon index , and evenness of the sequence data sorted by experimental group of piglets . the combined data evaluation for extraction procedures showed no significant differences in species richness for extraction procedure i , but richness increased in animals fed the high dietary zinc oxide concentration . vice versa , evenness was not significantly different for extraction procedure ii , but it showed increased evenness for the higher dietary zinc oxide concentration . the evaluation of data comprising extraction procedures and primer sets showed a numerical decrease in species richness for data from extraction procedure i and primer set 8f-534r , but the opposite was true for data from extraction procedure ii and primer set 968f-1401r . similarly , the shannon index failed to reach significant difference among experimental groups for data from extraction procedure i and primer set 8f-534r . data from extraction procedure ii and both primer sets yielded significant increases in species richness for animals fed the high dietary zinc oxide concentration , but no significant differences were observed for shannon index and evenness . comparative results for different extraction procedures and primer sets were also observed for many genera ( see supporting information , table s4 ) . the relative sequence abundance of clostridium spp . , veillonella spp . , and weissella spp . was numerically or significantly different for one or more of the studied factors . using extraction procedure ii , primer set 968f-1401r led to a numerical decrease . using extraction procedure i , primer set 968f-1401r for microbacterium spp . , the database showed almost identical relative sequence abundance . although the trend for increasing or decreasing relative sequence abundance was often similar among experimental groups , percentages differed for many combinations of extraction procedures and primer sets . as an example , if one would use extraction procedure i , the primer set 8f-534r would show a drastic and significant increase for streptococcus spp . and leuconostoc spp . in animals fed the high dietary zinc oxide concentration . if one would have employed the primer set 968f-1401r with the same extraction procedure , only a moderate nonsignificant increase would be detected for these genera . this study was carried out to investigate the effect of different dna extraction procedures and primer sets on pyrosequencing results regarding the composition of bacterial communities in the ileum of piglets . barcoded 16s rrna pcr amplicons have been employed in many different pyrosequencing studies over the last few years . thus , the analysis of the microbiota in the gut of humans , pigs , and rodents [ 25 , 26 ] as well as the analysis of cattle feces , plant viruses , forest soil fungi , soils [ 30 , 31 ] , hot springs , the atmosphere , sea food , or even human lymphocyte clonality relied on the method of using barcoded primer sets for the detection of microbial communities . although the barcoded amplicon method undoubtedly reduces the yet expensive use of massively parallel sequencing , no methodological study has been published on pre- and postsequencing parameters to the knowledge of the authors . the processing of sequence reads for low occurrence seemed justified , as the number of genera in unprocessed sequence data was more than twice as high as in processed sequence data , but the total number of deleted sequences was low . in addition , sequence reads with less than five sequences only occurred in a few samples . thus , using unprocessed sequence reads would have introduced a bias towards genera of rare occurrence . this was not justified , because it would have distorted a meaningful statistical analysis of the factors studied . the total number of unclassified sequences was in the range of 0.6% to 6.2% depending on primer set and extraction procedure which was considered as low and not contributing to the goals of this study . it is known that the yield of genomic dna from bacterial species depends on the type of extraction procedure employed [ 12 , 13 ] . although the total dna content of the master samples was very similar , the commercial silica - gel based extraction procedure led to approximately 3- to 5-fold higher numbers of total sequence reads than the bead beating method . bead beating may have disrupted plant material from feed and , thus , more plant derived pcr inhibitors may have been present in subsequent dna extracts . in fact , a longer amplification ( 3 cycles ) was observed during pcr optimization to reach a plateau for the 968f-1401r primer set compared to the 8f-534r primer set . however , as diluted pcr amplicons were used to generate the master samples for sequencing , the lower sequencing yield with the bead beating procedure can not be related to the presence of pcr inhibitors in the original dna extracts . a reduced sequence yield could also originate from poor quality of the pcr amplicons , which would lead to a reduced sequence yield in the dna library after processing ( blunt end preparation , ligation pcr ) , but read lengths were very similar for both extraction procedures . calf thymus dna has a gc content of only 42% , but pcr amplicons from the bead beating procedure and the primer set 8f-534r led to pcr amplicons with a gc content of 5055% . this combination generally also produced a twofold higher sequence yield than the 968f-1401r primers , which displayed the majority of sequences at 4550% gc . it is known that minor groove binding dyes such as sybr green i depend on gc content [ 36 , 37 ] and , thus , the higher gc content of pcr amplicons produced by the bead beating procedure may have led to an underestimation of the true dna content . the distribution of sequence reads between different bacterial orders was more uniform for the bead beating procedure than for the commercial extraction kit , because significantly higher proportions of the dominant lactobacillales were prevalent in dna extracts from the commercial extraction kit regardless of the chosen primer set . no clear distinction could be found between the more rigid gram - positive bacteria and the gram - negative bacteria , which have been reported to be easier to extract , as both extraction procedures differed in yields for several gram - positive ( lactobacillales versus clostridiales ) and gram - negative orders ( enterobacteriales versus pseudomonadales ) . however , in regard to bacterial genera known to adhere to intestinal epithelial cells or mucus , some differences were observed . thus , with the exception of campylobacterales ( mainly arcobacter ) , the bead beating method was superior for enterobacteriales ( mainly klebsiella ) , actinomycetales ( actinomyces ) , fusobacteriales ( mainly fusobacterium ) , neisseriales ( mainly neisseria ) , and erysipelotrichales ( only erysipelothrix ) . all the mentioned bacterial genera contain species that are known to adhere strongly to epithelial cells or mucus [ 3841 ] . although the commercial extraction procedure yielded a higher percentage for the dominating lactobacillales , among which lactobacillus spp . has a known adherence potential , the most pronounced differences regarding extraction procedures were found for weissella spp . , which are not known to adhere to epithelial cells . epithelial cells and mucus are shed continuously in the proximal parts of the small intestine and bacteria that adhere to epithelial cells are likely to be present in ileum digesta . thus , the thorough physical disruption of particles by bead beating may have enhanced the extraction of bacterial cells adhering to intestinal epithelial mucus or feed particles . finally , the enhanced detection of genes for 16s rrna chloroplasts from plants such as the major diet components soy and wheat indicates that the bead beating procedure successfully disrupted plant cell walls and must , therefore , be considered as the more thorough method regarding disintegration of sample particles . however , considering only the dominant bacterial genera above 0.1% of total sequences per sample , especially genera of the clostridiales order were better represented by the bead beating method and , thus , the richness ( amount of genera ) of dominant bacteria was higher . this has implications for barcoding pyrosequencing studies which cover high sample numbers , because less barcoded pcr amplicons per sample will be detected and , therefore , dominant bacteria will play a larger role in determining the bacterial composition . in conclusion , dna extraction procedures with bead beating seem to be superior , but due to the strong disintegration of particles by bead beating , removal of pcr inhibitors must be complete . in contrast to sequencing genomic dna of a few samples without any amplification , barcodes can be used in pyrosequencing studies to drastically increase the amount of samples on a single pyrosequencing plate . the drawback of the ability to sequence multiple samples is that an additional pcr is required for each sample in order to apply the respective tags to each pcr product . this procedure requires primer sets that naturally introduce a bias for the subsequent sequence analysis . this study used four commonly implemented primers that target the hypervariable regions v1-v3 ( 8f-534r ) and v6-v8 ( 968f-1401r ) of bacterial 16s rrna genes . thus , of the major orders , lactobacillales , fusobacteriales , burkholderiales , and campylobacterales rrna genes were better amplified by primers spanning the v1-v3 region , whereas clostridiales , actinomycetales , bacilliales , and neisseriales were better represented by primers spanning the v6-v8 region . no differences were observed for enterobacteriales , pseudomonadales , erysipelotrichales , or caulobacteriales . even within the dominant lactobacillales , significant and varying influences of primer sets were observed for four of seven dominant genera ( lactobacillus spp . however , amplification of members of the clostridiales order , which represented the second most abundant order , was more uniform as all genera were best amplified by primers spanning the v6-v8 region . these results confirm data from other studies on the variability of bacterial 16s rrna gene amplification using universal primers for microbial community analysis [ 17 , 4244 ] . this primer dilemma may be solved for pyrosequencing studies by using more than one primer pair to cover hypervariable regions of the 16s rrna gene . the authors have used this approach to study the influence of zinc oxide on porcine ileal bacterial communities by combining sequence reads of the two primer sets used in the present study on the basis of larger sequence number per single samples . it seems to be imperative for the design of barcoded pyrosequencing studies to examine the main bacterial composition in a given habitat in order to choose a primer set that covers most of the bacterial community . finally , the primer set targeting the hypervariable regions v1-v3 amplified a considerable proportion of 16s rrna genes of plant chloroplasts , reducing the amount of sequences of bacterial origin . although this may not apply to many habitats , all environments that contain significant amounts of plants in form of feed or roots should take notice of the possibility that pcr amplicons resulting from the 8f-534r set could be contaminated with plant chloroplast sequences . furthermore , deposited sequences attributed to uncultured deferribacterales by databases should be considered with caution , depending on the habitat . many methods that are used for analysis of biological samples from two or more different environments will lead to similar trends although absolute values may differ . according to the results of this study , that statement may not be true for pyrosequencing of barcoded 16s rrna gene amplicons . already on the primary methodological level , differences for species richness were observed among the low and high dietary zinc oxide experimental groups depending on the method of dna extraction . adding different primer sets to the analysis , one would conclude a nonsignificant decrease for species richness as well as moderate nonsignificant increase for the shannon index , if bead beating and primer set 8f-534r were used . using the same dna extraction method with the primer set 968f-1401r , the observed drastic increase in species richness and shannon index would lead to the conclusion that dietary zinc oxide has a major impact on bacterial communities in the ileum of piglets . for sarcina spp . , investigators using the commercial spin column method and primer set 8f-534r would not even detect this genus , while the bead beating method would indicate sarcina spp . to be a major component of the bacterial community , which is drastically reduced due to dietary zinc oxide . as this tendency was observed for other genera as well , the biological implications and drawn conclusions may be completely different . this empirical study has shown that the choice of extraction procedures and primer can severely influence the outcome of pyrosequencing studies . dna extraction seemed more complete using bead beating . a viable solution for pcr amplification could be the use of two or more primer sets to completely cover the bacterial diversity in complex samples . with respect to published studies on barcoded pyrosequencing of bacterial 16s rrna genes , the method and derived results should be regarded with care .

in this study , the effect of different dna extraction procedures and primer sets on pyrosequencing results regarding the composition of bacterial communities in the ileum of piglets was investigated . ileal chyme from piglets fed a diet containing different amounts of zinc oxide was used to evaluate a pyrosequencing study with barcoded 16s rrna pcr products . two dna extraction methods ( bead beating versus silica gel columns ) and two primer sets targeting variable regions of bacterial 16s rrna genes ( 8f-534r versus 968f-1401r ) were considered . the seed viewer software of the mg - rast server was used for automated sequence analysis . a total of 5.2 105 sequences were used for analysis after processing for read length ( 150 bp ) , minimum sequence occurrence ( 5 ) , and exclusion of eukaryotic and unclassified / uncultured sequences . dna extraction procedures and primer sets differed significantly in total sequence yield . the distribution of bacterial order and main bacterial genera was influenced significantly by both parameters . however , this study has shown that the results of pyrosequencing studies using barcoded pcr amplicons of bacterial 16s rrna genes depend on dna extraction and primer choice , as well as on the manner of downstream sequence analysis .

1. Introduction 2. Methods 3. Results 4. Discussion 5. Conclusions

combined with the technique of barcoded pcr amplicons , deep sequencing methods are able to process many samples at a relatively low cost per sequence [ 6 , 7 ] . however , as with any new technology , pitfalls exist . for barcoded pcr amplicon sequencing studies , nucleic acids must be extracted and the resulting dna extract should ideally represent the entire bacterial diversity in a given habitat . complex samples such as environmental samples from soil , waste treatment , or the gastrointestinal tract harbour not only diverse microbial communities , but also other components including mixtures of different carbohydrates , proteins , or minerals . gram - positive cell walls are generally more rigid than gram - negative cell walls , and the extraction of bacterial dna itself , therefore , becomes a balance between efficient cell lysis and the destruction of dna from already lysed cells . the most efficient rupturing of bacterial cell walls seems to be achieved by bead beating , although commercial kits such as the qiagen stool amp kit yield high amounts of stool dna without bead beating . for barcoded pcr amplicons , dna must be amplified in order to sequence multiple samples in a single pyrosequencing run . the hypervariable regions of the bacterial 16s rrna gene are generally the targets of choice , as the 16s rrna gene is a valuable phylogenetic marker but also has the advantage of being the most sequenced bacterial gene ; that is , sequences from pyrosequencing studies can be assigned against a large collection of reference sequences . however , it has been shown that there is no universal primer set that covers all known bacterial 16s rrna genes [ 1517 ] . the aim of this study was , therefore , to evaluate the impact of dna extraction , primer sets , and automated data evaluation on final results . a total of 12 ileal samples from 40- to 42-day - old piglets fed a standard starter diet supplemented with 200 or 3000 mg g zno ( n = 6 per group ) , respectively , were used for this study . subsequent to the euthanasia of the piglets , the gastrointestinal tracts were opened immediately , and the contents of the ileum were removed and stored at 80c . total nucleic acids were extracted from 1 g of ileal digesta by using a guanidinium thiocyanate ( 4 m ) containing lysis buffer at 90c for 2x 5 minutes , 2x 1 minute bead beating with acid washed glass beads ( 0.30.5 mm ) , subsequent phenol / chloroform ( 50 : 50 , v / v ) extraction , and isopropanol ( 98% ) precipitation . crude extracts were purified to pcr grade dna with commercial silica gel spin columns ( nucleospinkit tissue , machery - nagel , dueren , germany ) . dna extraction was performed with a commercial kit ( qiagen stool kit , qiagen , hilden , germany ) and 200 mg ileal digesta in triplicate according to the instructions of the manufacturer except for an increase in temperature during the lysis step to 90c . dna samples were diluted to 100 ng l , and 1 l was used in triplicate for 25 l pcr reactions . two primer sets ( s - d - bact-0008-a - s-20/s - d - bact-0534-a - a-17 and s - d - bact-0968-a - s-18/s - d - bact-1401-a - a-17 ) at a concentration of 0.3 m were used to amplify two regions of bacterial 16s rrna genes . a commercial master mix kit ( hotstartaq plus master mix ; qiagen , hilden ; with added sybr green i during cycle number optimization ) was used for pcr amplification under the following cycling conditions : 1x 15 min at 95c , 32x ( for the 8f-534r set ) or 35x ( for the 968f-1401r set ) 15 sec at 95c , 30 sec at 55c , 30 sec at 72c , and 1x 1 min 20c . the pcr products were purified with a commercial kit ( qiaquick nucleotide removal kit , qiagen , hilden , germany ) and the amount of dna was determined as described above . pyrosequencing was performed by agowa ( berlin , germany ) on a genome sequencer flx system using a titanium series picotiterplate , which was split in half to accommodate the two dna master samples from different extraction procedures . after removal of the sample barcodes and primer sequences , data files were uploaded to the mg - rast server [ 18 , 19 ] and processed by its seed software using silva ssu as reference databases . the phylogenetic profile of each sample was computed with the following parameters from the seed software : maximum e - value of 1e-5 , minimum percent identity of 98% , and minimum alignment length of 150 bases . sequences that were assigned as unclassified or of eukaryotic origin were not considered in the analysis process . for statistical interpretation , the next step in the analysis was the deletion of all data with four or less identical sequence reads per sample in order to increase the confidence of sequence reads and to reduce the bias through possible sequencing errors [ 21 , 22 ] . the remaining sequences were used to calculate the relative abundance of specific sequence reads in a sample . dna extraction , subsequent barcode pcr , and merging of 12 pcr products per extraction procedure yielded two master sample pools of 30 and 50 ng l , respectively . the 454-sequencing of two master samples yielded a total of 1.11 10 sequences with an average read length of 379 bases . after the correction for read length ( minimum 150 bases ) , 6.05 10 sequence reads were used for further analysis . on average , 24763 ( 19867 ) and 26092 ( 18054 ) sequence reads were present in the 200 mg g zno and 3000 mg g zno experimental group , respectively . figure 1 shows sets of curves on the distribution of length of sequence reads for single samples . however , the primer set 8f-534r led to a significant shift to more gc - rich sequences , when the extraction procedure i was used . the percentage of unclassified / uncultured sequences split by extraction procedure and primer set is shown in table s2 , supplementary material available online at http://dx.doi.org/10.1155/2014/548683 . after exclusion of unclassified / eukaryotic sequences as well as filtering for minimum occurrence ( 5 sequences ) , 4.2 10 sequences remained for further analysis . table 1 shows the total number of aligned sequences for both extraction procedures and primer sets , as well as the total number of assigned bacterial genera after filtering for low sequence occurrence . regarding the primer sets , the 8f-534r primer set generally led to more sequence reads and detected more bacterial genera compared to the 968f-1401r primer set with the extraction procedure ii . the removal of sequences with less than five reads per sample reduced the total amount of sequence reads only slightly , whereas a drastic reduction in assigned bacterial genera was observed ( see supporting information for data on unprocessed sequences , table s3 ) . however , the multivariate anova analysis revealed that there were highly significant interactions for extraction procedure and the choice of the primer set . significant differences were observed for most bacterial orders depending on the choice of the extraction procedure or primer set . table 3 shows the relative distribution of the 12 most prominent bacterial orders from a total of 25 orders that were detected . the extraction procedure ii showed numerically higher lactobacillales reads than the extraction procedure i. the 8f-534r primer set also had numerically higher amounts of lactobacillales reads than the 968f-1401r set , but significant differences were only found for the combination of the extraction procedure i and primer set 968f-1401r . on the contrary , the extraction procedure i generally led to a significantly higher relative abundance of clostridiales reads . on the genus level , a total of 154 bacterial genera were detected in processed sequence reads with the two extraction procedures and the two primer sets . of the total number of genera , 101 bacterial genera were detected by the silva database . table 4 shows the relative distribution of the major bacterial genera , which exceeded 0.1% of total reads in the database . regarding the extraction procedure , the combined amount of major genera for both primer sets was 26.8 ( 9.4 ) for the extraction procedure i versus 22.3 ( 2.3 ) genera for the extraction procedure ii . the most prominent differences between both extraction procedures were observed for genera of the clostridiales order , in which the extraction procedure ii led to fewer genera above 0.1% of the total sequences . regarding the primer sets , the combined amount of major genera for both extraction procedures was 22.8 ( 4.3 ) genera for primer set 8f-534r versus 26.7 ( 12.0 ) genera for primer set 968f-1401r . table 5 shows richness , shannon index , and evenness of the sequence data sorted by experimental group of piglets . the combined data evaluation for extraction procedures showed no significant differences in species richness for extraction procedure i , but richness increased in animals fed the high dietary zinc oxide concentration . the evaluation of data comprising extraction procedures and primer sets showed a numerical decrease in species richness for data from extraction procedure i and primer set 8f-534r , but the opposite was true for data from extraction procedure ii and primer set 968f-1401r . similarly , the shannon index failed to reach significant difference among experimental groups for data from extraction procedure i and primer set 8f-534r . data from extraction procedure ii and both primer sets yielded significant increases in species richness for animals fed the high dietary zinc oxide concentration , but no significant differences were observed for shannon index and evenness . comparative results for different extraction procedures and primer sets were also observed for many genera ( see supporting information , table s4 ) . although the trend for increasing or decreasing relative sequence abundance was often similar among experimental groups , percentages differed for many combinations of extraction procedures and primer sets . this study was carried out to investigate the effect of different dna extraction procedures and primer sets on pyrosequencing results regarding the composition of bacterial communities in the ileum of piglets . barcoded 16s rrna pcr amplicons have been employed in many different pyrosequencing studies over the last few years . thus , the analysis of the microbiota in the gut of humans , pigs , and rodents [ 25 , 26 ] as well as the analysis of cattle feces , plant viruses , forest soil fungi , soils [ 30 , 31 ] , hot springs , the atmosphere , sea food , or even human lymphocyte clonality relied on the method of using barcoded primer sets for the detection of microbial communities . although the barcoded amplicon method undoubtedly reduces the yet expensive use of massively parallel sequencing , no methodological study has been published on pre- and postsequencing parameters to the knowledge of the authors . the total number of unclassified sequences was in the range of 0.6% to 6.2% depending on primer set and extraction procedure which was considered as low and not contributing to the goals of this study . it is known that the yield of genomic dna from bacterial species depends on the type of extraction procedure employed [ 12 , 13 ] . although the total dna content of the master samples was very similar , the commercial silica - gel based extraction procedure led to approximately 3- to 5-fold higher numbers of total sequence reads than the bead beating method . however , as diluted pcr amplicons were used to generate the master samples for sequencing , the lower sequencing yield with the bead beating procedure can not be related to the presence of pcr inhibitors in the original dna extracts . a reduced sequence yield could also originate from poor quality of the pcr amplicons , which would lead to a reduced sequence yield in the dna library after processing ( blunt end preparation , ligation pcr ) , but read lengths were very similar for both extraction procedures . calf thymus dna has a gc content of only 42% , but pcr amplicons from the bead beating procedure and the primer set 8f-534r led to pcr amplicons with a gc content of 5055% . it is known that minor groove binding dyes such as sybr green i depend on gc content [ 36 , 37 ] and , thus , the higher gc content of pcr amplicons produced by the bead beating procedure may have led to an underestimation of the true dna content . the distribution of sequence reads between different bacterial orders was more uniform for the bead beating procedure than for the commercial extraction kit , because significantly higher proportions of the dominant lactobacillales were prevalent in dna extracts from the commercial extraction kit regardless of the chosen primer set . no clear distinction could be found between the more rigid gram - positive bacteria and the gram - negative bacteria , which have been reported to be easier to extract , as both extraction procedures differed in yields for several gram - positive ( lactobacillales versus clostridiales ) and gram - negative orders ( enterobacteriales versus pseudomonadales ) . however , in regard to bacterial genera known to adhere to intestinal epithelial cells or mucus , some differences were observed . thus , with the exception of campylobacterales ( mainly arcobacter ) , the bead beating method was superior for enterobacteriales ( mainly klebsiella ) , actinomycetales ( actinomyces ) , fusobacteriales ( mainly fusobacterium ) , neisseriales ( mainly neisseria ) , and erysipelotrichales ( only erysipelothrix ) . all the mentioned bacterial genera contain species that are known to adhere strongly to epithelial cells or mucus [ 3841 ] . has a known adherence potential , the most pronounced differences regarding extraction procedures were found for weissella spp . epithelial cells and mucus are shed continuously in the proximal parts of the small intestine and bacteria that adhere to epithelial cells are likely to be present in ileum digesta . thus , the thorough physical disruption of particles by bead beating may have enhanced the extraction of bacterial cells adhering to intestinal epithelial mucus or feed particles . finally , the enhanced detection of genes for 16s rrna chloroplasts from plants such as the major diet components soy and wheat indicates that the bead beating procedure successfully disrupted plant cell walls and must , therefore , be considered as the more thorough method regarding disintegration of sample particles . however , considering only the dominant bacterial genera above 0.1% of total sequences per sample , especially genera of the clostridiales order were better represented by the bead beating method and , thus , the richness ( amount of genera ) of dominant bacteria was higher . this has implications for barcoding pyrosequencing studies which cover high sample numbers , because less barcoded pcr amplicons per sample will be detected and , therefore , dominant bacteria will play a larger role in determining the bacterial composition . in conclusion , dna extraction procedures with bead beating seem to be superior , but due to the strong disintegration of particles by bead beating , removal of pcr inhibitors must be complete . this procedure requires primer sets that naturally introduce a bias for the subsequent sequence analysis . this study used four commonly implemented primers that target the hypervariable regions v1-v3 ( 8f-534r ) and v6-v8 ( 968f-1401r ) of bacterial 16s rrna genes . thus , of the major orders , lactobacillales , fusobacteriales , burkholderiales , and campylobacterales rrna genes were better amplified by primers spanning the v1-v3 region , whereas clostridiales , actinomycetales , bacilliales , and neisseriales were better represented by primers spanning the v6-v8 region . even within the dominant lactobacillales , significant and varying influences of primer sets were observed for four of seven dominant genera ( lactobacillus spp . however , amplification of members of the clostridiales order , which represented the second most abundant order , was more uniform as all genera were best amplified by primers spanning the v6-v8 region . these results confirm data from other studies on the variability of bacterial 16s rrna gene amplification using universal primers for microbial community analysis [ 17 , 4244 ] . this primer dilemma may be solved for pyrosequencing studies by using more than one primer pair to cover hypervariable regions of the 16s rrna gene . the authors have used this approach to study the influence of zinc oxide on porcine ileal bacterial communities by combining sequence reads of the two primer sets used in the present study on the basis of larger sequence number per single samples . it seems to be imperative for the design of barcoded pyrosequencing studies to examine the main bacterial composition in a given habitat in order to choose a primer set that covers most of the bacterial community . finally , the primer set targeting the hypervariable regions v1-v3 amplified a considerable proportion of 16s rrna genes of plant chloroplasts , reducing the amount of sequences of bacterial origin . although this may not apply to many habitats , all environments that contain significant amounts of plants in form of feed or roots should take notice of the possibility that pcr amplicons resulting from the 8f-534r set could be contaminated with plant chloroplast sequences . many methods that are used for analysis of biological samples from two or more different environments will lead to similar trends although absolute values may differ . according to the results of this study , that statement may not be true for pyrosequencing of barcoded 16s rrna gene amplicons . already on the primary methodological level , differences for species richness were observed among the low and high dietary zinc oxide experimental groups depending on the method of dna extraction . adding different primer sets to the analysis , one would conclude a nonsignificant decrease for species richness as well as moderate nonsignificant increase for the shannon index , if bead beating and primer set 8f-534r were used . using the same dna extraction method with the primer set 968f-1401r , the observed drastic increase in species richness and shannon index would lead to the conclusion that dietary zinc oxide has a major impact on bacterial communities in the ileum of piglets . , investigators using the commercial spin column method and primer set 8f-534r would not even detect this genus , while the bead beating method would indicate sarcina spp . to be a major component of the bacterial community , which is drastically reduced due to dietary zinc oxide . as this tendency was observed for other genera as well , the biological implications and drawn conclusions may be completely different . this empirical study has shown that the choice of extraction procedures and primer can severely influence the outcome of pyrosequencing studies . dna extraction seemed more complete using bead beating . with respect to published studies on barcoded pyrosequencing of bacterial 16s rrna genes , the method and derived results should be regarded with care .

every year millions of people move from their homes and resettle in other countries as an effect of globalization , war , natural disasters , politics and family relations . the most common reason for immigrating to sweden has been political , but family relations are also important ( 1 ) . bosnia and herzegovina is a part of the balkan region , and was geographically and politically bonded with the former yugoslavia until 1991 , at which time it became an independent state , after a period of war . many bosnians lost their lives in the war , and other survived by fleeing . between 1992 and 1995 , the people of bosnia and herzegovina experienced one of the most terrible wars seen in europe since the end of world war ii . violence and ethnic cleansing were deliberately used as a tool to drive people from the areas where they were born and had lived for generations ( 2 ) . the whole social structure was destroyed and there was no adequate social support system . in the same war , the civilians of bosnia and herzegovina were exposed to extreme threats and intense feelings of helplessness ( 3,4 ) . the traumatic events experienced by thousands of people during this conflict may have a lasting effect on the mental health of the country ( 5 ) . they have also had impact abroad as negative health consequences are especially high when relocation is due to severe conflicts , associated with violence and trauma ( 6 ) . since the 1970s , immigration has increased in sweden as well as in many other european countries . immigration is now the main source of an increasing of the number of swedish population . only in 2006 , 86,436 immigrants were granted residence permits in sweden , of which 25,096 were for protection or humanitarian reasons ( 7 ) . refugees , unlike many immigrants who have left their homes for economic reasons or to join family members already settled in another country , have fled in order to survive . most of these people have faced difficult transit experiences , culture shock , adjustment problems related to language and occupational change , and disruption in their sense of selfhood and community in the resettlement country ( 8) . additionally , a refugee has often suffered multiple losses such as severance from family and friends . trans cultural nursing is an essential aspect of healthcare today and this is a new concept and a separate area in nursing science ( 9 , 10 ) . in developing the theory , a major hurdle for health professionals was to discover meanings , practices and factors influencing care by religion , politics , economics , world view , environment , cultural values , history , language and gender . nurses and health professionals describe cultural competence as having the ability to understand cultural differences in order to provide quality care to a diversity of people . cultural competence involves nurses and health professionals continuously striving to provide effective care within the cultural confines of their patients . the central purpose of this theory is to discover and explain diverse and universal culturally based care factors influencing the health , well - being , illness or death of individuals or groups . the purpose and goal of the theory is to use research findings to provide culturally congruent , safe and meaningful care to clients of diverse or similar cultures . the three modes for congruent care , decisions and actions proposed in the theory are predicted to lead to health and well - being or to face illness and death . in sum , the culture care theory has been a major and significant contribution to establish and maintain the discipline of trans cultural nursing discipline over the past five decades . the holistic and particularistic features and the ethno nursing method have led to a new body of knowledge about culture and care phenomena . having knowledge of the patient 's cultural perspectives enables the nurse to provide more effective and appropriate care ( 9 , 10 ) . the language plays a central role in communication and interpretation of the culture , and it is important that nurses and health professionals first understand their own cultural values , attitudes , beliefs and practices that they have acquired from their own families before learning about other cultural ways ( 11 ) . this means that the way to express symptoms of disease and the expectations of the patient and on the patient by healthcare providers differ between regions , which may cause problems when an immigrant calls for medical attention in a new country ( 12 , 13 , 14 , 15 ) . the swedish health care system is expected to provide equal health care opportunities for all patients ( 16 ) . the swedish health and medical services act ( 1982 ) stipulate that health care must : be of good quality and take account of the patient 's need for safe care and treatment , be readily available , be based on respect for the self - determination and privacy of the patient andpromote good relationships between patients and health care providers . be of good quality and take account of the patient 's need for safe care and treatment , be readily available , be based on respect for the self - determination and privacy of the patient and promote good relationships between patients and health care providers . previous studies about the healthcare system among refugees showed that immigrants in sweden have significantly poorer health than native swedes ( 17 ) . and may therefore have increased need for healthcare services ( 18 , 19).previous studies made in sweden about refugees coming from burundi , colombia , iraq , kazakhstan , poland , kosovo and syria , showed that the main problems were related to feelings of being uninformed and being sent to various levels of care , which resulted in lack of trust , and feelings that no one took overall responsibility ( 20 ) . the aim of the present study was to describe the life of bosnian immigrants after arriving to sweden , with focus on contacts with healthcare system . our study is based on a qualitative design , as the study aimed to describe and analyze how patients experienced the swedish health system . inclusion criteria were subjects / persons coming from bosnia and herzegovina , who were more than 60 years old , had lived in sweden more than 10 years and had visited the healthcare canter more than twice during the past month . twenty subjects / persons were invited to participate in the study , of which 15 participants agreed to participate . three of the participants declined participation without explanation and two of participants moved back to bosnia and herzegovina during the study period . accordingly , fifteen persons participated in the study : eight women and seven men , aged between 65 and 86 years . all participants had lived in sweden between 13 and 21 years ( table 1 ) . the first author of the study ( ns ) made appointments for all interviews . demographic data of informants data was collected through individual face - to - face interviews by the first author ( ns ) using open - ended questions , following an interview guide inspired by kvale ( 21 ) . the opening question was can you please tell me about your life after arriving to sweden ? and could you please tell me about your experiences to being a patient in sweden ? the initial question were supplemented with other short questions like could you please tell me more about this ? or all contacts with the informants were arranged in collaboration with one key person in the bosnian association of gothenburg , located close to the place where the participants lived . information concerning the aim and background of the study was printed and distributed to the informants , and repeated to them orally before the interview . participants were encouraged to speak freely using their own words and the interviewer encouraged the informants to respond to questions as comprehensively as possible . the interviews were carried out in bosnian by the second author , who is bilingual . all interviews were translated first into swedish by the second author , and the translation was checked by a professional translator . the interviews lasted between 60 and 90 minutes and were taped , transcribed and transcribed verbatim . the audio - recorded interviews were transcribed verbatim and analyzed in accordance with graneheim and lundman ( 2004 ) . due to the nature of our study , a qualitative dynamic analysis method that stays close to data was needed . in this context a qualitative content analysis method in accordance with graneheim and lundman chose for analysis and interpretation of the collected data . this method is capable to condense a large amount of data to a limited number of themes , categories , subcategories and codes . furthermore , content analysis method makes it possible to include interpretations of a latent content the transcripts were read carefully in order to identify the informants ' experiences and conceptions of the migration and its effect . then , the analysis proceeded by extracting meaning units consisting of one or several words , sentences , or paragraphs containing aspects related to each other and addressing a specific topic in the material . then meaning units related to each other through their content and context were abstracted and grouped together into a condensed meaning unit , with a description close to the original text . the condensed text was further abstracted and labelled with a code . subcategories that focused on the same problem were brought together , in order to create more extensive conceptions , which addressed an obvious issue ( 22 ) . the results are presented with direct quotes from the interviews . according to the swedish law , there is no need for an ethical board review if written consent has been obtained from the participants and if there is no physical intervention involved in the study ( 23 ) . however , the study conformed to the principles outlined in the declaration of helsinki ( 24 ) . participants were informed that participation was voluntary and that confidentiality would be maintained . written informed consent was obtained from the participants . the analysis resulted in one category and four subcategories depending on how the participants described their lives in sweden . although this article is primarily concerned with the participant 's experiences in sweden , several questions concerned their lives in bosnia and herzegovina . informants in this study told us freely about their experiences before arriving in sweden , about their life in sweden and experiences during the visits to the healthcare centres . the interviews were then analyzed in terms of different themes or subcategories ( table 2 ) . overview of the categories and subcategories as a consequence of everything happening in the world today you can no longer decide where to live . to become refugees and immigrants in a foreign country is difficult and has its consequences . in the interviews it was found that older people found it especially hard to come to another country after leaving everything , their homes , their children , their relatives and friends , and all their property . the culture and environment were perceived as strange , which they all felt contributed to their health and well - being deteriorating . on arriving to sweden , all of the informants were placed in refugee centres and sent around sweden . it was terrible ; i did not feel well and considered to returning to bosnia.(p3 ) a woman described her time in the centre as follows : the doctors talked and talked , explained , discussed with me and themselves i just looked at them and did not know if i should laugh or cry.(p4 ) i was in a house with refugees from somalia . it was no fun.(p1 ) coming from a country that is at war , and all the problems associated with this situation was emphasized by all the informants in our study as reasons not to get involved and learn the swedish language . all had hoped that the war would end in a few months so that they could return to their home country . none of the informants knew more than a few swedish words and were thus unable to communicate in swedish . this also means that the informants lacked the social skills that could connect them with swedish society . one participant described his language experiences like this : knowing a language is a treasure . i often meet older foreign persons on the street who want to ask me something . they talk incessantly , but i understand nothing.(p2 ) all the informants described when they were out walking , how they were assisted in the swedish language by their children , grandchildren or friends . this meant that informants became more passive and did not care about their communication difficulties because there was always someone available who could help them with translation . at the same time it happened that the children were the ones who suffered most because they had to plan both for themselves and their families . communication difficulties were described as follows : my neighbors are swedes , two elderly women who are very nice . we would have had so much to talk about and it 'd be great fun , but now we cannot.(p5 ) language and communication barriers can lead to various difficulties and challenging situations . all informants had situations where they felt uncomfortable , laughed and felt as if they had lost their personal worth , a situation which is described in the following : two years ago i broke my hip , went to the hospital and met a swedish physician . i explained through the interpreter that i was a doctor , but he did not hear or did not want to hear.(p8 ) another participant described her situation like this : my son 's wife is swedish . the problem is that i ca n't speak swedish language.(p7 ) an interpreter was sometimes hired to deal with different situations where informants must communicate in swedish but could not . in the health care services , for example , three of the informants felt that it was much more sensitive to talk about their symptoms if another person was present . medical staff unassisted by a professional interpreter can not be sure that the patient is given the opportunity to express their views completely . however , during the interviews it was revealed that the interpretation situations caused a variety of problems . the interpreter did not speak the same language , did not translate correctly , sometimes they were not on time for an appointment , and it took a long time to get a translation . about their experiences with interpreters one participant said the following : once we needed an interpreter and he came . she is the best.(p11 ) about keeping times , one informant described the situation as follows : the interpreter did not come , but who cares?.(p10 ) among the respondents , there were some who had difficult experiences from the war in bosnia and herzegovina and did not want an interpreter . some of the informants did not want to accept that they speak serbo - croatian language , only the language of the country they come from . he said he spoke serbo - croatian , maybe he did , but i understood nothing . then came a croatian interpreter and it went well.(p9 ) one informant had the following to say about language : with yugoslavia 's disappearance the serbo - croatian language disappeared , and then there will be serbian , bosnian , slovenian , macedonian , croatian and other languages of the former yugoslavia.(p12 ) all respondents thought it obvious that children should help their parents who live in sweden but can not speak swedish even if it usually led to encroachment on the child 's privacy . one participant described the relationship with his / her children like this : my children and i live next door to each other . i 'm really happy to have my children here.(p15 ) one participant described his relationship with his children in sweden like this : asking for help from my children would mean that i intervene into their lives , but i must . i 'm sorry , but i have to.(p13 ) one participant said : my children call me daily . i have the nicest kids in the world.(p14 ) all respondents found it difficult to move from their home country , leaving everything , to a completely different country . this was experienced as something terrible . when it comes to being thankful that they were allowed to stay in sweden , emotions were mixed and informants had different opinions on the issue . we have always paid 50 % tax for our work in bosnia and herzegovina . today i have a pension which is 2000 sek per month . what should i be grateful for?(p4 ) we must adapt to swedish society and thus thank sweden and the swedes for all the help we received and are still getting here.(p8 ) even stronger feelings appeared on the question of debt of gratitude : who has something bad to say to the good country that took care of us ? if we lived in bosnia and herzegovina now , we would fight for our survival . as a consequence of everything happening in the world today you can no longer decide where to live . to become refugees and immigrants in a foreign country is difficult and has its consequences . in the interviews it was found that older people found it especially hard to come to another country after leaving everything , their homes , their children , their relatives and friends , and all their property . the culture and environment were perceived as strange , which they all felt contributed to their health and well - being deteriorating . on arriving to sweden , all of the informants were placed in refugee centres and sent around sweden . it was terrible ; i did not feel well and considered to returning to bosnia.(p3 ) a woman described her time in the centre as follows : the doctors talked and talked , explained , discussed with me and themselves i just looked at them and did not know if i should laugh or cry.(p4 ) i was in a house with refugees from somalia . it was no fun.(p1 ) coming from a country that is at war , and all the problems associated with this situation was emphasized by all the informants in our study as reasons not to get involved and learn the swedish language . all had hoped that the war would end in a few months so that they could return to their home country . none of the informants knew more than a few swedish words and were thus unable to communicate in swedish . this also means that the informants lacked the social skills that could connect them with swedish society . one participant described his language experiences like this : knowing a language is a treasure . i often meet older foreign persons on the street who want to ask me something . they talk incessantly , but i understand nothing.(p2 ) all the informants described when they were out walking , how they were assisted in the swedish language by their children , grandchildren or friends . this meant that informants became more passive and did not care about their communication difficulties because there was always someone available who could help them with translation . at the same time it happened that the children were the ones who suffered most because they had to plan both for themselves and their families . communication difficulties were described as follows : my neighbors are swedes , two elderly women who are very nice . we would have had so much to talk about and it 'd be great fun , but now we cannot.(p5 ) language and communication barriers can lead to various difficulties and challenging situations . all informants had situations where they felt uncomfortable , laughed and felt as if they had lost their personal worth , a situation which is described in the following : two years ago i broke my hip , went to the hospital and met a swedish physician . i explained through the interpreter that i was a doctor , but he did not hear or did not want to hear.(p8 ) another participant described her situation like this : my son 's wife is swedish . the problem is that i ca n't speak swedish language.(p7 ) an interpreter was sometimes hired to deal with different situations where informants must communicate in swedish but could not . in the health care services , for example , three of the informants felt that it was much more sensitive to talk about their symptoms if another person was present . medical staff unassisted by a professional interpreter can not be sure that the patient is given the opportunity to express their views completely . however , during the interviews it was revealed that the interpretation situations caused a variety of problems . the interpreter did not speak the same language , did not translate correctly , sometimes they were not on time for an appointment , and it took a long time to get a translation . about their experiences with interpreters one participant said the following : once we needed an interpreter and he came . she is the best.(p11 ) about keeping times , one informant described the situation as follows : the interpreter did not come , but who cares?.(p10 ) among the respondents , there were some who had difficult experiences from the war in bosnia and herzegovina and did not want an interpreter . some of the informants did not want to accept that they speak serbo - croatian language , only the language of the country they come from . he said he spoke serbo - croatian , maybe he did , but i understood nothing . we booked another appointment and waited another month which was no good . then came a croatian interpreter and it went well.(p9 ) one informant had the following to say about language : with yugoslavia 's disappearance the serbo - croatian language disappeared , and then there will be serbian , bosnian , slovenian , macedonian , croatian and other languages of the former yugoslavia.(p12 ) all respondents thought it obvious that children should help their parents who live in sweden but can not speak swedish even if it usually led to encroachment on the child 's privacy . one participant described the relationship with his / her children like this : my children and i live next door to each other . i 'm really happy to have my children here.(p15 ) one participant described his relationship with his children in sweden like this : asking for help from my children would mean that i intervene into their lives , but i must . i 'm sorry , but i have to.(p13 ) one participant said : my children call me daily . i have the nicest kids in the world.(p14 ) all respondents found it difficult to move from their home country , leaving everything , to a completely different country . when it comes to being thankful that they were allowed to stay in sweden , emotions were mixed and informants had different opinions on the issue . we have always paid 50 % tax for our work in bosnia and herzegovina . what should i be grateful for?(p4 ) we must adapt to swedish society and thus thank sweden and the swedes for all the help we received and are still getting here.(p8 ) even stronger feelings appeared on the question of debt of gratitude : who has something bad to say to the good country that took care of us ? if we lived in bosnia and herzegovina now , we would fight for our survival . on arriving to sweden , all of the informants were placed in refugee centres and sent around sweden . it was terrible ; i did not feel well and considered to returning to bosnia.(p3 ) a woman described her time in the centre as follows : the doctors talked and talked , explained , discussed with me and themselves i just looked at them and did not know if i should laugh or cry.(p4 ) i was in a house with refugees from somalia . coming from a country that is at war , and all the problems associated with this situation was emphasized by all the informants in our study as reasons not to get involved and learn the swedish language . all had hoped that the war would end in a few months so that they could return to their home country . none of the informants knew more than a few swedish words and were thus unable to communicate in swedish . this also means that the informants lacked the social skills that could connect them with swedish society . one participant described his language experiences like this : knowing a language is a treasure . i often meet older foreign persons on the street who want to ask me something . they talk incessantly , but i understand nothing.(p2 ) all the informants described when they were out walking , how they were assisted in the swedish language by their children , grandchildren or friends . this meant that informants became more passive and did not care about their communication difficulties because there was always someone available who could help them with translation . at the same time it happened that the children were the ones who suffered most because they had to plan both for themselves and their families . communication difficulties were described as follows : my neighbors are swedes , two elderly women who are very nice . we would have had so much to talk about and it 'd be great fun , but now we cannot.(p5 ) language and communication barriers can lead to various difficulties and challenging situations . all informants had situations where they felt uncomfortable , laughed and felt as if they had lost their personal worth , a situation which is described in the following : two years ago i broke my hip , went to the hospital and met a swedish physician . i explained through the interpreter that i was a doctor , but he did not hear or did not want to hear.(p8 ) another participant described her situation like this : my son 's wife is swedish . the problem is that i ca n't speak swedish language.(p7 ) an interpreter was sometimes hired to deal with different situations where informants must communicate in swedish but could not . in the health care services , for example , three of the informants felt that it was much more sensitive to talk about their symptoms if another person was present . medical staff unassisted by a professional interpreter can not be sure that the patient is given the opportunity to express their views completely . however , during the interviews it was revealed that the interpretation situations caused a variety of problems . the interpreter did not speak the same language , did not translate correctly , sometimes they were not on time for an appointment , and it took a long time to get a translation . about their experiences with interpreters one participant said the following : once we needed an interpreter and he came . she is the best.(p11 ) about keeping times , one informant described the situation as follows : the interpreter did not come , but who cares?.(p10 ) among the respondents , there were some who had difficult experiences from the war in bosnia and herzegovina and did not want an interpreter . some of the informants did not want to accept that they speak serbo - croatian language , only the language of the country they come from . he said he spoke serbo - croatian , maybe he did , but i understood nothing . we booked another appointment and waited another month which was no good . then came a croatian interpreter and it went well.(p9 ) one informant had the following to say about language : with yugoslavia 's disappearance the serbo - croatian language disappeared , and then there will be serbian , bosnian , slovenian , macedonian , croatian and other languages of the former yugoslavia.(p12 ) all respondents thought it obvious that children should help their parents who live in sweden but can not speak swedish even if it usually led to encroachment on the child 's privacy . one participant described the relationship with his / her children like this : my children and i live next door to each other . i 'm really happy to have my children here.(p15 ) one participant described his relationship with his children in sweden like this : asking for help from my children would mean that i intervene into their lives , but i must . i 'm sorry , but i have to.(p13 ) one participant said : my children call me daily . all respondents found it difficult to move from their home country , leaving everything , to a completely different country . this was experienced as something terrible . when it comes to being thankful that they were allowed to stay in sweden , emotions were mixed and informants had different opinions on the issue . we have always paid 50 % tax for our work in bosnia and herzegovina . today i have a pension which is 2000 sek per month . what should i be grateful for?(p4 ) we must adapt to swedish society and thus thank sweden and the swedes for all the help we received and are still getting here.(p8 ) even stronger feelings appeared on the question of debt of gratitude : who has something bad to say to the good country that took care of us ? if we lived in bosnia and herzegovina now , we would fight for our survival . we found that the change of scenery , culture and language influenced the well - being of the informants . during the war , the majority of informants were forced to leave their property , they were separated from their children and had to leave their work and social community thereby leaving language , their homeland and what they so far had experienced during their lives behind . language and communication difficulties were felt to be the major problems . based on our results , we could see that the informants perceived well - being in their home country when they lived in their own environment . there they lived with their own culture and spoke their own language . having a job , taking care of the family and taking part of the social community was perceived as very positive . from having an active and fulfilling life , all informants switched to a less active and more isolated life . all informants felt a great loss of all those belongings they had left in their previous world . according to the participants in the interviews , their well - being deteriorated when they became refugees in sweden . according to andersson ( 25 ) this may be the result of settling in a foreign country , which brings experiences of alienation ; of a lack of identity and rootlessness . despite the fact that the informants moved to another country , they had not left their culture and the social communication . none of the informants were able to understand the new language , or convey information in swedish . not being able to understand and make them understood was experienced as negative by all informants . all informants also regretted that they had never learned the swedish language . according to magnusson ( 26 ) the relationship between culture and communication is intimate and it is through communication that culture is passed on . culture itself influences how we express our feelings , as well as ourselves verbally and non verbally . without knowledge in swedish language the informants were not able to have swedish friends and without them the informants can not learn the swedish culture . the findings in our study are in the line with other studies which showed that some participants could not access information because they were unfamiliar with the structure of the swedish healthcare system . lacking linguistic skills and difficulties in communication with care providers , for example regarding information about their disease and involvement in treatment , caused frustration and increased the risk of misunderstandings , miscommunication and inequalities in healthcare provision ( 27 , 28 , 29 ) . the fact that they were forced to live in another country eliminated their cultural and historical background , which had a negative impact on their social situation . language limitations and communication difficulties meant that all informants were forced to seek help from their relatives or had to use of an interpreter when they visited various institutions . our findings are also in line with a study of chinese and vietnamese patients living in the usa . ngo - metzger et al ( 30 ) showed that using an interpreter could even exacerbate disparities in patient 's perception of their providers , despite receiving more information compared to those without an interpreter . thus , an interpreter could not substitute a language - concordant provider ( 30 ) . according to gerrish ( 27 ) the quality of care is affected if the hospital does not have the opportunity to hire a professional interpreter , and allows children to interpret for their parents or relatives . he further argues that such interpretations are filled with feelings , misunderstandings , wrong interpretations and misinterpretations of the diagnosis and treatment resulting in detrimental medical misunderstandings . on the other side hlander ( 31 ) patients because that healthcare professionals treat the patients group based on stereotypical images of how a patient from another country is supposed to act , think about care , need , so as the emphasis on cultural diversity might lead to exaggerated differences . one limitation of our study was that we interviewed the participants in their homes , and sometimes ( in the three cases ) the participants had guests . in two other cases children of the participants joined the participants during the interview period . these circumstances may have affected the answers and may have made the participants less open . the data in our study showed several major areas to improve access and quality of life for bosnian refugees in sweden . these areas are particularly concerned with language and cross - cultural communication and improvements could be made by organizing different meetings for swedes and immigrants from bosnia and herzegovina , organizing various courses in swedish for immigrants where they live , informing bosnian immigrants more about the swedish culture and the swedish health system and increasing the budget to train more interpreters in the swedish language . these are only a few areas that should be improved to make immigrants from bosnia and herzegovina feel better psychologically and physically . health care systems and patients are to a variable extent influenced by the local culture . this means that the way symptoms are described , the expectations of the patient , and the expectations that health care providers have on patients differ between regions , which may cause problems when an immigrant calls for medical attention in a new country . hence , health care system must adjust to the needs of ethnically diverse patients instead of the other way around . in order to provide trans cultural care , professional staff needs to know that historical , political and socioeconomic factors may influence ethnic minorities groups in sweden . effective and simple routines and facilities are also necessary when communicating with patients speaking a foreign language . health care staff needs to recognize that social problems might be medicalized and to develop a deeper understanding of the individual and how to meet individual needs in the light of immigrational and cultural background that might influence health .

background : we aimed to explore the background of refugees emigrating to sweden and their situation in the new country with special focus on their contacts with the swedish healthcare system.material and methods : our study has a qualitative design . data was collected between january and october 2013 during face - to - face interviews using open - ended questions . a qualitative content analysis was carried out in accordance with the graneheim and lundman method ( 2004 ) . the participants were 8 women and 7 men , aged between 65 and 86 years who had emigrated from bosnia and herzegovina . they had lived in sweden between 13 and 21 years.results:the findings revealed that the participants themselves experienced that change of scenery , culture and language influenced their own well - being . the most important finding was that language and communication difficulties are experienced as the major problems . these difficulties implied that all informants were forced to seek help from their children or to use an interpreter when they visited various healthcare institutions.conclusions:health care professionals need to be aware of the diverse needs of various ethnic groups in sweden , some of whom may carry traumatic experiences that could influence their health . in order to provide trans cultural care , a professional staff needs to know that historical , political and socioeconomic factors may influence ethnic minorities . health care staff needs to recognize that social problems might be medicalized . in particular this article emphasizes the problems associated with language .

1. INTRODUCTION 2. SUBJECTS AND METHODS 3. RESULTS 3.1 Life in Sweden 3.1.1. The refugee centre 3.1.2. The language barriers 3.1.3. The children's help 3.1.4. Having gratitude in Sweden 4. DISCUSSION 5. CONCLUSION

the most common reason for immigrating to sweden has been political , but family relations are also important ( 1 ) . bosnia and herzegovina is a part of the balkan region , and was geographically and politically bonded with the former yugoslavia until 1991 , at which time it became an independent state , after a period of war . between 1992 and 1995 , the people of bosnia and herzegovina experienced one of the most terrible wars seen in europe since the end of world war ii . in the same war , the civilians of bosnia and herzegovina were exposed to extreme threats and intense feelings of helplessness ( 3,4 ) . they have also had impact abroad as negative health consequences are especially high when relocation is due to severe conflicts , associated with violence and trauma ( 6 ) . since the 1970s , immigration has increased in sweden as well as in many other european countries . only in 2006 , 86,436 immigrants were granted residence permits in sweden , of which 25,096 were for protection or humanitarian reasons ( 7 ) . refugees , unlike many immigrants who have left their homes for economic reasons or to join family members already settled in another country , have fled in order to survive . most of these people have faced difficult transit experiences , culture shock , adjustment problems related to language and occupational change , and disruption in their sense of selfhood and community in the resettlement country ( 8) . additionally , a refugee has often suffered multiple losses such as severance from family and friends . in developing the theory , a major hurdle for health professionals was to discover meanings , practices and factors influencing care by religion , politics , economics , world view , environment , cultural values , history , language and gender . nurses and health professionals describe cultural competence as having the ability to understand cultural differences in order to provide quality care to a diversity of people . cultural competence involves nurses and health professionals continuously striving to provide effective care within the cultural confines of their patients . the central purpose of this theory is to discover and explain diverse and universal culturally based care factors influencing the health , well - being , illness or death of individuals or groups . the purpose and goal of the theory is to use research findings to provide culturally congruent , safe and meaningful care to clients of diverse or similar cultures . the three modes for congruent care , decisions and actions proposed in the theory are predicted to lead to health and well - being or to face illness and death . having knowledge of the patient 's cultural perspectives enables the nurse to provide more effective and appropriate care ( 9 , 10 ) . the language plays a central role in communication and interpretation of the culture , and it is important that nurses and health professionals first understand their own cultural values , attitudes , beliefs and practices that they have acquired from their own families before learning about other cultural ways ( 11 ) . this means that the way to express symptoms of disease and the expectations of the patient and on the patient by healthcare providers differ between regions , which may cause problems when an immigrant calls for medical attention in a new country ( 12 , 13 , 14 , 15 ) . the swedish health care system is expected to provide equal health care opportunities for all patients ( 16 ) . the swedish health and medical services act ( 1982 ) stipulate that health care must : be of good quality and take account of the patient 's need for safe care and treatment , be readily available , be based on respect for the self - determination and privacy of the patient andpromote good relationships between patients and health care providers . be of good quality and take account of the patient 's need for safe care and treatment , be readily available , be based on respect for the self - determination and privacy of the patient and promote good relationships between patients and health care providers . previous studies about the healthcare system among refugees showed that immigrants in sweden have significantly poorer health than native swedes ( 17 ) . and may therefore have increased need for healthcare services ( 18 , 19).previous studies made in sweden about refugees coming from burundi , colombia , iraq , kazakhstan , poland , kosovo and syria , showed that the main problems were related to feelings of being uninformed and being sent to various levels of care , which resulted in lack of trust , and feelings that no one took overall responsibility ( 20 ) . the aim of the present study was to describe the life of bosnian immigrants after arriving to sweden , with focus on contacts with healthcare system . our study is based on a qualitative design , as the study aimed to describe and analyze how patients experienced the swedish health system . inclusion criteria were subjects / persons coming from bosnia and herzegovina , who were more than 60 years old , had lived in sweden more than 10 years and had visited the healthcare canter more than twice during the past month . three of the participants declined participation without explanation and two of participants moved back to bosnia and herzegovina during the study period . accordingly , fifteen persons participated in the study : eight women and seven men , aged between 65 and 86 years . all participants had lived in sweden between 13 and 21 years ( table 1 ) . demographic data of informants data was collected through individual face - to - face interviews by the first author ( ns ) using open - ended questions , following an interview guide inspired by kvale ( 21 ) . the opening question was can you please tell me about your life after arriving to sweden ? and could you please tell me about your experiences to being a patient in sweden ? or all contacts with the informants were arranged in collaboration with one key person in the bosnian association of gothenburg , located close to the place where the participants lived . information concerning the aim and background of the study was printed and distributed to the informants , and repeated to them orally before the interview . participants were encouraged to speak freely using their own words and the interviewer encouraged the informants to respond to questions as comprehensively as possible . the interviews were carried out in bosnian by the second author , who is bilingual . the audio - recorded interviews were transcribed verbatim and analyzed in accordance with graneheim and lundman ( 2004 ) . due to the nature of our study , a qualitative dynamic analysis method that stays close to data was needed . in this context a qualitative content analysis method in accordance with graneheim and lundman chose for analysis and interpretation of the collected data . furthermore , content analysis method makes it possible to include interpretations of a latent content the transcripts were read carefully in order to identify the informants ' experiences and conceptions of the migration and its effect . subcategories that focused on the same problem were brought together , in order to create more extensive conceptions , which addressed an obvious issue ( 22 ) . according to the swedish law , there is no need for an ethical board review if written consent has been obtained from the participants and if there is no physical intervention involved in the study ( 23 ) . participants were informed that participation was voluntary and that confidentiality would be maintained . the analysis resulted in one category and four subcategories depending on how the participants described their lives in sweden . although this article is primarily concerned with the participant 's experiences in sweden , several questions concerned their lives in bosnia and herzegovina . informants in this study told us freely about their experiences before arriving in sweden , about their life in sweden and experiences during the visits to the healthcare centres . overview of the categories and subcategories as a consequence of everything happening in the world today you can no longer decide where to live . in the interviews it was found that older people found it especially hard to come to another country after leaving everything , their homes , their children , their relatives and friends , and all their property . the culture and environment were perceived as strange , which they all felt contributed to their health and well - being deteriorating . on arriving to sweden , all of the informants were placed in refugee centres and sent around sweden . (p1 ) coming from a country that is at war , and all the problems associated with this situation was emphasized by all the informants in our study as reasons not to get involved and learn the swedish language . all had hoped that the war would end in a few months so that they could return to their home country . this also means that the informants lacked the social skills that could connect them with swedish society . (p2 ) all the informants described when they were out walking , how they were assisted in the swedish language by their children , grandchildren or friends . at the same time it happened that the children were the ones who suffered most because they had to plan both for themselves and their families . (p5 ) language and communication barriers can lead to various difficulties and challenging situations . all informants had situations where they felt uncomfortable , laughed and felt as if they had lost their personal worth , a situation which is described in the following : two years ago i broke my hip , went to the hospital and met a swedish physician . in the health care services , for example , three of the informants felt that it was much more sensitive to talk about their symptoms if another person was present . medical staff unassisted by a professional interpreter can not be sure that the patient is given the opportunity to express their views completely . however , during the interviews it was revealed that the interpretation situations caused a variety of problems . (p10 ) among the respondents , there were some who had difficult experiences from the war in bosnia and herzegovina and did not want an interpreter . some of the informants did not want to accept that they speak serbo - croatian language , only the language of the country they come from . (p15 ) one participant described his relationship with his children in sweden like this : asking for help from my children would mean that i intervene into their lives , but i must . when it comes to being thankful that they were allowed to stay in sweden , emotions were mixed and informants had different opinions on the issue . we have always paid 50 % tax for our work in bosnia and herzegovina . if we lived in bosnia and herzegovina now , we would fight for our survival . as a consequence of everything happening in the world today you can no longer decide where to live . in the interviews it was found that older people found it especially hard to come to another country after leaving everything , their homes , their children , their relatives and friends , and all their property . the culture and environment were perceived as strange , which they all felt contributed to their health and well - being deteriorating . on arriving to sweden , all of the informants were placed in refugee centres and sent around sweden . (p3 ) a woman described her time in the centre as follows : the doctors talked and talked , explained , discussed with me and themselves i just looked at them and did not know if i should laugh or cry. (p1 ) coming from a country that is at war , and all the problems associated with this situation was emphasized by all the informants in our study as reasons not to get involved and learn the swedish language . this also means that the informants lacked the social skills that could connect them with swedish society . (p2 ) all the informants described when they were out walking , how they were assisted in the swedish language by their children , grandchildren or friends . at the same time it happened that the children were the ones who suffered most because they had to plan both for themselves and their families . (p5 ) language and communication barriers can lead to various difficulties and challenging situations . all informants had situations where they felt uncomfortable , laughed and felt as if they had lost their personal worth , a situation which is described in the following : two years ago i broke my hip , went to the hospital and met a swedish physician . in the health care services , for example , three of the informants felt that it was much more sensitive to talk about their symptoms if another person was present . medical staff unassisted by a professional interpreter can not be sure that the patient is given the opportunity to express their views completely . however , during the interviews it was revealed that the interpretation situations caused a variety of problems . about their experiences with interpreters one participant said the following : once we needed an interpreter and he came . (p10 ) among the respondents , there were some who had difficult experiences from the war in bosnia and herzegovina and did not want an interpreter . some of the informants did not want to accept that they speak serbo - croatian language , only the language of the country they come from . (p15 ) one participant described his relationship with his children in sweden like this : asking for help from my children would mean that i intervene into their lives , but i must . when it comes to being thankful that they were allowed to stay in sweden , emotions were mixed and informants had different opinions on the issue . we have always paid 50 % tax for our work in bosnia and herzegovina . if we lived in bosnia and herzegovina now , we would fight for our survival . on arriving to sweden , all of the informants were placed in refugee centres and sent around sweden . coming from a country that is at war , and all the problems associated with this situation was emphasized by all the informants in our study as reasons not to get involved and learn the swedish language . all had hoped that the war would end in a few months so that they could return to their home country . none of the informants knew more than a few swedish words and were thus unable to communicate in swedish . this also means that the informants lacked the social skills that could connect them with swedish society . (p2 ) all the informants described when they were out walking , how they were assisted in the swedish language by their children , grandchildren or friends . at the same time it happened that the children were the ones who suffered most because they had to plan both for themselves and their families . (p5 ) language and communication barriers can lead to various difficulties and challenging situations . all informants had situations where they felt uncomfortable , laughed and felt as if they had lost their personal worth , a situation which is described in the following : two years ago i broke my hip , went to the hospital and met a swedish physician . (p7 ) an interpreter was sometimes hired to deal with different situations where informants must communicate in swedish but could not . in the health care services , for example , three of the informants felt that it was much more sensitive to talk about their symptoms if another person was present . medical staff unassisted by a professional interpreter can not be sure that the patient is given the opportunity to express their views completely . however , during the interviews it was revealed that the interpretation situations caused a variety of problems . (p10 ) among the respondents , there were some who had difficult experiences from the war in bosnia and herzegovina and did not want an interpreter . some of the informants did not want to accept that they speak serbo - croatian language , only the language of the country they come from . (p15 ) one participant described his relationship with his children in sweden like this : asking for help from my children would mean that i intervene into their lives , but i must . all respondents found it difficult to move from their home country , leaving everything , to a completely different country . when it comes to being thankful that they were allowed to stay in sweden , emotions were mixed and informants had different opinions on the issue . we have always paid 50 % tax for our work in bosnia and herzegovina . if we lived in bosnia and herzegovina now , we would fight for our survival . we found that the change of scenery , culture and language influenced the well - being of the informants . during the war , the majority of informants were forced to leave their property , they were separated from their children and had to leave their work and social community thereby leaving language , their homeland and what they so far had experienced during their lives behind . language and communication difficulties were felt to be the major problems . based on our results , we could see that the informants perceived well - being in their home country when they lived in their own environment . there they lived with their own culture and spoke their own language . from having an active and fulfilling life , all informants switched to a less active and more isolated life . all informants felt a great loss of all those belongings they had left in their previous world . according to the participants in the interviews , their well - being deteriorated when they became refugees in sweden . despite the fact that the informants moved to another country , they had not left their culture and the social communication . none of the informants were able to understand the new language , or convey information in swedish . not being able to understand and make them understood was experienced as negative by all informants . all informants also regretted that they had never learned the swedish language . according to magnusson ( 26 ) the relationship between culture and communication is intimate and it is through communication that culture is passed on . without knowledge in swedish language the informants were not able to have swedish friends and without them the informants can not learn the swedish culture . the findings in our study are in the line with other studies which showed that some participants could not access information because they were unfamiliar with the structure of the swedish healthcare system . the fact that they were forced to live in another country eliminated their cultural and historical background , which had a negative impact on their social situation . language limitations and communication difficulties meant that all informants were forced to seek help from their relatives or had to use of an interpreter when they visited various institutions . according to gerrish ( 27 ) the quality of care is affected if the hospital does not have the opportunity to hire a professional interpreter , and allows children to interpret for their parents or relatives . on the other side hlander ( 31 ) patients because that healthcare professionals treat the patients group based on stereotypical images of how a patient from another country is supposed to act , think about care , need , so as the emphasis on cultural diversity might lead to exaggerated differences . one limitation of our study was that we interviewed the participants in their homes , and sometimes ( in the three cases ) the participants had guests . in two other cases children of the participants joined the participants during the interview period . the data in our study showed several major areas to improve access and quality of life for bosnian refugees in sweden . these areas are particularly concerned with language and cross - cultural communication and improvements could be made by organizing different meetings for swedes and immigrants from bosnia and herzegovina , organizing various courses in swedish for immigrants where they live , informing bosnian immigrants more about the swedish culture and the swedish health system and increasing the budget to train more interpreters in the swedish language . these are only a few areas that should be improved to make immigrants from bosnia and herzegovina feel better psychologically and physically . health care systems and patients are to a variable extent influenced by the local culture . this means that the way symptoms are described , the expectations of the patient , and the expectations that health care providers have on patients differ between regions , which may cause problems when an immigrant calls for medical attention in a new country . hence , health care system must adjust to the needs of ethnically diverse patients instead of the other way around . in order to provide trans cultural care , professional staff needs to know that historical , political and socioeconomic factors may influence ethnic minorities groups in sweden . health care staff needs to recognize that social problems might be medicalized and to develop a deeper understanding of the individual and how to meet individual needs in the light of immigrational and cultural background that might influence health .

historically , neutrophils were described as simple professional killers of invading pathogens to the human organism . in this regard , it was considered that only the release of various antimicrobial and cytotoxic proteins synthesized and distributed into different types of granules participated to the innate immune response mediated by neutrophils . granule types have been characterized to be readily mobilized upon an inflammatory stimulus at the plasma membrane in reverse order to their formation according to the formed - first - released - last model . indeed , in the different stages of neutrophil development , azurophil granules are formed first followed by specific granules , gelatinase granules , and , lastly , secretory vesicles , which are the most easily mobilized organelles in the mature neutrophils . due to recent progress , this classical view has been expanded by the acknowledgment that appropriately activated neutrophils constitute a substantial source of a variety of secreted cytokines supporting a direct contribution of these cells in the regulation framework of the adaptive immune response [ 35 ] . neutrophils not only are a source of de novo synthesized cytokines dependent on gene induction but also have the capacity to express cytokines at a basal level from preformed stores . however , precise intracellular localization of these packaged cytokines and mechanisms underlining their secretion remain largely elusive . the widely accepted assumption is that multiple secretory pathways coexist in neutrophils allowing the regulated release of diverse proinflammatory mediators . preformed cytokines are instantly released upon ligand - receptor signaling during the so - called regulated exocytosis process whereas de novo synthesized cytokines may be released after trafficking via recycling endosomes during the mechanism referred to as constitutive exocytosis these distinct processes selectively control the combination of granule proteins and cytokines released into the local microenvironment from neutrophils over a temporal and spatial range and are thus regulatory mechanisms important for the onset and resolution of inflammation enabling the development of an appropriate inflammatory response . it is now largely recognized that neutrophil - derived granule proteins and cytokines contribute to the maintenance of the inflammatory response and , when excessively secreted , to the ongoing process of tissue damage leading to the development of many chronic inflammatory disorders such as inflammatory bowel diseases , rheumatoid arthritis , chronic obstructive pulmonary disease , and atherosclerosis . determination of the regulatory mechanisms mediating the different patterns of cytokine trafficking and release may create opportunities to define new targets or strategies to selectively reduce cytokine secretion in clinical diseases . therefore , we selected relevant cytokines secreted by neutrophils , described to contribute to the development of chronic inflammatory diseases , in order to investigate their release in combination to degranulation upon stimulation with bacterial lipopolysaccharide ( lps ) . here , we propose an appealing model based on a linear fitting approach of cytokine secretion and degranulation giving a first basis for deeper understanding of the relationship between these two processes . it also provides a predictive view on the distribution of cytokines in neutrophils and offers an outstanding starting point to target future research on release mechanisms involved in inflammatory processes . peripheral blood of healthy volunteers was collected in edta - containing tubes ( bd vacutainer , bd biosciences , erembodegem , belgium ) . samples were collected in accordance with the good clinical and ethical practices , which have been approved by the ethics review panel ( erp ) of the university of luxembourg according to the comit national d'ethique de recherche ( cner ) from luxembourg . neutrophils were isolated from blood samples by polymorphprep separation procedure ( axis - shield , dundee , scotland ) according to manufacturer 's instructions . remaining erythrocytes in the neutrophil cell suspension were lysed for 10 min with red blood cell lysis buffer ( 155 mm nh4cl , 10 mm khco3 , 0.1 mm edta , and ph 7.4 ) . purity of isolated neutrophils was analyzed by the bd facscanto ii flow cytometer ( bd biosciences ) using two mixtures of selection markers cd66b - fitc / cd11b - pe / cd14-apc and cd15-fitc / cd16-pe / cd45-apc ( immunotools , friesoythe , germany ) on 10,000 events in the gated population of homogenous ( fsc - a versus ssc - a ) , single ( ssc - a versus ssc - h ) , and living cells ( negative cells for sytox blue staining ( invitrogen , gent , belgium ) ) . human neutrophils were cultured in x - vivo 15 medium with l - glutamine and gentamicin ( lonza ) at 37c and 5% co2 up to 24 h after purification . purified neutrophils were stimulated with bacterial lps from e. coli serotype o111:b4 ( sigma , bornem , belgium ) for simulating proinflammatory conditions . for kinetic studies of cytokine secretion and degranulation , neutrophils were stimulated with 100 ng / ml lps for 2 , 4 , 6 , 12 , and 24 h under serum - free conditions to avoid any serum component contamination , which could interfere with specific lps - induced cell responses . in accordance with the literature , the most relevant markers have been selected for degranulation analysis . degranulation was determined by measuring the expression of cd markers characteristic for azurophil granules ( cd63-pe ) , specific granules ( cd15-fitc , cd66b - fitc ) , gelatinase granules ( cd11b - pe ) , and secretory vesicles ( cd13-apc , cd14-apc , cd18-fitc , and cd45-apc ) at the plasma membrane by flow cytometry ( all antibodies are from bd biosciences except cd14-apc from immunotools ) . igg1-fitc , igg2a - pe ( bd biosciences ) , and igg1-apc antibodies ( immunotools ) were used as negative isotype controls to place the cells in the first decade of any plot , whereas cd45-fitc , cd45-pe , or cd45-apc ( bd biosciences ) single dye staining was used to set compensations . data analysis was performed by measuring the mean fluorescence intensity ( mfi ) for each cd marker with bd facsdiva software ( bd biosciences ) on the gated population of granulocytes ( fsc - a versus ssc - a ) , single ( ssc - a versus ssc - h ) , and living cells ( negative cells for sytox blue staining ( invitrogen ) ) . in total , 10,000 events were recorded per staining . the relative translocation of cd markers to the plasma membrane for each granule was determined by calculating the ratio between mfi of lps - stimulated cells and nonstimulated control from the same time point . density of human neutrophils was adjusted to 10 10 cells per condition for subsequent quantitative measurement of cytokine secretion by lps - stimulated cells , respectively . fresh supernatants were collected and used directly for cytometric bead array ( cba , bd biosciences ) analysis . the multiplex standard curve composed of mixed cytokine standards the following beads were used : ccl2 ( mcp1 , bead d8 ) , ccl3 ( mip1 , bead b9 ) , ccl4 ( mip1 , bead e4 ) , ccl5 ( rantes , bead d4 ) , il1a ( bead d6 ) , il1b ( bead b4 ) , il6 ( bead a7 ) , il8 ( cxcl8 , bead a9 ) , il12b ( bead e5 ) , and tnfa ( bead c4 ) . after 2 h of incubation , samples were rinsed with wash buffer and centrifuged . samples were washed again prior to flow cytometry analysis ( bd facscanto ii , bd biosciences ) . results were quantified using the standard curves and the flow cytometric analysis program ( fcap ) array software ( soft flow , minneapolis , usa ) . kinetic profiles of cytokine secretion and degranulation were imported into r statistical software ( https://www.r-project.org/ ) and a linear regression approach was applied . this approach was used to find the optimal proportionality factor , namely , the slope of the model , and provide methods to evaluate the significance of our models . all ratio values between lps - stimulated and nonstimulated control conditions ( stimulation points 0 , 2 , 4 , 6 , 12 , and 24 h ) from the time series of cytokine secretion and degranulation were log10 normalized to minimize scale effect . for each granule - specific cd marker , a linear model has been fitted with the secreted cytokines . for each model , anova analysis has been performed and the adjusted r - squared ( rsq ) value and the slope of the model were retained . models with a significant difference to the null model ( p value 0.05 ) and with a high adjusted rsq value between degranulation marker and secreted cytokine ( rsq r , with r being determined by simulations ; see section 2 ) were considered as underlying a strong similarity of pattern between a secreted cytokine and a mobilized degranulation marker . then , cytokines linked to the same degranulation marker were clustered . to visualize these clusters , time series of the degranulation marker and its relative cytokines of the cluster to permit comparison between time series from different scales , values from the secreted cytokines were divided by the slope of their linear model . to define the optimal rsq threshold , we simulated the fitting between pairs of granule marker and cytokine with controlled perturbations between them and choose the threshold by determining at which level of perturbation the rsq was drastically dropping . the simulations were designed as follows : time series of each granule - specific marker and cytokines were taken individually ( 19 time series in total ) and used to simulate matching time series with more or less perturbations . for the six points of each time series ( 0 , 2 , 4 , 6 , 12 , and 24 h ) , a random number between vi e and vi + e was drawn . vi is the ith element of the time series and e is a predefined constant . e controls the intensity of the perturbation : the highest e is the more different both profiles are expected to be . all values from 0 to 1 with a step of 0.1 were tested for e ( 0 , 0.1 , 0.2 , etc . . then , the rsq of the original profile versus simulated profile was computed as mentioned in linear fitting approach via r statistical software . this process was repeated 1000 times and , for each of the 19 original profiles , the average of the rsqs was computed and plotted against the e value . to define the optimal rsq threshold that defines a cut - off between linear fitting models with high rsq and low rsq , we clustered the distribution of averaged rsq into two groups , using a k - means approach ( a silhouette analysis of all clustering solution with 2 to 10 clusters confirmed that using 2 clusters was the best solution , data not shown ) . the last element of the cluster with the highest center , namely , 0.796 , was taken as rsq threshold for our analysis . statistical analyses were performed using the prism6 software ( graph pad software , la jolla , ca , usa ) . when normality and homogeneity of variances were ascertained , as determined by the f - test , student 's t - test analyses were performed to establish two group comparisons . since recent reports have implicated neutrophils in the development of chronic inflammatory disorders , we wanted to characterize the regulatory mechanisms in the release of neutrophil - derived products . in a first step , cytokine candidates found secreted by highly purified ( 98% ) neutrophils upon lps stimulation were selected for integration into our mathematical model . these cytokines have a particular relevance since they have been reported to contribute to the development of different chronic inflammatory diseases through the recruitment of diverse immune cells to the inflammatory site ( table 1 ) . to develop a reliable model that investigates the relationship between degranulation and cytokine secretion , neutrophils were treated for 0 h , 2 h , 4 h , 6 h , 12 h , and 24 h with lps 100 ng / ml since maximal peak of secretion for cytokines was reached at this concentration ( data not shown ) . . a basal secretion level of all cytokines was detected in supernatants from neutrophils under nonstimulated conditions . secretion of most of the cytokines released into the extracellular medium was augmented with increasing time of lps stimulation , except for il12 and ccl5 whose release was not significantly affected by lps treatment ( figure 1 ) . the secretion pattern was different for each cytokine in the way that different profiles could be identified . except for il1 , secretion levels of cytokines were maximal 6 h or 12 h after treatment with lps and decreased after 24 h. maximal cytokine secretion was observed at ( i ) 6 h lps for ccl3 and ( ii ) 12 h lps for tnf , il6 , il8 , ccl2 , il1 , and ccl4 . tnf , il1 , il1 , and ccl3 were only discretely secreted ( 150 pg / ml ) whereas il6 , ccl2 , and ccl4 were secreted at an intermediate level ( ~250600 pg / ml ) and il8 was highly secreted ( 30000 pg / ml ) . to collect data for the implementation of the model , the second series of experiments consisted in identifying the kinetic degranulation profile of the different granule types upon time - dependent lps stimulation . degranulation can be determined by the upregulation of granule membrane molecules as a consequence of membrane fusion from granules with the plasma membrane . therefore , lps - treated cells were analyzed for cell surface expression of several cd molecules known as degranulation markers . results showed that lps stimulation affected the ease of mobilization of intracellular granule types in neutrophils in a time - dependent manner , as reflected by increased translocation of cd markers to the cell surface ( figure 2 ) . in a temporal pattern , lps stimulation induced the release of specific granules as demonstrated by the redistribution of cd15 and cd66b to the plasma membrane . translocation of these cd markers towards the plasma membrane was scattered over a time interval of 4 h and 24 h. in a similar way , lps stimulation increased the presence of cd11b as well as cd13 , cd18 , and cd45 at the plasma membrane reflecting an increased release of gelatinase granules and secretory vesicles , respectively . as observed with cd15 and cd66b for the specific granules , increase of these cd markers maximal expression at the plasma membrane for all the cd markers was detected after 6 h or 12 h of lps stimulation ( figure 2 ) . it must be noted that lps was unable to trigger the mobilization of azurophil granules since cd63 expression was not changed at the plasma membrane . many approaches exist for the examination of time series of expression data ( e.g. , ) but none of them could be applied to analyze short - time series of secretion . for this reason , we used a novel model approach to explore the relationship between cytokine secretion and degranulation by their kinetic profiles ( figure 3 ) . we hypothesized that time series of secreted cytokines with similar pattern to time series of degranulation markers present at the plasma membrane should have proportional values at each time point of lps stimulation , so that a proportionality factor between the two profile curves can be defined . to address this question , we choose to use the linear regression approach , which fits best to our needs : it captures proportionality well , can be used with only one pair of profiles ( in our case , cytokine versus granule - specific marker ) , and includes measures to evaluate the results ( significance of the model and r - squared value , section 2 ) . all ratio values between lps - stimulated and nonstimulated control conditions from the time series of cytokine secretion and degranulation ( table 2 ) were log10 normalized . while the anova analyses the efficiency of this model ( i.e. , proportional kinetic profile curves are significantly different from the null model ) , the adjusted rsq value measures the correlation between the kinetic profiles ( i.e. , proximity to the linear fitting ) . the optimal rsq value was determined by simulations , in which predefined perturbations were introduced to our kinetic profiles ( section 2 ) . how augmenting perturbations ( e from 0 to 1 ) influenced the linear fitting of two time series , in our example , the linear fitting between the granule marker cd11b and the cytokine il8 , the original kinetic profile is depicted by the black line whereas the one with perturbations is represented by the red line . by plotting the average rsq values ( derived from 1000 repetitions of simulations ) against e values , k - means clustering can differentiate between high ( black ) and low ( red ) rsq ( figure 4(b ) ) . by setting these parameters nonsignificant outcomes with low rsq were eliminated , and the threshold for rsq was set to rsq 0.796 . an example of a significant linear fitting model is shown ( figure 4(c ) ) , in which the behaviour of il8 is correlated to cd11b . due to its p value of 0 and rsq of 0.91 , the correlation between il8 and cd11b fits to the model . the granule membrane molecules ( cd63 , cd66b , cd11b , and cd45 ) , specific of each type of granule , that are most highly upregulated have been targeted to show the linear fitting approach ( figure 5 ) . this method applied to the time series data showed that the secretion of three selected proinflammatory cytokines ( il8 , il6 , and il1 ) strongly correlated with the release of secretory vesicles , gelatinase granules , and specific granules ( figure 5 ) . the release of the cytokine il8 fitted to cd66b suggesting that secretion of this cytokine correlated to specific granules ( figure 5(b ) ) . moreover , time series of il1 , il8 , and il6 release were strongly correlated to the degranulation marker cd11b , showing a relationship between gelatinase granules and these cytokines ( figure 5(c ) ) . furthermore , secretory vesicles represented by the marker cd45 were fitted to il8 ( figure 5(d ) ) . since no significant cytokine correlation has been observed for cd63 , azurophil granules are probably not associated with cytokine secretion ( figure 5(a ) ) . for many years , the contributory impact of neutrophils to the development of chronic inflammation was not seriously taken into account since they have been considered as terminally differentiated cells synthesizing low amount of rna and protein . however , the vast number of neutrophils found at the site of infection can not be neglected due to the fact that their secreted amounts of granule proteins and cytokines exert a cumulative and synergistic effect on the inflammatory tissue environment . these proinflammatory soluble mediators are highly decisive for the onset of inflammatory processes and the activation and the recruitment of various immune cells to the infection site . however , little is known about the combination in which cytokines and granule proteins are secreted by neutrophils . in the present report , we therefore aimed to predict the spatiotemporal regulation of proinflammatory mediator release in neutrophils . for this purpose , lps has been used as stimulus agent since it has been well described to induce the secretion of granule contents and cytokines [ 17 , 38 ] . once the model is established , it could constitute an important tool to investigate other stimulation conditions ( fmlf , tnf , il8 , or combination of stimuli ) in order to mimic different microenvironmental conditions ( e.g. , healthy and pathological diseases ) and help to improve our knowledge of inflammatory processes . our study is the first to propose an original approach allowing the establishment of a relationship between cytokine secretion and degranulation in neutrophils . we choose to use the linear fitting approach to integrate data generated from own experiments and obtained from lps - mediated short - time series of degranulation and cytokine secretion . other approaches , such as pearson correlation , are based only on the average of all values correlated . in contrast , our model is able to reliably predict time - specific associations between the two dynamic functions in neutrophils , respecting each time point of stimulation . according to our results , a number of cytokines could be fitted to the different types of neutrophil granules . these granules have been characterized to be mobilized towards the plasma membrane in a hierarchically and more precisely reverse order to their formation according to the formed - first - released - last model . our model illustrates the fact that neutrophil - derived cytokines and granules are released in a hierarchical sequence in accordance with their roles during the microbial elimination processes and inflammatory response ( figure 6(a ) ) . in this study , the linear fitting approach ( i ) gives us information about the concurrent behavior of cytokine secretion and degranulation upon inflammation , thus underlining the key role of both functions in the regulation of inflammatory responses and ( ii ) can represent an attractive method to investigate the possible mobilization or localization of cytokines in the different types of granules . given that cytokines can exert pleiotropic functions , some of them are probably localized in different types of granules as suggested by our model . in this sense , we found that il8 correlated to secretory vesicles ( figure 6(a ) ) , which are the most easily mobilized organelles in mature neutrophils . who reported that il8 is stored in cytoplasmic granules in resting peripheral blood neutrophils and , thus , can be rapidly mobilized and released by the cells . il8 can be secreted into the extracellular milieu from intracellular stores or by de novo synthesis via the classical secretory pathway . two phases of secretion have been described : an early secretory phase which is directly induced by lps and a late secretory phase which results from lps - stimulated release of other proinflammatory mediators such as tnf and il1 . the fact that il8 is stored in different types of granules could allow its secretion over a large time interval . large amount of il8 released could be explained by a positive feedback loop generated by mmp-9 on the il8-induced neutrophil activity . indeed , it has been previously reported that mmp-9 released from gelatinase granules is able to process il8 which is stored in the same granules as mmp-9 before secretion as shown by our linear fitting approach . il8 cleaved by mmp-9 can enhance neutrophil degranulation , in comparison to nondegraded il8 , and thus increase the quantity of mmp-9 and il8 released leading to an amplification of this system . our results also show a significant fitting between kinetics of il6 secretion and the release of gelatinase granules . in line with this observation , terebuh et al . showed by immunohistochemical staining of neutrophils that il6 might be localized in gelatinase granules and secretory vesicles finally , in our model , il1 could be fitted to gelatinase degranulation as previously postulated . il1 is secreted by a nonclassical secretory pathway ( independent of endoplasmic reticulum and golgi apparatus ) . different release models have been suggested for il1 but the mechanisms associated are poorly understood and still controversially discussed . our data indicate that the process of il1 secretion involves a trafficking via granules which could be related to gelatinase granules . in contrast to other granules , degranulation of azurophil granules seems unchanged by lps suggesting that either ( i ) this granule type may require further cell activation to induce its mobilization towards the plasma membrane or ( ii ) the upregulation of cd63 at the plasma membrane might not be significant enough to detect since azurophil granules are rather poor in receptors in contrast to secretory vesicles . for this reason , no cytokine could probably be fitted to cd63 in human neutrophils . in this view , late release of azurophil granule contents can be explained by the involvement of these proteins in neutrophil extracellular trap formation . in this regard , the role of these granules during cytokine secretion appears very restricted . this assumption is supported by our results showing that azurophil granules are not able to translocate to the plasma membrane upon lps stimulation in neutrophils . intracellular localization of cytokines in neutrophils remains largely elusive due to the fact that reliable staining for electron microscopy is facing challenges as the low intracellular amount of cytokines and other techniques used to document subcellular organelle location of cytokines have limited resolution ( e.g. , subcellular fractionation ) . in comparison to the modelling approach proposed by rrvig et al . , which is based on proteomic and mrna array data to predict localization of proteins in granules , our approach is complementary by including functional data analysis . hence , the linear fitting between degranulation and cytokine release in lps - treated neutrophils represents an attractive method to investigate the possible localization of cytokines in the different types of granules even if additional experiments are required to confirm the intracellular localization of cytokines . furthermore , since our linear fitting approach has been adapted to investigate secretion kinetics , it can easily be extrapolated for the analysis of other short - time series deriving from other cell types , disease , or developmental states , for example , protein arrays or proteomics data . our linear fitting approach primarily constitutes a tool aiming to investigate regulatory mechanisms during neutrophil exocytosis but can also serve as basis to identify regulatory proteins by the supplementary analysis of proteins involved in exocytosis ( e.g. , snare and rab proteins ) and the construction of a dynamic regulatory network .

neutrophils participate in the maintenance of host integrity by releasing various cytotoxic proteins during degranulation . due to recent advances , a major role has been attributed to neutrophil - derived cytokine secretion in the initiation , exacerbation , and resolution of inflammatory responses . because the release of neutrophil - derived products orchestrates the action of other immune cells at the infection site and , thus , can contribute to the development of chronic inflammatory diseases , we aimed to investigate in more detail the spatiotemporal regulation of neutrophil - mediated release mechanisms of proinflammatory mediators . purified human neutrophils were stimulated for different time points with lipopolysaccharide . cells and supernatants were analyzed by flow cytometry techniques and used to establish secretion profiles of granules and cytokines . to analyze the link between cytokine release and degranulation time series , we propose an original strategy based on linear fitting , which may be used as a guideline , to ( i ) define the relationship of granule proteins and cytokines secreted to the inflammatory site and ( ii ) investigate the spatial regulation of neutrophil cytokine release . the model approach presented here aims to predict the correlation between neutrophil - derived cytokine secretion and degranulation and may easily be extrapolated to investigate the relationship between other types of time series of functional processes .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Conclusion

historically , neutrophils were described as simple professional killers of invading pathogens to the human organism . in this regard , it was considered that only the release of various antimicrobial and cytotoxic proteins synthesized and distributed into different types of granules participated to the innate immune response mediated by neutrophils . granule types have been characterized to be readily mobilized upon an inflammatory stimulus at the plasma membrane in reverse order to their formation according to the formed - first - released - last model . indeed , in the different stages of neutrophil development , azurophil granules are formed first followed by specific granules , gelatinase granules , and , lastly , secretory vesicles , which are the most easily mobilized organelles in the mature neutrophils . due to recent progress , this classical view has been expanded by the acknowledgment that appropriately activated neutrophils constitute a substantial source of a variety of secreted cytokines supporting a direct contribution of these cells in the regulation framework of the adaptive immune response [ 35 ] . the widely accepted assumption is that multiple secretory pathways coexist in neutrophils allowing the regulated release of diverse proinflammatory mediators . preformed cytokines are instantly released upon ligand - receptor signaling during the so - called regulated exocytosis process whereas de novo synthesized cytokines may be released after trafficking via recycling endosomes during the mechanism referred to as constitutive exocytosis these distinct processes selectively control the combination of granule proteins and cytokines released into the local microenvironment from neutrophils over a temporal and spatial range and are thus regulatory mechanisms important for the onset and resolution of inflammation enabling the development of an appropriate inflammatory response . it is now largely recognized that neutrophil - derived granule proteins and cytokines contribute to the maintenance of the inflammatory response and , when excessively secreted , to the ongoing process of tissue damage leading to the development of many chronic inflammatory disorders such as inflammatory bowel diseases , rheumatoid arthritis , chronic obstructive pulmonary disease , and atherosclerosis . determination of the regulatory mechanisms mediating the different patterns of cytokine trafficking and release may create opportunities to define new targets or strategies to selectively reduce cytokine secretion in clinical diseases . therefore , we selected relevant cytokines secreted by neutrophils , described to contribute to the development of chronic inflammatory diseases , in order to investigate their release in combination to degranulation upon stimulation with bacterial lipopolysaccharide ( lps ) . here , we propose an appealing model based on a linear fitting approach of cytokine secretion and degranulation giving a first basis for deeper understanding of the relationship between these two processes . it also provides a predictive view on the distribution of cytokines in neutrophils and offers an outstanding starting point to target future research on release mechanisms involved in inflammatory processes . samples were collected in accordance with the good clinical and ethical practices , which have been approved by the ethics review panel ( erp ) of the university of luxembourg according to the comit national d'ethique de recherche ( cner ) from luxembourg . neutrophils were isolated from blood samples by polymorphprep separation procedure ( axis - shield , dundee , scotland ) according to manufacturer 's instructions . remaining erythrocytes in the neutrophil cell suspension were lysed for 10 min with red blood cell lysis buffer ( 155 mm nh4cl , 10 mm khco3 , 0.1 mm edta , and ph 7.4 ) . purity of isolated neutrophils was analyzed by the bd facscanto ii flow cytometer ( bd biosciences ) using two mixtures of selection markers cd66b - fitc / cd11b - pe / cd14-apc and cd15-fitc / cd16-pe / cd45-apc ( immunotools , friesoythe , germany ) on 10,000 events in the gated population of homogenous ( fsc - a versus ssc - a ) , single ( ssc - a versus ssc - h ) , and living cells ( negative cells for sytox blue staining ( invitrogen , gent , belgium ) ) . human neutrophils were cultured in x - vivo 15 medium with l - glutamine and gentamicin ( lonza ) at 37c and 5% co2 up to 24 h after purification . purified neutrophils were stimulated with bacterial lps from e. coli serotype o111:b4 ( sigma , bornem , belgium ) for simulating proinflammatory conditions . for kinetic studies of cytokine secretion and degranulation , neutrophils were stimulated with 100 ng / ml lps for 2 , 4 , 6 , 12 , and 24 h under serum - free conditions to avoid any serum component contamination , which could interfere with specific lps - induced cell responses . degranulation was determined by measuring the expression of cd markers characteristic for azurophil granules ( cd63-pe ) , specific granules ( cd15-fitc , cd66b - fitc ) , gelatinase granules ( cd11b - pe ) , and secretory vesicles ( cd13-apc , cd14-apc , cd18-fitc , and cd45-apc ) at the plasma membrane by flow cytometry ( all antibodies are from bd biosciences except cd14-apc from immunotools ) . igg1-fitc , igg2a - pe ( bd biosciences ) , and igg1-apc antibodies ( immunotools ) were used as negative isotype controls to place the cells in the first decade of any plot , whereas cd45-fitc , cd45-pe , or cd45-apc ( bd biosciences ) single dye staining was used to set compensations . the relative translocation of cd markers to the plasma membrane for each granule was determined by calculating the ratio between mfi of lps - stimulated cells and nonstimulated control from the same time point . density of human neutrophils was adjusted to 10 10 cells per condition for subsequent quantitative measurement of cytokine secretion by lps - stimulated cells , respectively . fresh supernatants were collected and used directly for cytometric bead array ( cba , bd biosciences ) analysis . samples were washed again prior to flow cytometry analysis ( bd facscanto ii , bd biosciences ) . kinetic profiles of cytokine secretion and degranulation were imported into r statistical software ( https://www.r-project.org/ ) and a linear regression approach was applied . this approach was used to find the optimal proportionality factor , namely , the slope of the model , and provide methods to evaluate the significance of our models . all ratio values between lps - stimulated and nonstimulated control conditions ( stimulation points 0 , 2 , 4 , 6 , 12 , and 24 h ) from the time series of cytokine secretion and degranulation were log10 normalized to minimize scale effect . for each granule - specific cd marker , a linear model has been fitted with the secreted cytokines . for each model , anova analysis has been performed and the adjusted r - squared ( rsq ) value and the slope of the model were retained . models with a significant difference to the null model ( p value 0.05 ) and with a high adjusted rsq value between degranulation marker and secreted cytokine ( rsq r , with r being determined by simulations ; see section 2 ) were considered as underlying a strong similarity of pattern between a secreted cytokine and a mobilized degranulation marker . to visualize these clusters , time series of the degranulation marker and its relative cytokines of the cluster to permit comparison between time series from different scales , values from the secreted cytokines were divided by the slope of their linear model . to define the optimal rsq threshold , we simulated the fitting between pairs of granule marker and cytokine with controlled perturbations between them and choose the threshold by determining at which level of perturbation the rsq was drastically dropping . the simulations were designed as follows : time series of each granule - specific marker and cytokines were taken individually ( 19 time series in total ) and used to simulate matching time series with more or less perturbations . for the six points of each time series ( 0 , 2 , 4 , 6 , 12 , and 24 h ) , a random number between vi e and vi + e was drawn . then , the rsq of the original profile versus simulated profile was computed as mentioned in linear fitting approach via r statistical software . to define the optimal rsq threshold that defines a cut - off between linear fitting models with high rsq and low rsq , we clustered the distribution of averaged rsq into two groups , using a k - means approach ( a silhouette analysis of all clustering solution with 2 to 10 clusters confirmed that using 2 clusters was the best solution , data not shown ) . since recent reports have implicated neutrophils in the development of chronic inflammatory disorders , we wanted to characterize the regulatory mechanisms in the release of neutrophil - derived products . these cytokines have a particular relevance since they have been reported to contribute to the development of different chronic inflammatory diseases through the recruitment of diverse immune cells to the inflammatory site ( table 1 ) . to develop a reliable model that investigates the relationship between degranulation and cytokine secretion , neutrophils were treated for 0 h , 2 h , 4 h , 6 h , 12 h , and 24 h with lps 100 ng / ml since maximal peak of secretion for cytokines was reached at this concentration ( data not shown ) . except for il1 , secretion levels of cytokines were maximal 6 h or 12 h after treatment with lps and decreased after 24 h. maximal cytokine secretion was observed at ( i ) 6 h lps for ccl3 and ( ii ) 12 h lps for tnf , il6 , il8 , ccl2 , il1 , and ccl4 . to collect data for the implementation of the model , the second series of experiments consisted in identifying the kinetic degranulation profile of the different granule types upon time - dependent lps stimulation . degranulation can be determined by the upregulation of granule membrane molecules as a consequence of membrane fusion from granules with the plasma membrane . therefore , lps - treated cells were analyzed for cell surface expression of several cd molecules known as degranulation markers . results showed that lps stimulation affected the ease of mobilization of intracellular granule types in neutrophils in a time - dependent manner , as reflected by increased translocation of cd markers to the cell surface ( figure 2 ) . in a temporal pattern , lps stimulation induced the release of specific granules as demonstrated by the redistribution of cd15 and cd66b to the plasma membrane . translocation of these cd markers towards the plasma membrane was scattered over a time interval of 4 h and 24 h. in a similar way , lps stimulation increased the presence of cd11b as well as cd13 , cd18 , and cd45 at the plasma membrane reflecting an increased release of gelatinase granules and secretory vesicles , respectively . many approaches exist for the examination of time series of expression data ( e.g. , ) but none of them could be applied to analyze short - time series of secretion . for this reason , we used a novel model approach to explore the relationship between cytokine secretion and degranulation by their kinetic profiles ( figure 3 ) . we hypothesized that time series of secreted cytokines with similar pattern to time series of degranulation markers present at the plasma membrane should have proportional values at each time point of lps stimulation , so that a proportionality factor between the two profile curves can be defined . to address this question , we choose to use the linear regression approach , which fits best to our needs : it captures proportionality well , can be used with only one pair of profiles ( in our case , cytokine versus granule - specific marker ) , and includes measures to evaluate the results ( significance of the model and r - squared value , section 2 ) . all ratio values between lps - stimulated and nonstimulated control conditions from the time series of cytokine secretion and degranulation ( table 2 ) were log10 normalized . , proportional kinetic profile curves are significantly different from the null model ) , the adjusted rsq value measures the correlation between the kinetic profiles ( i.e. , proximity to the linear fitting ) . how augmenting perturbations ( e from 0 to 1 ) influenced the linear fitting of two time series , in our example , the linear fitting between the granule marker cd11b and the cytokine il8 , the original kinetic profile is depicted by the black line whereas the one with perturbations is represented by the red line . due to its p value of 0 and rsq of 0.91 , the correlation between il8 and cd11b fits to the model . the granule membrane molecules ( cd63 , cd66b , cd11b , and cd45 ) , specific of each type of granule , that are most highly upregulated have been targeted to show the linear fitting approach ( figure 5 ) . this method applied to the time series data showed that the secretion of three selected proinflammatory cytokines ( il8 , il6 , and il1 ) strongly correlated with the release of secretory vesicles , gelatinase granules , and specific granules ( figure 5 ) . the release of the cytokine il8 fitted to cd66b suggesting that secretion of this cytokine correlated to specific granules ( figure 5(b ) ) . moreover , time series of il1 , il8 , and il6 release were strongly correlated to the degranulation marker cd11b , showing a relationship between gelatinase granules and these cytokines ( figure 5(c ) ) . since no significant cytokine correlation has been observed for cd63 , azurophil granules are probably not associated with cytokine secretion ( figure 5(a ) ) . for many years , the contributory impact of neutrophils to the development of chronic inflammation was not seriously taken into account since they have been considered as terminally differentiated cells synthesizing low amount of rna and protein . however , the vast number of neutrophils found at the site of infection can not be neglected due to the fact that their secreted amounts of granule proteins and cytokines exert a cumulative and synergistic effect on the inflammatory tissue environment . these proinflammatory soluble mediators are highly decisive for the onset of inflammatory processes and the activation and the recruitment of various immune cells to the infection site . in the present report , we therefore aimed to predict the spatiotemporal regulation of proinflammatory mediator release in neutrophils . for this purpose , lps has been used as stimulus agent since it has been well described to induce the secretion of granule contents and cytokines [ 17 , 38 ] . once the model is established , it could constitute an important tool to investigate other stimulation conditions ( fmlf , tnf , il8 , or combination of stimuli ) in order to mimic different microenvironmental conditions ( e.g. , healthy and pathological diseases ) and help to improve our knowledge of inflammatory processes . our study is the first to propose an original approach allowing the establishment of a relationship between cytokine secretion and degranulation in neutrophils . we choose to use the linear fitting approach to integrate data generated from own experiments and obtained from lps - mediated short - time series of degranulation and cytokine secretion . according to our results , a number of cytokines could be fitted to the different types of neutrophil granules . our model illustrates the fact that neutrophil - derived cytokines and granules are released in a hierarchical sequence in accordance with their roles during the microbial elimination processes and inflammatory response ( figure 6(a ) ) . in this study , the linear fitting approach ( i ) gives us information about the concurrent behavior of cytokine secretion and degranulation upon inflammation , thus underlining the key role of both functions in the regulation of inflammatory responses and ( ii ) can represent an attractive method to investigate the possible mobilization or localization of cytokines in the different types of granules . given that cytokines can exert pleiotropic functions , some of them are probably localized in different types of granules as suggested by our model . in this sense , we found that il8 correlated to secretory vesicles ( figure 6(a ) ) , which are the most easily mobilized organelles in mature neutrophils . who reported that il8 is stored in cytoplasmic granules in resting peripheral blood neutrophils and , thus , can be rapidly mobilized and released by the cells . two phases of secretion have been described : an early secretory phase which is directly induced by lps and a late secretory phase which results from lps - stimulated release of other proinflammatory mediators such as tnf and il1 . the fact that il8 is stored in different types of granules could allow its secretion over a large time interval . indeed , it has been previously reported that mmp-9 released from gelatinase granules is able to process il8 which is stored in the same granules as mmp-9 before secretion as shown by our linear fitting approach . our results also show a significant fitting between kinetics of il6 secretion and the release of gelatinase granules . showed by immunohistochemical staining of neutrophils that il6 might be localized in gelatinase granules and secretory vesicles finally , in our model , il1 could be fitted to gelatinase degranulation as previously postulated . in contrast to other granules , degranulation of azurophil granules seems unchanged by lps suggesting that either ( i ) this granule type may require further cell activation to induce its mobilization towards the plasma membrane or ( ii ) the upregulation of cd63 at the plasma membrane might not be significant enough to detect since azurophil granules are rather poor in receptors in contrast to secretory vesicles . for this reason , no cytokine could probably be fitted to cd63 in human neutrophils . in this regard , the role of these granules during cytokine secretion appears very restricted . intracellular localization of cytokines in neutrophils remains largely elusive due to the fact that reliable staining for electron microscopy is facing challenges as the low intracellular amount of cytokines and other techniques used to document subcellular organelle location of cytokines have limited resolution ( e.g. in comparison to the modelling approach proposed by rrvig et al . , which is based on proteomic and mrna array data to predict localization of proteins in granules , our approach is complementary by including functional data analysis . hence , the linear fitting between degranulation and cytokine release in lps - treated neutrophils represents an attractive method to investigate the possible localization of cytokines in the different types of granules even if additional experiments are required to confirm the intracellular localization of cytokines . furthermore , since our linear fitting approach has been adapted to investigate secretion kinetics , it can easily be extrapolated for the analysis of other short - time series deriving from other cell types , disease , or developmental states , for example , protein arrays or proteomics data . our linear fitting approach primarily constitutes a tool aiming to investigate regulatory mechanisms during neutrophil exocytosis but can also serve as basis to identify regulatory proteins by the supplementary analysis of proteins involved in exocytosis ( e.g.

coronary spasm is closely associated with endothelial dysfunction and plays a potential role in the progression of atherosclerosis.1 , 2 , 3 whereas patients with coronary spasm have better prognosis for cardiovascular events than those with moreserious coronary artery disease ( cad ) , coronary atherosclerotic stenosis correlates with cardiovascular events in patients with coronary vasospastic angina ( vsa).4 , 5 , 6 statins reduce the risk of cardiovascular events in obstructive cad7 , 8 , 9 , 10 , 11 and also ameliorate endothelial dysfunction.12 , 13 , 14 in a prospective , randomized study , we showed previously that the addition of fluvastatin at 30 mg / day to conventional medical therapy with calciumchannel blockers ( ccbs ) for 6 months significantly reduced acetylcholine ( ach)provoked coronary spasm in vsa patients free of coronary organic stenosis.15 however , there is no evidence at present that statin therapy improves the prognosis of vsa patients . the purpose of this study was to assess the effects of statin therapy on clinical outcome of vsa patients free of coronary organic stenosis . the present study was a retrospective observational study that enrolled 1877 consecutive japanese patients who had typical or atypical anginalike chest pain and underwent achprovocation testing at our hospital between january 1991 and december 2010 . we excluded 117 patients for the following reasons : acute myocardial infarction ( ami ; n=20 ) , cardiomyopathy ( n=75 ) , brugada syndrome ( n=10 ) , and other miscellaneous conditions ( n=12 ) . to investigate the prognosis of patients free of significant atherosclerotic stenosis thus , 640 vsa patients from the remaining 1402 free of significant organic stenosis were included for analysis ( figure 1 ) . we defined significant atherosclerotic stenosis ( hereafter referred to as organic stenosis ) as 75% stenosis ( 5175% narrowing of the luminal diameter ) of the right , left anterior descending , or left circumflex coronary artery and the major branches , or 50% stenosis ( 2650% narrowing of the luminal diameter ) of the left main trunk , as defined by the american heart association ( aha ) classification ( by visual estimation).16 flow chart of the patient recruitment process . the achprovocation test was conducted as described previously in the vsa guideline by the japanese circulation society.17 achinduced coronary spasm was defined as total or subtotal obstruction within the borders of a single isolated coronary segment , or severe diffuse vasoconstriction observed in more than 2 adjacent coronary segments of epicardial coronary arteries associated with transient myocardial ischemia , as evidenced by ischemic stsegment changes on the electrocardiogram ( ecg ) , as described previously.6 , 18 the study protocol was approved by the human ethics review committee of kumamoto university ( kumamoto , japan ) , and a signed consent form was obtained from each subject . we defined the risk factors for cad as current smoking , hypertension , dyslipidemia , diabetes mellitus , and family history of ischemic heart disease ( ihd ) , as described previously.6 , 18 selection of medical treatments for vsa was left to the discretion of each attending physician . followup data were collected from the medical records , the patients , their families , or the attending physicians . the primary endpoint was defined as major adverse cardiac events ( mace ) , including cardiac death , hospitalization for ami , and unstable angina . the secondary endpoint was allcause mortality during the followup period that began from the date of the diagnosis of vsa to the date of the first event or until december 2012 . we defined cardiac death as sudden death or death associated with ami , and ami as the presence of severe chest discomfort lasting > 30 minutes associated with stsegment changes and elevated cardiac enzyme levels . unstable angina was defined as recurrence or worsening of chest discomfort or pain associated with ischemic ecg changes . spasminduced ami and unstable angina were diagnosed by transient ischemic ecg changes during spontaneous nitrateresponsive angina episode , with at least one of the following : ( 1 ) absence of culprit lesion , spontaneous coronary spasm relieved by administration of intracoronary nitroglycerin , or achprovoked coronary spasm on coronary angiography or ( 2 ) absence of ischemic findings suggestive of significant atherosclerotic stenosis on stress testing ( exercise ecg and/or myocardial scintigraphy ) . data of normally distributed continuous variables are expressed as meansd , whereas those with skewed distribution are presented as median values ( interquartile range ; iqr ) , and categorical variables as frequencies and percentages . group comparisons were analyzed by the unpaired t test or mann whitney u test for continuous variables , the chisquare test or fisher 's exact test for categorical variables , and the logrank test for macefree survival curves , as appropriate . cox proportional hazards regression was used to compute hazard ratios ( hrs ) and 95% ci as estimates for the endpoint . hrs were adjusted for clinical characteristics , angiographic findings , and medications according to univariate analysis for the endpoint . all variables with p<0.10 on the univariate analysis were entered into the multivariate model using the stepwise backward selection method , and p<0.05 was set for inclusion in the multivariate model . in addition , to reduce the effect of treatment selection bias and possible confounders , we performed adjustment for significant differences in the baseline characteristics of patients with propensity score matching . the predicted probability of use of statins was calculated by fitting a logistic regression model , using all clinically relevant variables such as age , sex , hypertension , current smoking , family history of ihd , total cholesterol , chest pain at rest only , and use of ccb , angiotensin ii receptor blockers ( arb ) , nicorandil , and aspirin . one patient on statins was matched to 1 patient not treated with statins using nearestneighbor matching within a caliper width of 0.01 without replacement . all statistical analyses were performed with the statistical package for social sciences software ( version 23.0 ; ibm corp , armonk , ny ) . the present study was a retrospective observational study that enrolled 1877 consecutive japanese patients who had typical or atypical anginalike chest pain and underwent achprovocation testing at our hospital between january 1991 and december 2010 . we excluded 117 patients for the following reasons : acute myocardial infarction ( ami ; n=20 ) , cardiomyopathy ( n=75 ) , brugada syndrome ( n=10 ) , and other miscellaneous conditions ( n=12 ) . to investigate the prognosis of patients free of significant atherosclerotic stenosis thus , 640 vsa patients from the remaining 1402 free of significant organic stenosis were included for analysis ( figure 1 ) . we defined significant atherosclerotic stenosis ( hereafter referred to as organic stenosis ) as 75% stenosis ( 5175% narrowing of the luminal diameter ) of the right , left anterior descending , or left circumflex coronary artery and the major branches , or 50% stenosis ( 2650% narrowing of the luminal diameter ) of the left main trunk , as defined by the american heart association ( aha ) classification ( by visual estimation).16 flow chart of the patient recruitment process . the achprovocation test was conducted as described previously in the vsa guideline by the japanese circulation society.17 achinduced coronary spasm was defined as total or subtotal obstruction within the borders of a single isolated coronary segment , or severe diffuse vasoconstriction observed in more than 2 adjacent coronary segments of epicardial coronary arteries associated with transient myocardial ischemia , as evidenced by ischemic stsegment changes on the electrocardiogram ( ecg ) , as described previously.6 , 18 the study protocol was approved by the human ethics review committee of kumamoto university ( kumamoto , japan ) , and a signed consent form was obtained from each subject . we defined the risk factors for cad as current smoking , hypertension , dyslipidemia , diabetes mellitus , and family history of ischemic heart disease ( ihd ) , as described previously.6 , 18 selection of medical treatments for vsa was left to the discretion of each attending physician . followup data were collected from the medical records , the patients , their families , or the attending physicians . the primary endpoint was defined as major adverse cardiac events ( mace ) , including cardiac death , hospitalization for ami , and unstable angina . the secondary endpoint was allcause mortality during the followup period that began from the date of the diagnosis of vsa to the date of the first event or until december 2012 . we defined cardiac death as sudden death or death associated with ami , and ami as the presence of severe chest discomfort lasting > 30 minutes associated with stsegment changes and elevated cardiac enzyme levels . unstable angina was defined as recurrence or worsening of chest discomfort or pain associated with ischemic ecg changes . spasminduced ami and unstable angina were diagnosed by transient ischemic ecg changes during spontaneous nitrateresponsive angina episode , with at least one of the following : ( 1 ) absence of culprit lesion , spontaneous coronary spasm relieved by administration of intracoronary nitroglycerin , or achprovoked coronary spasm on coronary angiography or ( 2 ) absence of ischemic findings suggestive of significant atherosclerotic stenosis on stress testing ( exercise ecg and/or myocardial scintigraphy ) . data of normally distributed continuous variables are expressed as meansd , whereas those with skewed distribution are presented as median values ( interquartile range ; iqr ) , and categorical variables as frequencies and percentages . group comparisons were analyzed by the unpaired t test or mann whitney u test for continuous variables , the chisquare test or fisher 's exact test for categorical variables , and the logrank test for macefree survival curves , as appropriate . cox proportional hazards regression was used to compute hazard ratios ( hrs ) and 95% ci as estimates for the endpoint . hrs were adjusted for clinical characteristics , angiographic findings , and medications according to univariate analysis for the endpoint . all variables with p<0.10 on the univariate analysis were entered into the multivariate model using the stepwise backward selection method , and p<0.05 was set for inclusion in the multivariate model . in addition , to reduce the effect of treatment selection bias and possible confounders , we performed adjustment for significant differences in the baseline characteristics of patients with propensity score matching . the predicted probability of use of statins was calculated by fitting a logistic regression model , using all clinically relevant variables such as age , sex , hypertension , current smoking , family history of ihd , total cholesterol , chest pain at rest only , and use of ccb , angiotensin ii receptor blockers ( arb ) , nicorandil , and aspirin . one patient on statins was matched to 1 patient not treated with statins using nearestneighbor matching within a caliper width of 0.01 without replacement . all statistical analyses were performed with the statistical package for social sciences software ( version 23.0 ; ibm corp , armonk , ny ) . achprovoked coronary spasm without significant atherosclerotic stenosis was observed in 640 of 1402 patients ( figure 1 ) . table 1 shows the clinical characteristics of the vsa patients of the statin group ( n=168 ) and nostatin group ( n=472 ) . vsa patients of the statin group tended to be older , female , less likely to be smokers , and to have family history of ihd and chest pain at rest only and more likely to have hypertension , dyslipidemia , higher total cholesterol levels , higher lowdensity lipoprotein ( ldl)cholesterol levels , higher highdensity lipoprotein ( hdl)cholesterol levels , lower creactive protein ( crp ) , and arb , nicorandil , and aspirin use compared to those of the nostatin group . dyslipidemia on admission was identified in 160 of 168 ( 95.2% ) vsa patients of the statin group , but in only 125 of 472 ( 26.5% ) vsa patients of the nostatin group . as shown in table 1 , ldlcholesterol levels were significantly higher in vsa patients of the statin group than those of the other group ( 127.641.6 vs 112.429.9 mg / dl ; p<0.001 ) . the remaining 8 of 168 ( 4.8% ) patients were prescribed statins according to the vsa guideline by the japanese circulation society.17 clinical characteristics in the vsa patients with and without statins data are meansd , median ( interquartile range ) , or n ( % ) . ace indicates angiotensinconverting enzyme ; ach , acetylcholine ; arb , angiotensin ii receptor blockers ; bmi , body mass index ; ccb , calciumchannel blocker ; ckd , chronic kidney disease ; crp , creactive protein ; egfr , estimated glomerular filtration rate ; hdl , highdensity lipoprotein ; ihd , ischemic heart disease ; ldl , lowdensity lipoprotein ; vsa , vasospastic angina . after performing propensity score matching for the entire cohort , 128 matched pairs of patients were identified . for the logistic regression model to estimate propensity score , hosmerlemeshow goodness of fit chisquare was 2.875 with a p value of 0.942 and the area under the curve of receiver operating characteristic curve was 0.776 . there were no significant differences in clinically relevant variables between patients of the 2 treatment groups , except for incidence of dyslipidemia . table 2 provides detailed information of statin therapy . among 168 patients treated with statins , 26 ( 15.5% ) were treated with atorvastatin , 17 ( 10.1% ) with pitavastatin , 8 ( 4.8% ) with rosuvastatin , 74 ( 44.0% ) with pravastatin , 5 ( 3.0% ) with simvastatin , and 38 ( 22.6% ) with fluvastatin . in the matchedcohort , 17 ( 13.3% ) patients were treated with atorvastatin , 13 ( 10.2% ) with pitavastatin , 8 ( 6.3% ) with rosuvastatin , 57 ( 44.5% ) with pravastatin , 4 ( 3.1% ) with simvastatin , and 29 ( 22.7% ) with fluvastatin . details of statin therapy the primary endpoints were identified in the number of patients indicated in table 3 and figure 2 . during a median followup period of 60 months ( iqr , 4660 ) , 24 ( 3.8% ) patients of the entire cohort experiecned the primary endpoints , including cardiac death in 3 , acute myocardial infarction in 2 , unstable angina in 18 of the vsa patients not on statins , and unstable angina in a single patient of the statin group . manifestations of cardiac events in vsa patients were identified in 15 of all 24 patients ( 62.5% ) with mace . furthermore , 14 ( 58.3% ) patients were diagnosed as spasminduced mi or unstable angina , and the culprit lesion was identified on coronary angiography in 1 ( 4.2% ) patient . in patients of the statin group , 1 was diagnosed with spasminduced unstable angina . manifestations of cardiac events in the remaining 9 patients could not be assessed because detailed information about events was not available . meier survival curve indicated worse macefree 5year survival rates in patients of the nostatin group compared to those of the statin group ( 94.3% vs 99.4% ; p=0.012 ; figure 2a ) . clinical outcome of vsa patients of the statin and nostatin groups data are n ( % ) . mace indicates major adverse cardiac events ; mi , myocardial infarction ; vsa , vasospastic angina . meier curves for macefree survival for vasospastic angina patients of ( a ) the entire statins and nostatins groups and ( b ) the matched cohort . table 4 shows the results of univariate and multivariate cox proportional hazards analyses for factors that correlated with mace during followup . multivariate cox hazard regression analysis identified the use of statin as a significant negative correlate of mace ( hr , 0.11 ; 95% ci , 0.020.84 ; p=0.033 ) and stsegment elevation during angina attack as a significant positive correlate of mace ( hr , 5.28 ; 95% ci , 2.1912.7 ; p<0.001 ) . results of univariate and multivariate cox proportional hazards analyses of data of the entire cohort for factors associated with mace ace indicates angiotensinconverting enzyme ; arb , angiotensin ii receptor blockers ; bmi , body mass index ; ccb , calciumchannel blocker ; ckd , chronic kidney disease ; egfr , estimated glomerular filtration rate ; hr , hazard ratio ; ihd , ischemic heart disease ; mace , major adverse cardiac events . analysis of the matched cohort showed 2 cases with cardiac death and 7 with unstable angina among patients of the nostatin group ( table 3 ) . meier survival curve indicated significantly lower incidence of mace in the statin group compared to the nostatin group ( 100% vs 91.7% ; p=0.002 ; figure 2b ) . because of the limited number of events , it was considered that the data of the matched cohort could not be subjected to univariate and multivariate cox hazard regression analyses ; any such analysis would overestimate mace prognostic factors . achprovoked coronary spasm without significant atherosclerotic stenosis was observed in 640 of 1402 patients ( figure 1 ) . table 1 shows the clinical characteristics of the vsa patients of the statin group ( n=168 ) and nostatin group ( n=472 ) . vsa patients of the statin group tended to be older , female , less likely to be smokers , and to have family history of ihd and chest pain at rest only and more likely to have hypertension , dyslipidemia , higher total cholesterol levels , higher lowdensity lipoprotein ( ldl)cholesterol levels , higher highdensity lipoprotein ( hdl)cholesterol levels , lower creactive protein ( crp ) , and arb , nicorandil , and aspirin use compared to those of the nostatin group . dyslipidemia on admission was identified in 160 of 168 ( 95.2% ) vsa patients of the statin group , but in only 125 of 472 ( 26.5% ) vsa patients of the nostatin group . as shown in table 1 , ldlcholesterol levels were significantly higher in vsa patients of the statin group than those of the other group ( 127.641.6 vs 112.429.9 mg / dl ; p<0.001 ) . the remaining 8 of 168 ( 4.8% ) patients were prescribed statins according to the vsa guideline by the japanese circulation society.17 clinical characteristics in the vsa patients with and without statins data are meansd , median ( interquartile range ) , or n ( % ) . ace indicates angiotensinconverting enzyme ; ach , acetylcholine ; arb , angiotensin ii receptor blockers ; bmi , body mass index ; ccb , calciumchannel blocker ; ckd , chronic kidney disease ; crp , creactive protein ; egfr , estimated glomerular filtration rate ; hdl , highdensity lipoprotein ; ihd , ischemic heart disease ; ldl , lowdensity lipoprotein ; vsa , vasospastic angina . after performing propensity score matching for the entire cohort , 128 matched pairs of patients were identified . for the logistic regression model to estimate propensity score , hosmerlemeshow goodness of fit chisquare was 2.875 with a p value of 0.942 and the area under the curve of receiver operating characteristic curve was 0.776 . there were no significant differences in clinically relevant variables between patients of the 2 treatment groups , except for incidence of dyslipidemia . table 2 provides detailed information of statin therapy . among 168 patients treated with statins , 26 ( 15.5% ) were treated with atorvastatin , 17 ( 10.1% ) with pitavastatin , 8 ( 4.8% ) with rosuvastatin , 74 ( 44.0% ) with pravastatin , 5 ( 3.0% ) with simvastatin , and 38 ( 22.6% ) with fluvastatin . in the matchedcohort , 17 ( 13.3% ) patients were treated with atorvastatin , 13 ( 10.2% ) with pitavastatin , 8 ( 6.3% ) with rosuvastatin , 57 ( 44.5% ) with pravastatin , 4 ( 3.1% ) with simvastatin , and 29 ( 22.7% ) with fluvastatin . the primary endpoints were identified in the number of patients indicated in table 3 and figure 2 . during a median followup period of 60 months ( iqr , 4660 ) , 24 ( 3.8% ) patients of the entire cohort experiecned the primary endpoints , including cardiac death in 3 , acute myocardial infarction in 2 , unstable angina in 18 of the vsa patients not on statins , and unstable angina in a single patient of the statin group . manifestations of cardiac events in vsa patients were identified in 15 of all 24 patients ( 62.5% ) with mace . furthermore , 14 ( 58.3% ) patients were diagnosed as spasminduced mi or unstable angina , and the culprit lesion was identified on coronary angiography in 1 ( 4.2% ) patient . in patients of the statin group , 1 was diagnosed with spasminduced unstable angina . manifestations of cardiac events in the remaining 9 patients could not be assessed because detailed information about events was not available . meier survival curve indicated worse macefree 5year survival rates in patients of the nostatin group compared to those of the statin group ( 94.3% vs 99.4% ; p=0.012 ; figure 2a ) . clinical outcome of vsa patients of the statin and nostatin groups data are n ( % ) . mace indicates major adverse cardiac events ; mi , myocardial infarction ; vsa , vasospastic angina . meier curves for macefree survival for vasospastic angina patients of ( a ) the entire statins and nostatins groups and ( b ) the matched cohort . table 4 shows the results of univariate and multivariate cox proportional hazards analyses for factors that correlated with mace during followup . multivariate cox hazard regression analysis identified the use of statin as a significant negative correlate of mace ( hr , 0.11 ; 95% ci , 0.020.84 ; p=0.033 ) and stsegment elevation during angina attack as a significant positive correlate of mace ( hr , 5.28 ; 95% ci , 2.1912.7 ; p<0.001 ) . results of univariate and multivariate cox proportional hazards analyses of data of the entire cohort for factors associated with mace ace indicates angiotensinconverting enzyme ; arb , angiotensin ii receptor blockers ; bmi , body mass index ; ccb , calciumchannel blocker ; ckd , chronic kidney disease ; egfr , estimated glomerular filtration rate ; hr , hazard ratio ; ihd , ischemic heart disease ; mace , major adverse cardiac events . analysis of the matched cohort showed 2 cases with cardiac death and 7 with unstable angina among patients of the nostatin group ( table 3 ) . meier survival curve indicated significantly lower incidence of mace in the statin group compared to the nostatin group ( 100% vs 91.7% ; p=0.002 ; figure 2b ) . because of the limited number of events , it was considered that the data of the matched cohort could not be subjected to univariate and multivariate cox hazard regression analyses ; any such analysis would overestimate mace prognostic factors . statin therapy is the standard treatment for obstructive cad ; however , it remains to be elucidated whether this therapy improves the prognosis of vsa patients free of significant atherosclerotic coronary stenosis . the major finding of the present study was that statin therapy correlated with a lower rate of cardiovascular events in vsa patients free of significant stenosis . to the best of our knowledge , this is the first largepopulation study to investigate the prognostic effects and therapeutic implications of statin therapy in vsa patients free of significant coronary organic stenosis . in the present study , the kaplan meier survival curve indicated better 5year macefree survival rates in vsa patients of the statin group , compared to those of the nostatin group ( 99.4% vs 94.3% , respectively ) and matched cohort ( 100% vs 91.7% , respectively ) . although previous studies showed better prognosis of vsa patients free of organic stenosis than those with organic stenosis,4 , 5 , 6 the results of this study would make better impact on the prognosis of vsa patients free of organic stenosis . for example , 1 metaanalysis demonstrated the prognostic impact of statin therapy in patients at low risk of vascular disease.19 recently , chow et al.20 reported that statin therapy was associated with significant reduction in mortality in individuals with nonobstructive cad . thus , the results demonstrated that statin therapy could improve the longterm clinical outcome , including cardiovascular events , even in patients with nonobstructive cad , and the findings of the present study were consistently in agreement with these previous studies . statins reduce the risk of cardiovascular events by lowering ldlcholesterol levels.10 , 21 furthermore , previous studies showed that aggressive statin therapy provided better protection against coronary artery plaque formation and prevented cardiovascular events , compared with moderate treatment.7 , 8 in the present study , baseline ldlcholesterol levels were significantly higher in patients of the statins group than those of the nostatins group ( 127.641.6 vs 112.429.9 mg / dl , respectively ; p<0.001 ) , probably explaining the need for starting treatment with statin . although ldlcholesterol levels were not measured during followup , statin therapy seems to improve the prognosis of vsa patients by lowering ldlcholesterol levels and preventing plaque formation . in addition , because our study included patients with mildtomoderate organic coronary stenosis ( 2550% stenosis ) , in addition to those with no coronary stenosis , such patients might have had a greater benefit from statin therapy against atherosclerosis progression . most patients of the present study received lowintensity statin therapy probably because japanese are known to have higher responsiveness to statin treatment compared with caucasian.22 the 2013 american college of cardiology / aha guidelines recommend the use of higher dose and aggressive statin therapy in highrisk patients with clinically confirmed atherosclerotic cardiovascular disease without titration to a specific ldlc target for secondary prevention of cardiovascular diseases.23 based on the recommendation , higher dose of aggressive statin therapy might be needed in vsa patients . prospective , randomized studies should be performed to investigate the relation between statin intensity ( in terms of ldlcholesterol target levels ) and prognosis of vsa patients . although the present study identified the manifestations of cardiac events , more than half of the patients had spasminduced unstable angina . therefore , it seems that statins do not only reduce the risk of cardiovascular events in vsa patients through the prevention of coronary artery plaque progression , but also by direct suppression of coronary spasm . yasue et al.15 demonstrated that fluvastatin specifically suppressed vasoconstrictor response in the spasm segment , probably through its pleiotropic effects of amelioration of endothelial dysfunction , suppression of inflammation , and inhibition of the rho a / rhokinase pathway . their conclusion was based on the observation of decreased endothelial nitric oxide bioactivity and increased levels of inflammation markers in patients with coronary spasm.1 , 24 , 25 interestingly , previous studies from our laboratories have also demonstrated that statins significantly increased endothelial nitric oxide synthase mrna level , mrna stability , and endothelial nitric oxide activity . considered together , the above studies strongly suggest that statins seem to improve endothelial dysfunction and suppress coronary spasm.26 furthermore , it is also reported that statins therapy results in significant falls in serum levels of crp , a marker of inflammation.15 ridker et al.27 also demonstrated that statin therapy lowered levels of ldlc and crp , suggesting that the low levels of ldlc and crp after statin therapy are associated with low incidence of cardiovascular events . in the present study , crp levels were significantly lower in vsa patients treated with statins than those without ( 0.06 [ 0.030.14 ] vs 0.08 [ 0.050.23 ] , respectively ; p=0.005 ) . however , because ldlc and crp were measured only at the beginning of the followup , comparison of data obtained during and at end of followup are needed to confirm the antiinflammatory effect of statins . vascular smooth muscle cell hyperresponsiveness is one of the mechanisms of coronary artery spasm.28 this suggests that activation of the rho a / rhokinase pathway could be involved in the pathogenesis of coronary spasm . in fact , previous studies reported that statin therapy suppresses coronary spasm through inhibition of the rho a / rhokinase pathway.29 , 30 the present study has several limitations . the causeeffect relationship between medical treatment and pathological condition could not be assessed fully because the selection of medical treatments for vsa was left to the discretion of the attending physicians . however , the use of the propensity score matching reduced the effect of treatmentselection bias and possible confounders . nevertheless , other confounding factors that were not investigated in the present study could have affected the risk for cardiovascular events , and thus overestimation of the effects of statin therapy on coronary spasm attributed to the limited number of events can not be ruled out . a randomized second , the low absolute number of events in the present study limits the precision of the estimate of prognostic factors . for this reason third , the information about medical treatment and compliance was not sufficient . because we obtained such information only at the beginning of the followup , we could not assess the relationship between changes in medications during the followup period and the clinical outcome , thus limiting the value of analysis of prognosis and the main conclusion of the study . similarly , because we obtained the data of ldlcholesterol and crp levels only at the beginning of followup , we could not establish the exact mechanism of statin therapy , suggesting that lowering ldlcholesterol or crp levels plays at least some role in lowering incidence of coronary spasm . thus , a prospective study is needed to confirm the roles of ldlcholesterol and crp levels in the observed effects of statin therapy . fourth , it is possible that statin therapy was not tolerated by at least some of the patients of the nostatin group . withdrawal of statin therapy might have an impact on the conclusion made regarding the effectiveness of statin therapy . the present study demonstrated the impact of statin therapy on clinical outcome in vsa patients free of coronary atherosclerotic stenosis . the results indicated that statin therapy correlated with a lower rate of cardiovascular events in vsa patients free of significant organic stenosis . based on these findings , we conclude that statin therapy not only prevents the progression of coronary atherosclerotic plaque formation , but also seems to suppress coronary spasm through improvement of endothelial function and , consequently , reduces the likelihood of cardiovascular events in patients with coronary spasm . this study was supported , in part , by grantsinaid for scientific research ( no . 15k09089 ) from the ministry of education , culture , sports , science and technology of japan .

backgroundstatin therapy reduces the risk of cardiovascular events in patients with obstructive coronary artery disease . the aim of the present study was to determine the effects of statins on the prognosis of patients with coronary vasospastic angina ( vsa ) free of significant atherosclerotic stenosis.methods and resultsafter exclusion of 475 from 1877 consecutive patients who underwent an acetylcholineprovocation test between january 1991 and december 2010 , data of 640 vsa patients without significant organic stenosis of the remaining 1402 were analyzed retrospectively . propensity score matching was performed to reduce the effect of treatmentselection bias and possible confounders . the primary endpoint was major adverse cardiac events ( mace ) , including cardiac death , nonfatal myocardial infarction , and unstable angina . among the study population , dyslipidemia on admission was identified in 160 of 168 ( 95.2% ) patients of the statin group compared with only 125 of 472 ( 26.5% ) of the nostatin group . of the 640 patients , 24 ( 3.8% ) developed mace . multivariate cox hazard regression analysis identified statin therapy as a significant negative predictor of mace ( hazard ratio , 0.11 ; 95% ci , 0.020.84 ; p=0.033 ) . in the propensityscore matched cohorts ( n=128 each ) , kaplan meier survival curve showed a better 5year macefree survival rate for patients of the statin group compared to the nostatin group ( 100% vs 91.7% , respectively ; p=0.002).conclusionsstatin therapy correlated with a lower rate of cardiovascular events in vsa patients free of significant organic stenosis . statins seems to improve the prognosis of vsa patients free of significant organic stenosis .

Introduction Methods Study Population and AChProvocation Test Data Collection Followup Data Statistical Analysis Results Clinical Characteristics of VSA Patients of the Statin and Nostatin Groups Types and Doses of Statins Clinical Outcomes and Factors Associated With MACE Discussion Conclusions Sources of Funding Disclosures

coronary spasm is closely associated with endothelial dysfunction and plays a potential role in the progression of atherosclerosis.1 , 2 , 3 whereas patients with coronary spasm have better prognosis for cardiovascular events than those with moreserious coronary artery disease ( cad ) , coronary atherosclerotic stenosis correlates with cardiovascular events in patients with coronary vasospastic angina ( vsa).4 , 5 , 6 statins reduce the risk of cardiovascular events in obstructive cad7 , 8 , 9 , 10 , 11 and also ameliorate endothelial dysfunction.12 , 13 , 14 in a prospective , randomized study , we showed previously that the addition of fluvastatin at 30 mg / day to conventional medical therapy with calciumchannel blockers ( ccbs ) for 6 months significantly reduced acetylcholine ( ach)provoked coronary spasm in vsa patients free of coronary organic stenosis.15 however , there is no evidence at present that statin therapy improves the prognosis of vsa patients . the purpose of this study was to assess the effects of statin therapy on clinical outcome of vsa patients free of coronary organic stenosis . the present study was a retrospective observational study that enrolled 1877 consecutive japanese patients who had typical or atypical anginalike chest pain and underwent achprovocation testing at our hospital between january 1991 and december 2010 . to investigate the prognosis of patients free of significant atherosclerotic stenosis thus , 640 vsa patients from the remaining 1402 free of significant organic stenosis were included for analysis ( figure 1 ) . we defined significant atherosclerotic stenosis ( hereafter referred to as organic stenosis ) as 75% stenosis ( 5175% narrowing of the luminal diameter ) of the right , left anterior descending , or left circumflex coronary artery and the major branches , or 50% stenosis ( 2650% narrowing of the luminal diameter ) of the left main trunk , as defined by the american heart association ( aha ) classification ( by visual estimation).16 flow chart of the patient recruitment process . the achprovocation test was conducted as described previously in the vsa guideline by the japanese circulation society.17 achinduced coronary spasm was defined as total or subtotal obstruction within the borders of a single isolated coronary segment , or severe diffuse vasoconstriction observed in more than 2 adjacent coronary segments of epicardial coronary arteries associated with transient myocardial ischemia , as evidenced by ischemic stsegment changes on the electrocardiogram ( ecg ) , as described previously.6 , 18 the study protocol was approved by the human ethics review committee of kumamoto university ( kumamoto , japan ) , and a signed consent form was obtained from each subject . the primary endpoint was defined as major adverse cardiac events ( mace ) , including cardiac death , hospitalization for ami , and unstable angina . spasminduced ami and unstable angina were diagnosed by transient ischemic ecg changes during spontaneous nitrateresponsive angina episode , with at least one of the following : ( 1 ) absence of culprit lesion , spontaneous coronary spasm relieved by administration of intracoronary nitroglycerin , or achprovoked coronary spasm on coronary angiography or ( 2 ) absence of ischemic findings suggestive of significant atherosclerotic stenosis on stress testing ( exercise ecg and/or myocardial scintigraphy ) . in addition , to reduce the effect of treatment selection bias and possible confounders , we performed adjustment for significant differences in the baseline characteristics of patients with propensity score matching . the present study was a retrospective observational study that enrolled 1877 consecutive japanese patients who had typical or atypical anginalike chest pain and underwent achprovocation testing at our hospital between january 1991 and december 2010 . to investigate the prognosis of patients free of significant atherosclerotic stenosis thus , 640 vsa patients from the remaining 1402 free of significant organic stenosis were included for analysis ( figure 1 ) . we defined significant atherosclerotic stenosis ( hereafter referred to as organic stenosis ) as 75% stenosis ( 5175% narrowing of the luminal diameter ) of the right , left anterior descending , or left circumflex coronary artery and the major branches , or 50% stenosis ( 2650% narrowing of the luminal diameter ) of the left main trunk , as defined by the american heart association ( aha ) classification ( by visual estimation).16 flow chart of the patient recruitment process . the achprovocation test was conducted as described previously in the vsa guideline by the japanese circulation society.17 achinduced coronary spasm was defined as total or subtotal obstruction within the borders of a single isolated coronary segment , or severe diffuse vasoconstriction observed in more than 2 adjacent coronary segments of epicardial coronary arteries associated with transient myocardial ischemia , as evidenced by ischemic stsegment changes on the electrocardiogram ( ecg ) , as described previously.6 , 18 the study protocol was approved by the human ethics review committee of kumamoto university ( kumamoto , japan ) , and a signed consent form was obtained from each subject . we defined the risk factors for cad as current smoking , hypertension , dyslipidemia , diabetes mellitus , and family history of ischemic heart disease ( ihd ) , as described previously.6 , 18 selection of medical treatments for vsa was left to the discretion of each attending physician . the primary endpoint was defined as major adverse cardiac events ( mace ) , including cardiac death , hospitalization for ami , and unstable angina . spasminduced ami and unstable angina were diagnosed by transient ischemic ecg changes during spontaneous nitrateresponsive angina episode , with at least one of the following : ( 1 ) absence of culprit lesion , spontaneous coronary spasm relieved by administration of intracoronary nitroglycerin , or achprovoked coronary spasm on coronary angiography or ( 2 ) absence of ischemic findings suggestive of significant atherosclerotic stenosis on stress testing ( exercise ecg and/or myocardial scintigraphy ) . in addition , to reduce the effect of treatment selection bias and possible confounders , we performed adjustment for significant differences in the baseline characteristics of patients with propensity score matching . table 1 shows the clinical characteristics of the vsa patients of the statin group ( n=168 ) and nostatin group ( n=472 ) . vsa patients of the statin group tended to be older , female , less likely to be smokers , and to have family history of ihd and chest pain at rest only and more likely to have hypertension , dyslipidemia , higher total cholesterol levels , higher lowdensity lipoprotein ( ldl)cholesterol levels , higher highdensity lipoprotein ( hdl)cholesterol levels , lower creactive protein ( crp ) , and arb , nicorandil , and aspirin use compared to those of the nostatin group . dyslipidemia on admission was identified in 160 of 168 ( 95.2% ) vsa patients of the statin group , but in only 125 of 472 ( 26.5% ) vsa patients of the nostatin group . as shown in table 1 , ldlcholesterol levels were significantly higher in vsa patients of the statin group than those of the other group ( 127.641.6 vs 112.429.9 mg / dl ; p<0.001 ) . the remaining 8 of 168 ( 4.8% ) patients were prescribed statins according to the vsa guideline by the japanese circulation society.17 clinical characteristics in the vsa patients with and without statins data are meansd , median ( interquartile range ) , or n ( % ) . in the matchedcohort , 17 ( 13.3% ) patients were treated with atorvastatin , 13 ( 10.2% ) with pitavastatin , 8 ( 6.3% ) with rosuvastatin , 57 ( 44.5% ) with pravastatin , 4 ( 3.1% ) with simvastatin , and 29 ( 22.7% ) with fluvastatin . during a median followup period of 60 months ( iqr , 4660 ) , 24 ( 3.8% ) patients of the entire cohort experiecned the primary endpoints , including cardiac death in 3 , acute myocardial infarction in 2 , unstable angina in 18 of the vsa patients not on statins , and unstable angina in a single patient of the statin group . manifestations of cardiac events in vsa patients were identified in 15 of all 24 patients ( 62.5% ) with mace . furthermore , 14 ( 58.3% ) patients were diagnosed as spasminduced mi or unstable angina , and the culprit lesion was identified on coronary angiography in 1 ( 4.2% ) patient . in patients of the statin group , 1 was diagnosed with spasminduced unstable angina . meier survival curve indicated worse macefree 5year survival rates in patients of the nostatin group compared to those of the statin group ( 94.3% vs 99.4% ; p=0.012 ; figure 2a ) . clinical outcome of vsa patients of the statin and nostatin groups data are n ( % ) . mace indicates major adverse cardiac events ; mi , myocardial infarction ; vsa , vasospastic angina . multivariate cox hazard regression analysis identified the use of statin as a significant negative correlate of mace ( hr , 0.11 ; 95% ci , 0.020.84 ; p=0.033 ) and stsegment elevation during angina attack as a significant positive correlate of mace ( hr , 5.28 ; 95% ci , 2.1912.7 ; p<0.001 ) . results of univariate and multivariate cox proportional hazards analyses of data of the entire cohort for factors associated with mace ace indicates angiotensinconverting enzyme ; arb , angiotensin ii receptor blockers ; bmi , body mass index ; ccb , calciumchannel blocker ; ckd , chronic kidney disease ; egfr , estimated glomerular filtration rate ; hr , hazard ratio ; ihd , ischemic heart disease ; mace , major adverse cardiac events . analysis of the matched cohort showed 2 cases with cardiac death and 7 with unstable angina among patients of the nostatin group ( table 3 ) . meier survival curve indicated significantly lower incidence of mace in the statin group compared to the nostatin group ( 100% vs 91.7% ; p=0.002 ; figure 2b ) . because of the limited number of events , it was considered that the data of the matched cohort could not be subjected to univariate and multivariate cox hazard regression analyses ; any such analysis would overestimate mace prognostic factors . table 1 shows the clinical characteristics of the vsa patients of the statin group ( n=168 ) and nostatin group ( n=472 ) . vsa patients of the statin group tended to be older , female , less likely to be smokers , and to have family history of ihd and chest pain at rest only and more likely to have hypertension , dyslipidemia , higher total cholesterol levels , higher lowdensity lipoprotein ( ldl)cholesterol levels , higher highdensity lipoprotein ( hdl)cholesterol levels , lower creactive protein ( crp ) , and arb , nicorandil , and aspirin use compared to those of the nostatin group . dyslipidemia on admission was identified in 160 of 168 ( 95.2% ) vsa patients of the statin group , but in only 125 of 472 ( 26.5% ) vsa patients of the nostatin group . as shown in table 1 , ldlcholesterol levels were significantly higher in vsa patients of the statin group than those of the other group ( 127.641.6 vs 112.429.9 mg / dl ; p<0.001 ) . the remaining 8 of 168 ( 4.8% ) patients were prescribed statins according to the vsa guideline by the japanese circulation society.17 clinical characteristics in the vsa patients with and without statins data are meansd , median ( interquartile range ) , or n ( % ) . in the matchedcohort , 17 ( 13.3% ) patients were treated with atorvastatin , 13 ( 10.2% ) with pitavastatin , 8 ( 6.3% ) with rosuvastatin , 57 ( 44.5% ) with pravastatin , 4 ( 3.1% ) with simvastatin , and 29 ( 22.7% ) with fluvastatin . during a median followup period of 60 months ( iqr , 4660 ) , 24 ( 3.8% ) patients of the entire cohort experiecned the primary endpoints , including cardiac death in 3 , acute myocardial infarction in 2 , unstable angina in 18 of the vsa patients not on statins , and unstable angina in a single patient of the statin group . manifestations of cardiac events in vsa patients were identified in 15 of all 24 patients ( 62.5% ) with mace . furthermore , 14 ( 58.3% ) patients were diagnosed as spasminduced mi or unstable angina , and the culprit lesion was identified on coronary angiography in 1 ( 4.2% ) patient . in patients of the statin group , 1 was diagnosed with spasminduced unstable angina . meier survival curve indicated worse macefree 5year survival rates in patients of the nostatin group compared to those of the statin group ( 94.3% vs 99.4% ; p=0.012 ; figure 2a ) . mace indicates major adverse cardiac events ; mi , myocardial infarction ; vsa , vasospastic angina . multivariate cox hazard regression analysis identified the use of statin as a significant negative correlate of mace ( hr , 0.11 ; 95% ci , 0.020.84 ; p=0.033 ) and stsegment elevation during angina attack as a significant positive correlate of mace ( hr , 5.28 ; 95% ci , 2.1912.7 ; p<0.001 ) . results of univariate and multivariate cox proportional hazards analyses of data of the entire cohort for factors associated with mace ace indicates angiotensinconverting enzyme ; arb , angiotensin ii receptor blockers ; bmi , body mass index ; ccb , calciumchannel blocker ; ckd , chronic kidney disease ; egfr , estimated glomerular filtration rate ; hr , hazard ratio ; ihd , ischemic heart disease ; mace , major adverse cardiac events . analysis of the matched cohort showed 2 cases with cardiac death and 7 with unstable angina among patients of the nostatin group ( table 3 ) . meier survival curve indicated significantly lower incidence of mace in the statin group compared to the nostatin group ( 100% vs 91.7% ; p=0.002 ; figure 2b ) . statin therapy is the standard treatment for obstructive cad ; however , it remains to be elucidated whether this therapy improves the prognosis of vsa patients free of significant atherosclerotic coronary stenosis . the major finding of the present study was that statin therapy correlated with a lower rate of cardiovascular events in vsa patients free of significant stenosis . to the best of our knowledge , this is the first largepopulation study to investigate the prognostic effects and therapeutic implications of statin therapy in vsa patients free of significant coronary organic stenosis . in the present study , the kaplan meier survival curve indicated better 5year macefree survival rates in vsa patients of the statin group , compared to those of the nostatin group ( 99.4% vs 94.3% , respectively ) and matched cohort ( 100% vs 91.7% , respectively ) . although previous studies showed better prognosis of vsa patients free of organic stenosis than those with organic stenosis,4 , 5 , 6 the results of this study would make better impact on the prognosis of vsa patients free of organic stenosis . thus , the results demonstrated that statin therapy could improve the longterm clinical outcome , including cardiovascular events , even in patients with nonobstructive cad , and the findings of the present study were consistently in agreement with these previous studies . statins reduce the risk of cardiovascular events by lowering ldlcholesterol levels.10 , 21 furthermore , previous studies showed that aggressive statin therapy provided better protection against coronary artery plaque formation and prevented cardiovascular events , compared with moderate treatment.7 , 8 in the present study , baseline ldlcholesterol levels were significantly higher in patients of the statins group than those of the nostatins group ( 127.641.6 vs 112.429.9 mg / dl , respectively ; p<0.001 ) , probably explaining the need for starting treatment with statin . although ldlcholesterol levels were not measured during followup , statin therapy seems to improve the prognosis of vsa patients by lowering ldlcholesterol levels and preventing plaque formation . most patients of the present study received lowintensity statin therapy probably because japanese are known to have higher responsiveness to statin treatment compared with caucasian.22 the 2013 american college of cardiology / aha guidelines recommend the use of higher dose and aggressive statin therapy in highrisk patients with clinically confirmed atherosclerotic cardiovascular disease without titration to a specific ldlc target for secondary prevention of cardiovascular diseases.23 based on the recommendation , higher dose of aggressive statin therapy might be needed in vsa patients . although the present study identified the manifestations of cardiac events , more than half of the patients had spasminduced unstable angina . therefore , it seems that statins do not only reduce the risk of cardiovascular events in vsa patients through the prevention of coronary artery plaque progression , but also by direct suppression of coronary spasm . their conclusion was based on the observation of decreased endothelial nitric oxide bioactivity and increased levels of inflammation markers in patients with coronary spasm.1 , 24 , 25 interestingly , previous studies from our laboratories have also demonstrated that statins significantly increased endothelial nitric oxide synthase mrna level , mrna stability , and endothelial nitric oxide activity . in the present study , crp levels were significantly lower in vsa patients treated with statins than those without ( 0.06 [ 0.030.14 ] vs 0.08 [ 0.050.23 ] , respectively ; p=0.005 ) . however , the use of the propensity score matching reduced the effect of treatmentselection bias and possible confounders . nevertheless , other confounding factors that were not investigated in the present study could have affected the risk for cardiovascular events , and thus overestimation of the effects of statin therapy on coronary spasm attributed to the limited number of events can not be ruled out . fourth , it is possible that statin therapy was not tolerated by at least some of the patients of the nostatin group . the present study demonstrated the impact of statin therapy on clinical outcome in vsa patients free of coronary atherosclerotic stenosis . the results indicated that statin therapy correlated with a lower rate of cardiovascular events in vsa patients free of significant organic stenosis . based on these findings , we conclude that statin therapy not only prevents the progression of coronary atherosclerotic plaque formation , but also seems to suppress coronary spasm through improvement of endothelial function and , consequently , reduces the likelihood of cardiovascular events in patients with coronary spasm .

over the past several years , we have developed several animal models for acute , subacute , and chronic exposure to specific drugs of abuse , with emphasis on cocaine , morphlne and heroin , and alcohol . one of these models , which we have developed , validated , and used extensively in our studies , is binge -pattern cocaine administration mimicking the most common pattern of human abuse . in studies from our laboratory in which animals were allowed to self - administer cocaine with presentation of high , as well as moderate , and the usual low doses of cocaine , and with extended access ( 10 hours ) provided , we found that animals will escalate their use of cocaine . in fact , by 5 days of extended access to high doses of cocaine rats will self - administer more than twice the dose which we had usually used in our chronic binge - pattern cocaine administration ( 15 mg / kg x3 , that is 45 mg / kg / day ) . we have extended this binge - pattern , using both the steady - dose and escalating - dose binge - pattern administration of cocaine . we have been able to study various behavioral factors , as well as impact on gene expression , comparing these two models . one of the most important early findings from our laboratory ( and others ) on gene expression has been the finding of significant increased preprodynorphin gene expression in the striatum of rodents after acute , subacute , and chronic cocaine administration ( eg , refs 20,21 ) . this is especially important since we and others have shown that dynorphin peptides , which are the natural endogenous opioid ligands of the kappa - opioid receptors , serve to modulate dopaminergic tone and countermodulate cocaine - induced dopaminergic surges . in a recent study , we examined the effects of steady - dose versus escalating - dose binge - pattern cocaine administration upon striatal preprodynorphin messenger ribonucleic acid ( mrna ) levels , and also on behavioral stereotypy . we found that both steady - dose and escalating - dose binge cocaine administration resulted in increased preprodynorphin mrna levels in the caudate - putamen , but not in the nucleus accumbens . these are similar to all our earlier studies of the impact of acute , subacute , and chronic cocaine administrations . in this study , there were no significant differences in preprodynorphin mrna levels when escalating doses ( up to 30 mg / kg x 3 , or a total of 90 mg / day ) were administered during the last five days of 14-day chronic dosing , compared with a total of 45 mg / day , the steady dose these data showed that the enhancement of gene expression of dynorphin response to cocaine has probably reached its maximum level at a dose of 45 mg / kg / day of cocaine , and may or may not be dose - dependent at lower doses . further , in this study it was found that cocaine significantly affected body weight in both paradigms , and that both resulted in expression of behavioral stereotypy . however , of note , one component of stereotypy , that is , intense rapid head movements , was found to be both doseand time - dependent , with more profound effects in the escalating - dose model . extending our much earlier studies in the rat , the effects of the natural kappa - opioid receptor agonist , dynorphin a ( l-17 ) , on both basal striatal dopamine levels and on cocaine - induced increases in striatal dopamine levels , as well as on cocaine - induced conditioned place preference , was studied in c57bl/6j mice . in earlier studies conducted in the rat , we had shown that dynorphin applied directly into the striatum causes a dose - dependent reduction in dopaminergic levels . in this recent study , dynorphin , at four different doses , was infused into the caudate - putamen , and dopamine levels were quantitatively measured , using high - performance liquid chromatography , in the extracellular fluid obtained during in vivo microdialysis in that brain region . also , the effect of a relatively high dose of dynorphin a on increases in dopamine levels caused by 15 mg / kg of cocaine was measured using in vivo microdialysis . in related studies , the effect of this dose of dynorphin a on cocaine - induced conditioned place preference was studied . we found that dynorphin significantly decreased basal dopamine levels in a dose - dependent manner and by more than 60% at the highest dose . further , this effect was blocked by preinjection with a selective kappa - opioid receptor antagonist , nor - binaltorphimine ( nor - bni ) . further , it was found that the highest dose of dynorphin studied ( 4.4 nanomolar ) resulted in a complete block of the cocaineinduced increases in dopamine levels , and also attenuated locomotor activity induced by 15 mg / kg of cocaine , and blocked the formation of cocaine - induced conditioned place preference . these findings suggest that a dynorphin agonist might be helpful in managing cocaine and other stimulant dependency by preventing cocaine or other stimulant - induced dopamine surges . however , on the other hand , any significant lowering of basal dopaminergic tone could lead to dysphoria , and thus more craving for a drug of abuse such as cocaine . therefore , it has made our laboratory suggest that a potentially effective kappa - opioid receptor - directed compound for management of cocaine addiction would probably be a kappa partial agonist , that is , with modest agonist activity , but also antagonist activity , which should render stable basal dopaminergic tones , yet significantly attenuate cocaineor other stimulant - induced dopamine surges , as well as liking of cocaine . in related studies , zhang et al studied a related potent synthetic kappa - agonist , r-84760 , on cocaine - induced increases in striatal dopamine levels in cocaine - induced conditioned place preference in c57bl/6j mice . r-84760 is a novel nonpeptidic potent synthetic selective kappa - opioid receptor agonist that has been studied to a limited extent in humans for other indications . it was found that , similarly to dynorphin itself , this compound would effect a dose - dependent reduction in dopaminergic tone , as measured during in vivo microdialysis in the striatum . also , it was shown that , like dynorphin , a low dose ( 0.1 mg / kg ) of r-84760 would block cocaineinduced increases in the dopamine levels . also , it was found that similarly low doses of r-84760 would completely prevent the development of cocaine - induced conditioned place preference and would attenuate locomotor activity in the conditioning chamber . further , it was documented that these effects of r-84760 on lowering dopaminergic tone and cocaine - induced surges were completely blocked by a selective kappa - antagonist , norbni . thus , these effects were documented to be mediated exclusively by the kappa - opioid receptor . in different studies , we further explored the impact of extended - access ( 10 hours ) versus short - access ( 3 hours ) and also highversus low - dose cocaine impact on self - administration , cocaine - induced reinstatement , and on brain mrna levels . it was again found that the escalation of cocaine self - administration under long - access conditions was greater than under short - access , and was dose - dependent . further , we showed that such longaccess , with animals who were allowed self - administration for 10 hours at high doses , resulted in an increased susceptibility to drug - induced relapse . there were also differences in neurobiological indices , specifically levels of gene expression in those animals who were allowed to have long access and high doses , compared with short access . there were significant increases in proenkephalin gene expression in the caudate - putamen following longaccess and high - dose self - administration . further , it was found that dopamine d2 receptor mrna levels in the caudate - putamen and nucleus accumbens were significantly correlated with cocaine reinstatement . however , there was no significant correlation between neuropeptide mrna levels and cocaine - induced reinstatement . body weight progressively declined in the long - access self - administering rats . in parallel to these findings , food consumption was also significantly reduced in each group during self - administration , but the reduction in food intake was much greater in the long - access rats . during the 10-day extinction period , food consumption was significantly greater in the long - access , high - dose rats compared with both the short - access and the low - dose rats , and , in fact , food consumption during extinction in the high - dose group was significantly greater than pre - self - administration baseline levels . these findings are similar to observations made by our group in human cocaine addicts in a controlled research setting . they have negative implications for some groups of people , where the desire for thinness , or the desire for attaining the self - image of thinness , may contribute to continued cocaine ( or other stimulant ) self - administration . the many findings from these long - access , high - dose cocaine self - administration rodent studies , both our more recent ones , as well as our earlier ones , along with the studies from other groups , particularly those of koob and of miczek , suggest that the findings may not only be relevant potentially for the human situation , but provide new insights for further study both in laboratory - based and human research paradigms . we have conducted studies in collaboration with the laboratory of dr paul greengard in which we have studled the impact of change of a single amino acid in an important signal transduction protein on ( i ) both dopaminergic responses to binge - pattern cocaine ; as well as ( ii ) acquisition ; and then ( iii ) persistence and amounts of self - administration of cocaine . these studies were conducted in four separate lines of mutant mice , each with a mutation code for alanine introduced into the gene for the protein darpp-32 at four sites of phosphorylation . the four sites of phosphorylation chosen were : ( i ) the protein kinase a site , threonine 34a ; ( ii ) the cyclin - dependent kinase-3 site , threonine 75a ; ( iii ) the kinase ck2 site , serine 97a ; and finally , ( iv ) the kinase ck1 site , serine 130a . in each case , animals were bred so that both the mutant strain , as compared with the wildtype strain , could be studied , with the single amino acid change introduced into one of these four sites of critical phosphorylation involved in different pathways of the dopamine d1 receptor signal transduction through the darpp-32 cascade pathway acquisition of self - administration required significantly more time in the threonine 34a-/- mice . however , once self - administration was established , both threonine 34a and the serine 130a darpp-32 mutant mice administered significantly more cocaine than did their wild - type controls . this became especially apparent after training each of these strains on a high dose of cocaine ( 1 mg / kg ) and then starting the self - administration studies for each strain using an even higher dose of cocaine per injection ( 2 mg / kg ) , but then progressing downward in concentration to 1.0 , .05 , and .01 mg / kg per injection . as the dose was reduced below 1.0 mg / kg per injection , both the threonine 34a and the serine 130a mice significantly increased lever pressing to obtain more cocaine than did their matched wild - type controls . such an increase during reduction of cocaine concentration was not seen in either the threonine 75a or the serine 97a mice . this suggests that although somewhat slower to acquire self - administration , both the threonine 34 site and the serine 130 site of darpp-32 phosphorylation are important for the persistence of , and though not studied , possibly also to relapse to , cocaine self - administration . further , and in support of these findings , studies using microdialysis in the freely - moving mice could be carried out in three of the four strains ( the fourth strain was not available in adequate numbers for study . ) when this was performed , it was found that the same two strains that administered more cocaine , that is , the threonine 34a and the serine 130a , experienced a much lower rise in extracellular fluid dopamine after each of three binge cocaine injections than did the control mixed wild - type animals . further , this did not happen in the threonine 75a ; these animals had a much higher level of dopamine achieved after each dose of binge cocaine , and these were animals that showed no difference between the single amino acid change mutant strain and the wild - type strain . these findings suggest that a single amino acid change of a critical phosphorylation site may alter the behaviors of self - administration ; they also give further support to the concepts of many groups , that a lower dopaminergic tone either at rest , or achieved after any normal ( for instance , a liked or desired food ) or abnormal ( for instance , cocaine ) self - administration , may result in a lesser increase in dopamine tone . thus , such animals ( or possibly people ) could be expected to seek more activation of this pleasure - related dopaminergic system , and thus have a greater vulnerability to developing an addiction . we have conducted studies in which morphine was self - administered by animals and was available 18 hours / session / day . in these studies , animals were allowed to select a more concentrated or less concentrated morphine solution and once stable choice was established , the concentrations were increased . the animals allowed such a choice both escalated their morphine use to a much greater extent than did steady - dose animals . after 14 days the animals were self - administering extremely large amounts of morphine in the extended - access and escalating high - dose model . these studies showed that the average daily morphine self - administration increased from 22.5 mg on day 1 up to 66.4 mg by day 14 . in addition to our neurobiological studies of drug addiction by more traditional methods , such as gene expression , we have been collaborating with dr virginia pickel 's laboratory in the use of immunogold electron microscopy ( em ) to study drug - induced receptor trafficking . in these studies we have been exploring the effects of chronic intermittent self - administration of escalating doses of morphine on ionotropic glutamate receptor subunit trafficking in postsynaptic ( ie , dendritic ) sites in neurons , a process that is emerging as a critical cellular substrate of neural plasticity . because immunogold em can be used to localize receptors near intracellular organelles , as well as presumably functional areas of the plasma membrane , this approach provides a more functional view than many of the more conventional methods of measuring receptor levels . we have been using immunogold em to study glutamate receptor localization in neurons in portions of limbic - autonomic brain areas , namely the reciprocally connected nucleus tractus solitarius ( nts ) and central ( cea ) and basolateral ( bla ) nuclei of the amygdala , a brain circuit that may play a critical role in homeostatic adaptations associated with repetitive drug use . we have reported that the n - methyl - d - aspartate ( nmda)-nrl receptor subunit is decreased on the dendritic plasma membrane of nts neurons in animals self - administering morphine , compared with control animals not exposed to morphine . further , morphine self - administering rats showed regiondependent changes in the subcellular location of the ampa - glur1 receptor subunit in the amygdala . specifically , there was an increase in ampa - glur1 labeling on the dendritic plasma membrane of bla neurons and a concomitant decrease in dendritic ampaglur1 in cea neurons from animals self - administering morphine compared with control animals . these findings suggest that chronic opiate self - administration is associated with a redistribution of postsynaptic plasma membrane glutamate receptor subunits that play an important role in neural plasticity in brain circuitry regulating homeostatic processes . these adaptations may be an important neural substrate for alterations in drug reward , autonomic function , and behavioral processes , each of which may be associated with the acquisition and persistence of an addiction . in four separate earlier studies from our laboratory we have shown that chronic ( 14 days ) binge - pattern cocaine administration increases mu - opioid receptor mrna levels and also increases density of mu - opioid receptors in specific brain regions where there are abundant dopaminergic terminals from neurons located in the ventral tegmental area . in recent studies , bailey and our group have shown that early withdrawal from chronic binge cocaine administration results in a recurrence of an increase in mu - opioid receptor mrna levels in the rat frontal cortex , but only in this region . in further studies , bailey found that there is a persistent upregulation of mu - opioid receptors following long - term withdrawal from escalating - dose binge - pattern cocaine . in these studies , animals were treated with our new modified paradigm of escalating - dose binge cocaine over 14 days , which also results in an increase of mu - opioid receptor density , but with no increase in endogenous endorphin levels . following 14 days of withdrawal , there was still a highly significant increase in mu - opioid receptor density , and primarily in specific brain regions , again where there are dopaminergic terminals from the ventral tegmental area neurons and in fields in close proximity to both muopioid receptor mrna levels in the neurons producing mu - opioid receptors and presenting them on the cell surface . in a further set of studies , bailey explored changes in the kappa - opioid receptors following 14-day withdrawal from escalating - dose binge - pattern cocaine . here , very different findings were made . whereas in multiple studies from our laboratory we have found both increases in gene expression of dynorphin , and increases in kappaopioid receptor densities , and a correlated increase in kappa - opioid receptor mrna levels , with kappa , unlike mu - opioid receptors , which are found to be persistently increased in density following 14 days of withdrawal from binge - pattern escalating - dose cocaine , in this study there was lowering of kappa - opioid receptors in two specific brain regions in animals in long - term withdrawal from cocaine . such a decrease in density was not found in other regions , but also with no persistence of increase in density . these selective brain regions of decrease in kappa - opioid receptor might contribute , in part , to the biological substrate for the development of dysphoria , which is usually observed in drug - free former cocainedependent individuals . in our recent studies , we have also further explored the relative role of dopamine d1 and dopamine d2 receptors in various specific neurobiological changes , or neural plasticity , resulting from chronic exposure to cocaine . since it has been well established that dopamine plays a major role in the rewarding properties of cocaine , and since it has been established for a long time that one of cocaine 's prlmary sites of action is the presynaptic reuptake transporter for dopamine , where cocaine , by blocking reuptake , effects a flooding of perlsynaptlc space with dopamine , we have tried to dissect out the relative role of dopamine d1-like versus dopamine d2-like receptors in some of the resultant changes , both in behaviors , but also in gene expression and neuropeptide levels . during the last 5 years , we have completed further studies of the effects of selective dopamine d1-like and also dopamine d2-like receptor antagonists during acute binge - pattern cocaine administration on corticotropin - releasing factor ( crf ) mrna levels and pro - opiomelanocortin ( pomc ) mrna levels in the hypothalamus . in earlier studies , we have found that both dopamine d1-like and also dopamine d2-like antagonists attenuate the chronic binge - pattern cocaine - induced increase in adrenocorticotropin hormone ( acth ) and corticosterone levels . further , we have shown that an attenuation of cocalne - induced changes in stress hormone levels similarly occurs in animals with complete deletion of the darpp-32 protein , which is involved directly in dopamine d1 receptor signal transduction . in our recent studies , we again found that both dopamine d1-like and dopamine d2-like antagonists attenuated the elevation of corticosterone levels by acute , as well as in our earlier studies of chronic , binge - pattern cocaine . the previously identified acute binge cocaine - induced increases in hypothalamic crf mrna levels were not found in rats pretreated either with a dopamine d1-like or d2-llke antagonist . further , we found that neither the dopamine d1-like or dopamine d2-like receptor antagonists alone , in the absence of cocaine , altered mrna levels of crf in the hypothalamus . thus , these results further support our earlier concept , that both dopamine d1 receptors and dopamine d2 receptors mediate acute as well as chronic cocaine 's stimulatory effects on the hypothalamic - pituitary - adrenal ( hpa ) axis . since neurobiological evidence has suggested that there are functional interactions between the dopaminergic and opioid systems regulating preproenkephalin and preprodynorphin expression in the striatum , and since there is increasing evidence there may be direct connections between the dopaminergic system in the striatum and the stress - responsive components of the hypothalamus , we also raised the question of whether dopamine d1-like or dopamine d2-like antagonists could play a role in regulation of pomc mrna levels in the hypothalamus . in this part of these studies , it was found that dopamine d2-like receptor blockade increased the pomc mrna levels in the hypothalamus , a site with a different function than pomc mrna levels in the anterior pituitary . these findings suggest that activation of the dopamine d2 receptor may play a tonic inhibitory tone on hypothalamic pomc gene expression . however , neither dopamine d2 blockade nor acute binge cocaine altered pomc mrna levels in the amygdala , the anterior pituitary , or the neurolntermedlate level of the pituitary . also , dopamine d1 receptor blockade had no impact on hypothalamic pomc expression . thus , these results both suggest a possible specific role for dopamine d2 in at least acute cocaine effects on hypothalamic pomc gene expression . to further our studies on the relative role of the d1-like and d2-like ( and also d3-like , which are d2-like ) dopamine receptors in the setting of drug abuse , and , specifically the impact of binge - pattern cocaine administration , we have conducted studies using d1-/- or d3-/- selective dopamine receptor gene deletion mice . in these studies , we examined mu - opioid receptor gene expression in response to binge - pattern cocaine . we found that , at basal state , there was a significant increase in mu - opioid receptor mrna levels in the frontal cortex of both the d1-/- and d3-/- dopamine receptor gene deletion mice , as compared with each of their wild - type controls . however , there were no differences in basal levels of mu - oploid gene expression in the nucleus accumbens or in the caudate - putamen in these gene deletion mice . strikingly , and in an opposite direction from some of our earlier findings in wild - type rat models , acute binge cocaine 15 mg / kg x 3 doses resulted in the restoration of frontal cortex mu - opioid receptor mrna levels in the gene deletion mice to the levels of those in wildtype mice . further , in the nucleus accumbens core , after acute binge cocaine , there was an actual decrease in mu - opioid receptor levels in the d1-/- mutant mice , whereas in that brain region there was an increase in mu - opioid receptor gene expression in d3-/- mice . the opposite pertained in the caudate - putamen , with an increase in mu - opioid receptor levels after binge cocaine in the caudate - putamen of the d1-/- mice and a decrease in the dopamine d3-/- mice . in addition , a decrease in basal orexin mrna levels was found in the lateral hypothalamus of the d3-/- mice , which did not change with cocaine . these findings suggested that both d1 and d3 receptors are involved in mu - opioid receptor gene regulation in the frontal cortex , and also that d1 and d3 receptors may play opposite roles in the effects of cocaine on mu - opioid receptor gene expression in two striatal areas , the caudate - putamen and the nucleus accumbens core . in the control wild - type mice for the d1 receptor gene deletion , binge - pattern cocaine , as expected , increased mu - opioid receptor gene expression . however , in the wild - type controls for the dopamine d3 receptor knockout mice , there was a very modest , but not significant , increase in mu - opioid gene expression after binge cocaine , which was unexpected . these findings were made both in the caudate - putamen and in the nucleus accumbens core , suggesting that in the actual breeding of the wild - type animals , the controls for the d3 knockout groups may have been substantially different from the wild - type mice which were the controls to the d1 knockout mice . of particular note was the finding of increased basal levels of mu - oploid gene expression in both the d1 and d3 knockout mice , though only in the frontal cortex . these curious findings need to be studied further in d1 and d3 gene deletion mice , and also in dlfferent strains of wild - type mice . most of the other studies of the impact of drug - induced stress on many different parameters , with emphasis on documentations of specific changes or evidence of neuroplasticlty , have been conducted in rat models . in one sequence of studies , we examined the effects of acute morphine administration ; chronic intermittent escalating - dose morphine ( from 7.5 mg / kg / day on day 1 up to 120 mg / kg / day on day 10 ) ; and spontaneous 12-hour withdrawal from chronic morphine administration , using the escalating dose 10-day paradigm . there were no changes in mu - opioid receptor mrna levels in the lateral hypothalamus , the nucleus accumbens core , the caudate - putamen , or the amygdala following acute single injection of morphine , nor after chronic 10-day intermittent escalatlng - dose morphine . however , after 12 hours of withdrawal from 10-day chronic morphine administration , several indices documenting stress response in the hpa axis were found , including increased pomc mrna levels in the anterior pituitary , coupled with increases in acth levels , and also increased mu - opioid receptor mrna levels in the lateral hypothalamus , the nucleus accumbens core , and the caudate - putamen . the changes in mu - opioid receptor gene expression suggest both a rebound from the abrupt withdrawal from large doses of the exogenous opioid morphine , as well as changes integral to the hpa stress - responsive axis , as well as in the hypothalamus . several studies from other laboratories have demonstrated a role of lateral hypothalamic orexin ( hypocretin ) activation in drug - related positive reward , as well as in withdrawal effects ; therefore gene expression of this peptide was also studied . it has been established by others that around half of the lateral hypothalamic orexin neurons concomitantly express mu - opioid receptors . in parallel to the increase in mu - opioid receptor gene expression found in the lateral hypothalamus in acute morphine withdrawal , similarly the levels of orexin mrna in the lateral hypothalamus were also found to be increased . no changes were found in the lateral hypothalamic levels of preprodynorphin mrna , a gene which is known to be usually coexpressed with orexin in that hypothalamic region . these findings suggest that many different responses to the stress of morphine withdrawal occur , or , alternatively , changes which occur in the setting of withdrawal may drive the hpa axis activation and stress of withdrawal , just as we have found to be the case in our clinical studies . further , they suggest that in the lateral hypothalamus activation of orexin gene expression occurs in parallel to mu - oploid receptor gene expression . in a subsequent series of studies , a similar but somewhat different opioid administration paradigm was used . in these studies , heroin , the most common human opiate of abuse , was used , coupled with a chronic , intermittent escalating - dose administration paradigm and conducted with doses of heroin ranging on day 1 from 7.5 mg / kg up to 60 mg / kg by day 10 ( it should be noted that in this intermittent morphine escalating - dose paradigm , the starting dose was the same for heroin and morphine ( 7.5 mg / kg ) , but after 10 days , the escalation was up to 120 mg / kg when morphine was used , and 60 mg / kg when heroin was used . one group of animals was then studled at the end of chronic escalating heroin administration ; other animals were studied during early f 2-hour withdrawal from such chronic heroin exposure ; and a third group was studied after late 10 days of withdrawal from chronic heroin exposure . in this study , it was found that arglnine vasopressin mrna levels were significantly increased during early spontaneous withdrawal , and , of several brain regions examined , only in the amygdala . further , separate studies showed that arglnine vasopressin mrna levels were increased not only in early spontaneous withdrawal from heroin in the amygdala , but also following foot - shock in rats withdrawn from heroin self - administration . such findings were not made in the self - admlnistration control , heroin - nave rats . this increase in arglnine vasopressin mrna levels was no longer observed following 10 days of withdrawal from chronic heroin . as in earlier studies , pomc mrna levels in the anterior pituitary were found to be increased , both 30 mm after chronic heroin administration , which probably is a sign of very early withdrawal , as well as at 12 hours of withdrawal from heroin . similarly , acth levels were increased in early withdrawal , coupled with a significant increase in plasma corticosterone , after 12 hours of withdrawal . although the levels of both acth and corticosterone at the end of the chronic heroin administration , and thus 30 min , after the last dose , were somewhat greater than those in the salinetreated controls , these changes were not significant . in much earlier basic clinical research studies , performed in a stress - minimized research unit , documented that plasma levels of acth and cortisol became elevated before any signs and symptoms of opioid abstinence were observed or reported following very - low - dose opioid antagonist administration in opioid - dependent persons , suggesting that hpa axis activation drives , in part , the stress of opioid withdrawal , rather than reflecting a response to that stress . in separate , but related , studies , a model of heroin self - administration was used . the dose of heroin administration was 0.05 mg / kg per infusion , and 7 dally short - access ( 3-hour ) sessions were used . since vasopressin mrna elevations had been observed in animal models of heroin withdrawal , these studies were designed to look at the effects of a vasopressin receptor ( v1b receptor ) antagonist , ssr149415 , in that setting . administration of this compound was before the first extinction , or drug withdrawal , session . the vasopressin receptor antagonist dose - dependently attenuated foot - shock - induced reinstatement and blocked heroin - induced reinstatement . all these data suggest that arginine vasopressin activation may occur during withdrawal from opiates , and suggest that this peptide also may contribute to relapse or reinstatement . further , it is shown that , in the stress of withdrawal , when foot - shock is added , there is a significant increase in gene expression , and thus probably in the arginine vasopressin peptide . most important , the data suggest that a vasopressin antagonist might attenuate either stress ( in these experiments , withdrawal - induced and foot - shock - induced ) , and also drug - induced reinstatement and relapse to opiate self - administration or use . in other separate studies , possible alterations of arginine vasopressin mrna levels in the amygdala were studied in animals undergoing acute withdrawal from cocaine . in these studies , our model of steady - dose binge - pattern ( 15 mg / kg every hour x 3 hours with no cocaine for 22 hours ) administration for 14 days was used , followed by acute withdrawal ( 3 hours ) , subacute withdrawal ( 24 hours ) , and long - term withdrawal ( 10 days ) . it was found that , although there were no changes in arginine vasopressin mrna levels in the amygdala immediately following 14 days of cocaine administration , there were increases in arginine vasopressin mrna levels in acute withdrawal ( 3 hours ) from cocaine . further , it was found that the selective opioid receptor antagonist naloxone blocked this increase . as found in previously reported studies from our laboratory , chronic cocaine did not result in increased mu - opioid mrna levels in the amygdala , nor did acute withdrawal from cocaine in these studies . at 24 hours of withdrawal , significant increases in arginine vasopressin mrna levels in the amygdala were still observed . however , these levels had returned to normal after 10 days of withdrawal . as found in our previous studies , adaptation or tolerance to the cocaine effects on the hpa axis activation as expected , naloxone produced modest elevations in acth levels in cocane - nave rats ; naloxone did not have such an effect in the acute or subacute cocaine - withdrawn animals . there were no changes in arginine vasopressin , or pomc , or mu - opioid receptor mrna levels in the hypothalamus following chronic cocaine administration , and acute withdrawal from cocaine . these findings suggested that opioid receptors may mediate the increase in arginine vasopressin in the amygdala during acute cocaine withdrawal , and suggest a potential role for arginine vasopressin in the amygdala in some of the adversive effects of withdrawal from cocaine as well as in withdrawal from opiates . a recent set of laboratory - based studies in rats affirm , and further suggest a mechanism , for observations which we have made in two separate clinical studies , around 7 years apart , and in two parts of the world . we have determined that steady - state methadone may attenuate or eliminate the liking of cocaine , and may do so by a mu - opioid receptor - mediated mechanism in several earlier studies , as discussed above , we have shown that chronic binge - pattern cocaine administration results in an increase in mu - opioid receptor density in multiple , but not all , brain regions , and specifically in regions where there are abundant dopaminergic terminals from dopamine neurons in the ventral tegmental area and substantia nigra compecta . further , we have shown that acute and subacute , but not chronic , cocaine administration results in an increase in mu - opioid receptor mrna levels . in these recent studies , rats were implanted with either salineor methadone - filled osmotic minipumps and then conditioned with 1 , 5 , or 20 mg / kg cocaine intraperltoneally . animals with the 20 mg / kg / day or 55 mg / kg / day methadone - filled osmotic pumps did not express cocaine - induced place preference . however , methadone pumps at two doses ( 30 and 55 mg / kg / day ) did not alter intravenous self - administration of cocaine using a continuous schedule of reinforcement with different doses of cocaine ( 0.1 , 0.5 , and 2.0 mg / kg / infusion ) studied . mu - opioid receptor mrna levels were measured in animals treated with cocaine as part of conditioning for place preference . as in earlier studies , it was shown that this subacute cocaine administration resulted in increased mu - opioid receptor mrna levels in the nucleus accumbens core and in the frontal cortex 10 days after cocaine conditioning . however , this increase in mu - opioid receptor mrna levels was attenuated or eliminated by the steady - dose infusion of methadone . earlier studies have shown that the dose of 55 mg / kg / day subcutaneously by pump in the rat results in a plasma level similar to that in patients seen in methadone maintenance . these studies showed that , although high doses of methadone delivered by pump did not alter the direct reinforcing effects of cocaine as seen in self - administration , those doses of methadone did block both spontaneous and cocaine - induced seeking or liking 10 days after cocaine conditioning . further , we have suggested that this may be through the mechanism of methadone attenuating or preventing the relative endorphin deficiency resulting from the increased mu - opioid receptor density preceded by increased mu - opioid receptor gene expression , but with no concomitant increase in the endogenous opioids that bind to the mu receptor , that is , no increase in beta - endorphin or in the enkephalin peptides . these studies also build upon the early and also much more recent findings that , despite the fact that up to 70% of all persons in the middle atlantic states , as well as currently in tel aviv , israel , have concomitant dependence upon cocaine , when presenting for treatment for longstanding dependence on heroin , after 1 year or more of methadone treatment , as expected , the numbers using heroin dropped precipitously , to less than 20% of patients using heroin at any time ( as contrasted to heroin use by all patients 3 to 6 times a day prior to entry ) . this was accompanied by the more surprising findings that during steady - dose methadone maintenance treatment , the percentage of persons dependent on cocaine drops down to less than 20% , and those using any cocaine to less than 30 % . although these beneficial results of methadone maintenance on managing cocaine addiction were always attributed to the counseling and other psychosocial benefits derived from a good methadone maintenance program , we have , over the last decade , hypothesized that a pharmacological mechanism also is in place , a hypothesis based on our findings that binge cocaine increases acutely mu - opioid receptor gene expression and on a chronic basis , mu - opioid receptor density , and further , that a relative endorphin deficiency thus develops in humans , since there is no concomitant increase of beta - endorphin or enkephalins , as may be directly documented by stress - responsive metyrapone testing . these findings suggest that possibly an opioid agonist such as methadone , or possibly a partial agonist , such as buprenorphine , might be able to be effectively used to treat very severe , long - term , cocaine - dependent persons who have not responded to any other available current treatment . since there are no effective targeted pharma - cotherapies for cocaine addiction , the potential target of the mu - opioid receptor , with now a neurobiological basis for such treatment , might be warranted . in other studies , conducted by a collaboration with our colleagues at the karolinska institute in stockholm , sweden , yet another potential target for future therapeutic use , a noclceptin / orphanin fq receptor agonist ( ro64 - 6198 ) was found to reduce alcohol self - administration , and , further , and importantly , to prevent relapse to alcohol drinking in a rat model . other orphanin - nociceptin ( orl-1 ) receptor agonists may be found to have effectiveness in treatment of alcoholism and possibly other specific addictive diseases , which involve interactions between the dopaminergic system and different components of the opioid and opioid - like system . corticotropin - releasing factor ( crf ) , synthesized and released in the hypothalamus , passes through the portal blood system to the anterior pituitary , where it effects processing and release of the single gene product of the pomc gene ( reviewed in ref 7 ) . this large peptide is then further processed to yield many biologically active and important neuropeptides , including the major stress - responsive and glucocorticoid - regulated peptide , acth , as well as the longest ( 31 amino acids ) of the endogenous opioids , and a primary ligand of the endogenous mu - opioid receptor , beta - endorphin . acth and beta - endorphin are released in equimolar amounts from the anterior pituitary sites in humans ( who , unlike rodents , do not possess an intermediate lobe in the pituitary except transiently during pregnancy . ) acth impacts directly upon the adrenal cortex to bring about the processing and release of the major glucocorticoid in humans , cortisol , in addition to altering and enhancing the biotransformation and release of several other steroid hormones . there is some evidence that there may be retrograde passage of these two neuropeptides back into the hypothalamic region , which in human and nonhuman primates , but not in rodents , lies partially outside the brain barrier . glucocorticoids have been documented for a very long time to negatively regulate the hpa axis in a negative - feedback mode , with cortisol being the primary glucocorticoid in humans , non - human primates and guinea pigs , and corticosterone , the primary glucocorticoid having this effect in rats and mice . thus , cortisol acts at both the hypothalamic sites of crf production and at the anterior pituitary sites of pomc processing and release , to transiently attenuate or inhibit the release of these hormones . a 24-hour circadian rhythm is thus achieved , with the lowest levels of crf , acth , beta - endorphin and thus cortisol in the late afternoon and early evening in humans , and with levels rising again in the early morning hours , the opposite times pertain in rodents , with highest hormone levels at night , at the beginning of the activity period . based on early findings of volavka , our group and a few others years ago began to study the possible role of the endogenous opioid system , in particular , the mu - opioid receptor system , in also modulating the hpa axis . in several studies we have shown that the hpa axis is inhibited by the mu - opioid receptor system ( reviewed in refs 5,7,8 ) . in one study from our group , we looked at high and very high doses of two different selective mu - opioid receptor antagonists , both of which can be administered intravenously in humans , naloxone and nalmefene . studies using nonhuman primate membranes and , more recently , studies using cloned human genes in proper molecular - cellular constructs , have shown that , in contrast to rodents , naloxone binds almost exclusively to the mu - opioid receptor and acts as an antagonist . nalmefene , on the other hand , binds to both mu - and kappa - opioid receptors . very recently , in collaboration with the group of bidlack , we have shown that the kappa opioid receptor effect of nalmefene is that of a partial agonist ( that is , with some agonist and some antagonist properties ) , whereas the mu component is pure mu - opioid receptor antagonist . since we have studied both of these compounds in several earlier clinical research studies , we elected to use high and very high doses of each , to be sure that the ceiling of the effective doses in humans was exceeded . we found , as we and others had shown before , that naloxone activates the hpa axis by disinhibition and causes significant increases in both acth and cortisol . of great interest in this study , however , was the finding that nalmefene causes a significantly greater activation of the hpa axis , with higher resultant peripheral levels of acth and cortisol . our more recent studies , in which we found that the kappa component of nalmefene is a partial agonist , suggest that whereas the mu antagonists act at mu - opioid receptors of the hypothalamic and anterior pituitary sites , and through the mechanism of disinhibition bring about the increased release of crf and acth and beta - endorphin , the kappa partial agonist component of nalmefene may act directly to enhance release of crf and/or of the pomc peptides , acth and beta - endorphin , thus directly activating the hpa stress - responsive axis , which has been suggested by several workers in preclinical studies . this possibility has not , however , been well studied with any of the very few selective kappa agonists which have ever been introduced to human use , and only a few additional studies of these kappa agonists or partial agonists have been conducted in nonhuman primates . in earlier studies , it has been shown that activation of the hpa axis , with increased levels of plasma acth and cortisol , occurs after administration of alcohol or cocaine , and many groups have made similar findings in animal models . further , we have shown that tolerance develops to this hpa activation effect of both cocaine and alcohol . in other studies , we have suggested that activation of the hpa axis is sought by the rat or mouse , and by the human . in human studies conducted , in collaboration , by o'malley at yale in a clinical research setting , naltrexone , a selective mu - opioid antagonist with some kappa antagonist activity , was administered for 1 week to alcoholics and compared with placebo administered for one week to a similar group . then a laboratory session was conducted in which limited alcohol self - administration was permitted for up to 2 hours . we found , just as in the numerous field trials , that alcoholics receiving naltrexone drank significantly fewer drinks . because of the naltrexone disinhibition of the hypothalamic - pituitary sites of the hpa axis , there was a significant increase in levels of acth and cortisol in alcoholics treated with naltrexone after consumption of fewer than two drinks , whereas the much larger amounts of alcohol consumed by the alcoholics receiving placebo resulted in no significant activation of this axis . further , on responding to specific questionnaires , the alcoholics receiving naltrexone , and who had consumed only a small amount of alcohol , but had experienced modest activation of the hpa axis , felt no further craving , or desire to drink alcohol , and this decrease in craving was correlated to the increase of serum cortisol levels . the opposite pertained in those alcoholics receiving a placebo , who had consumed more alcohol , but had no activation of the hpa axis , and no increase in cortisol , a significant urge to drink alcohol persisted . many of our earlier studies have shown that short - acting opiates , opposite from the effects of cocaine and alcohol in the hpa axis , profoundly attenuate or suppress the hpa axis , resulting in lowered levels of acth and cortisol after opiate administration . however , after tolerance and physical dependence have developed , in the setting of withdrawal from opiates , profound activation of the hpa axis occurs with increases in levels of acth and cortisol . the neuroendocrine changes of opiate withdrawal look very similar to the normal response to a specific mu opioid receptor antagonist , such as naltrexone , when given to a healthy volunteer . therefore , it is not surprising , as we had predicted , that most opiate addicts will not willingly accept chronic daily naltrexone or other opioid antagonist treatment once experienced , whereas alcoholics would accept such treatment , and might be directly benefited . giving an opioid antagonist to any opiate - dependent person is contraindicated , because profound activation of the stress - responsive axis will occur and creates a very adversive and noxious experience . in many of our earlier studies , we have shown that during chronic methadone maintenance treatment , which provides steady perfusion with a synthetic ligand of the mu - opioid receptor , complete normalization of the hpa axis occurs , including normalization of basal levels of hormones , as well as responsivity in various functional tests . to dissect further the relative contribution of the glucocorticoid system contrasted to the mu - opioid receptor endogenous ligands , that is , beta - endorphins and enkephalins , we have conducted further studies using metyrapone . in humans , metyrapone blocks the final step of cortisol synthesis , that is , 11--hydroxylation . in the single oral dose test using metyrapone , the synthesis of cortisol is blocked for about 8 hours , and then returns to normal . therefore , one can measure the levels of acth ( which also reflect the equimolar release and levels of beta - endorphin ) following metyrapone administration which are elevated because with cortisol synthesis blocked , and the normal negative feedback is transiently cut off . in healthy human beings , with normal endogenous opioid systems , the mu - opioid receptor system responds to bring a check , or brake , to the increased release and levels of acth ( and beta - endorphin ) . however , we had shown in several earlier studies that in medication - free , drug - free former heroin addicts , there is no such mu - opio!d receptor - mediated brake , and thus hyper - responsivity to metyrapone testing is observed ( reviewed in refs 5,7 ) . further , we had reported that in abstinent cocaine addicts a similar hyper - responsivity to metyrapone testing exists . this hyper - responsivity , therefore , suggests a relative endorphin deficiency , which our laboratory -based studies also support . as discussed above , we have found that chronic binge cocaine administration causes an increase in gene expression in the mu - opioid receptor , as well as an increase in density in mu - opioid receptors , in specific brain regions with abundant dopaminergic terminals , and , further , in recent studies , we have found that this increase in mu - opioid receptor density persists for a protracted period of time after last cocaine exposure . however , we have also shown that there is no increase of the endogenous opioids that bind at the mu receptor . thus a relative endorphin deficiency develops ( or possibly was present a priori on a genetic or environmentally - induced basis ) . subsequently , frost and colleagues , using positron emission tomography ( pet ) showed similarly the mu - opioid receptor density being increased in recently - abstinent cocaine addicts , and further more recently have shown that this increase persists for protracted periods of time into successful cocaine abstinence . thus , a relative endorphin deficiency has been documented both in humans as well as in rodent models , in humans directly shown by testing of the stress - responsive system . in several studies , we have found that metyrapone responsivity is abnormal in opiate addicts , but becomes normalized in methadone maintenance patients ( reviewed in refs 5,7 ) . we also have shown that abnormal hyper - responsivity occurs in cocaine addicts . in a more recent study we also conducted studies in a subgroup of methadone maintenance patients who continued during 6-month treatment or more to meet the criteria of cocaine dependence . this group was maintained on moderate doses of methadone ( 60 to 90 mg / day ) . as discussed above , an early clinical study from our laboratory , a very recent clinical study from our laboratory , and a recent laboratory - based study have all suggested that increasing the dose of methadone may decrease cocaine addiction in maintenance patients with dual - dependency , and further , in the rodent model , that the addition of steady - state methadone may prevent alterations in mu - opioid receptor gene expression and attenuate or prevent conditioned place preference to cocaine . in another set of studies reported in the last decade we have re - explored the glucocorticoid negative feedback both in methadone - maintained former heroin addicts , as well as those with ongoing cocaine dependence . in all our earlier studies , we found , surprisingly , that all of the methadone - maintained patients had normal suppression to dexamethasone and , in this study , we also used two lower doses than the usual suppression dose , that is , 0.5 and .125 mg and found that all subjects suppressed completely ( as reviewed in refs 5,7,57 ) . although not significant , the glucocorticoid feedback effects in the cocaine - dependent , methadone - maintained patients , and also in the otherwise well - stabilized methadone - maintained patients appeared to be greater than the normal volunteers in the late afternoon , suggesting that there may be a modestly altered , or enhanced , negative feedback by glucocorticolds , in at least some subjects . this , in turn , may contribute to the observed attenuation of both basal and cocaine - induced responsivity of the hpa axis in humans and in rodents in other studies from our laboratory and others . in another study , we examined the effect of corticotropin - releasing factor in methadone - maintained versus control subjects . in this study , we found differences between long - term well - stabilized methadone - maintained subjects as compared with normal control subjects . in this study , two doses of crf were used ; one lower than the usual dose ( 0.5 jug / kg ) and one dose higher ( 2.0 mg / kg ) than usually used in the neuroendocrine diagnostic procedure ( 100 jug , irrespective of weight ) . there was no difference in hormonal measurements between the two groups following placebo administration , nor during low - dose hcrf administration . however , following high - dose crf administration , the methadone - maintained patients displayed a significantly greater increase in plasma acth levels than did the normal volunteers . this suggested that in long - term methadone - maintained patients some abnormalities in hpa axis responsivity may pertain , in this case , a greater sensitivity of the anterior pituitary to crf stimulation . in turn , these findings suggest that the basal and peak levels of crf may be slightly reduced in stable methadone maintenance patients , possibly related to the increased sensitivity to negative feedback by glucococorticoids , as discussed above , or due to the steady but high and exogenous opioid tone in patients in treatment with the long - acting mu agonists . further studies to explore this altered sensitivity in other persons with specific addictive diseases , not in treatment , as well as in treatment , are in progress . in another series of studies , we have been able to pursue in humans findings which we and others had made in rodents , that is , that dynorphin , the natural endogenous opioid ligand of the kappa - opioid receptor , may directly act to alter ( lower ) dopaminergic tone . we have been able to access dynorphin a(1 - 13 ) , a natural - sequenced dynorphin four residues shorter than the natural dynorphin a(1 - 17 ) for research use under an investigator - initiated investigational new drug application ( ind ) approved by the us food and drug administration . building upon the established biological fact that , in humans , prolactin release is almost exclusively under dopaminergic tone , and thus , that a lowering of dopamine in the tuberoinfundibular dopaminergic region results in a rise in prolactin levels , we conducted studies first in healthy volunteers using two different doses of intravenously - administered dynorphin a(1 - 13 ) ( 120 g / kg and 500 g / kg ) . since in humans some of the hypothalamus lies outside the blood - brain barrier , we assumed that the peptide dynorphin would be able to act on this tuberoinfundibular dopaminergic system . when we conducted these studies in a stress - minimized environment of our rockefeller hospital clinical research center , we found that peripheral administration of dynorphin a(l13 ) gave a prompt dose - dependent increase in serum prolactin levels , which then returned to normal within 120 minutes . this duration of action was much longer than we predicted , based on our in vitro biotransformation studies in which we established the probable half -life of dynorphin a(1 - 13 ) in human blood . of interest , with respect to the possible effect of dynorphin on the hpa axis , we found no increment in acth or crf following peripheral dynorphin administration . to document whether the dynorphin effect was modulated by the endogenous opioid system , we conducted studies using the lower dose of dynorphin a(1 - 13 ) following pretreatment with either naloxone or nalmefene , both selective mu - opioid receptor antagonists , and one ( nalmefene ) with partial kappa - opioid agonist activity we found pretreatment with either of these compounds attenuated the rise in serum prolactin . in these initial studies , a further very provocative ( but not completely unexpected given the physiological differences ) observation was made , an unusual finding in our human studies of addictive diseases , specifically that female subjects had modestly higher basal levels of prolactin than males , but when given dynorphin , gave a significantly exaggerated response , with higher levels of prolactin achieved after dynorphin administration and thus the reduction of dopamine in the tuberoinfundibular region . our subsequent studies of dynorphin effects now must be done always considering males and females separately . in a second set of studies , we have addressed the question of whether or not the dynorphin responsivity , with respect to lowering dopaminergic tone , will occur similarly in healthy long - term well - stabilized methadonemaintained subjects . two doses of dynorphin again were used for study in both a new group of healthy volunteer subjects and in a group of long - term stable methadonemaintained patients . again , in the healthy volunteer subjects , a dose - dependent rise in serum prolactin was observed after dynorphin administration . similarly , in the methadone - maintained patients ( receiving 80 to 120 mg / day of methadone ) , a dose - dependent rise in prolactin occurs . because years ago ( published in 1978 by our group ) , we had shown that methadone itself , acting as a mu opioid receptor agonist , acts to lower dopaminergic tone , causes increase in serum prolactin , which occurs at time of peak plasma levels of methadone ( that is , around 2 to 4 hours after oral methadone dose ) , in the dynorphin studies , we withheld the methadone dose until 60 minutes after the dynorphin was given . in these subjects , as in our much earlier studies , we showed a second and separate brisk rise in prolactin levels , beginning at 2 hours after methadone administration and remaining elevated at 5 hours after methadone administration . again , in the methadone - maintained patients , as in both groups of healthy volunteer subjects , there was a dose - dependent dynorphin - induced rise in prolactin levels which returned to basal levels by 90 to 120 minutes . thus , in this study , we were able to observe both the dynorphin- and methadone - induced lowering of tuberoinfundibular dopaminergic tone , resulting in both rises in serum prolactin levels . in yet another series of studies , we had observed that when given to healthy volunteers nalmefene caused a small but modest rise in serum prolactin levels . therefore , we entered into a collaboration with bidlack , and in that collaboration addressed directly the issue of whether the kappa opioid receptor activity of nalmefene is antagonist , or possibly , as we hypothesized , partial agonist . it was found clearly that nalmefene possesses kappa - opioid receptor partial agonist activity in in vitro studies using appropriate molecular cellular constructs . it was reconfirmed that the mu opioid receptor action of nalmefene is only that of antagonism ; the kappa opioid receptor action is both agonism ( partial agonist ) and antagonism . further , we were able to show that nalmefene effects a modest elevation of prolactin levels , suggesting a modest lowering of dopaminergic tone . this suggests , however , that nalmefene or other mu - opioid receptor antagonists , which have kappa - partial agonism ( probably also true for naltrexone ) may have augmented benefit for management of alcoholism , and possibly even for treatment for stimulant , such as cocaine , dependency , since a modest lowering of dopaminergic tone could be helpful in decreasing or attenuating the reward effect , whereas the inhibition of the mu - opioid receptor regulation of the stress - responsive hpa axis could provide modest activation of this axis , which we have directly documented to be sought by alcoholics , and in our animal modeling suggests is also sought by the cocaine self - administering animals . in these basic clinical research studies , we have again found an extremely important role of the mu - opioid receptor system , as well as identifying a previously not - appreciated role of the kappa - opioid receptor system in modulation of the human stress - responsive hpa axis . our genetics work , including our work in physiogenetics , has not been discussed herein , but has been reviewed elsewhere , as discussed above . all these findings have taught us that physiogenetics may occur , that is , difference in our response to our own proteins , peptides , neurotransmitters , or steroids , based on a polymorphism of a receptor or some polymorphism of the ligand or the pathway producing the ligand . further , such studies , in the future , may give us increasing insights into targets for therapeutics , as well as providing a basis for effective primary prevention of specific addictive diseases .

the articulated goals of dialogues in clinical neuroscience are to serve as an interface between clinical neuropsychiatry and the neurosciences by providing state - of - the - art information and original insights into relevant clinical , biological , and therapeutic aspects . my laboratory , the laboratory of the biology of addictive diseases at the rockefeller university , has for years been focused on bi - directional translational research , that is , learning by careful observations and study in patient populations with the disorders under study , in this case primarily specific addictive diseases , and then using that knowledge to create improved animal models or other laboratory - based research paradigms , while , at the same time , taking research findings made at the bench into the clinic as promptly as that is appropriate and feasible . in this invited review , therefore , the focus will be on perspectives of our laboratory of the biology of addictive diseases and related national institutes of health / national institute on drug abuse research center , including laboratory - based molecular neurobiological research , research using several animal models designed to mimic human patterns of drug abuse and addiction , as well as basic clinical research , intertwined with treatment - related research .

Laboratory-based molecular neurobiological and neurochemical studies related to cocaine and opiate addiction, and potential new approaches to treatment thereof Role of stress responsivity in the acquisition and persistence of specific addicitive diseases, and the impact of chronic exposure to drugs of abuse and withdrawal therefrom on components of the stress-responsive system, along with identification of potential new targets for therapeutic intervention Basic clinical research related to specific addictive diseases, with emphasis on stress responsivity: all research focused on treatment improvement

in studies from our laboratory in which animals were allowed to self - administer cocaine with presentation of high , as well as moderate , and the usual low doses of cocaine , and with extended access ( 10 hours ) provided , we found that animals will escalate their use of cocaine . one of the most important early findings from our laboratory ( and others ) on gene expression has been the finding of significant increased preprodynorphin gene expression in the striatum of rodents after acute , subacute , and chronic cocaine administration ( eg , refs 20,21 ) . in this study , there were no significant differences in preprodynorphin mrna levels when escalating doses ( up to 30 mg / kg x 3 , or a total of 90 mg / day ) were administered during the last five days of 14-day chronic dosing , compared with a total of 45 mg / day , the steady dose these data showed that the enhancement of gene expression of dynorphin response to cocaine has probably reached its maximum level at a dose of 45 mg / kg / day of cocaine , and may or may not be dose - dependent at lower doses . further , in this study it was found that cocaine significantly affected body weight in both paradigms , and that both resulted in expression of behavioral stereotypy . however , of note , one component of stereotypy , that is , intense rapid head movements , was found to be both doseand time - dependent , with more profound effects in the escalating - dose model . extending our much earlier studies in the rat , the effects of the natural kappa - opioid receptor agonist , dynorphin a ( l-17 ) , on both basal striatal dopamine levels and on cocaine - induced increases in striatal dopamine levels , as well as on cocaine - induced conditioned place preference , was studied in c57bl/6j mice . in this recent study , dynorphin , at four different doses , was infused into the caudate - putamen , and dopamine levels were quantitatively measured , using high - performance liquid chromatography , in the extracellular fluid obtained during in vivo microdialysis in that brain region . therefore , it has made our laboratory suggest that a potentially effective kappa - opioid receptor - directed compound for management of cocaine addiction would probably be a kappa partial agonist , that is , with modest agonist activity , but also antagonist activity , which should render stable basal dopaminergic tones , yet significantly attenuate cocaineor other stimulant - induced dopamine surges , as well as liking of cocaine . during the 10-day extinction period , food consumption was significantly greater in the long - access , high - dose rats compared with both the short - access and the low - dose rats , and , in fact , food consumption during extinction in the high - dose group was significantly greater than pre - self - administration baseline levels . the many findings from these long - access , high - dose cocaine self - administration rodent studies , both our more recent ones , as well as our earlier ones , along with the studies from other groups , particularly those of koob and of miczek , suggest that the findings may not only be relevant potentially for the human situation , but provide new insights for further study both in laboratory - based and human research paradigms . we have conducted studies in collaboration with the laboratory of dr paul greengard in which we have studled the impact of change of a single amino acid in an important signal transduction protein on ( i ) both dopaminergic responses to binge - pattern cocaine ; as well as ( ii ) acquisition ; and then ( iii ) persistence and amounts of self - administration of cocaine . when this was performed , it was found that the same two strains that administered more cocaine , that is , the threonine 34a and the serine 130a , experienced a much lower rise in extracellular fluid dopamine after each of three binge cocaine injections than did the control mixed wild - type animals . because immunogold em can be used to localize receptors near intracellular organelles , as well as presumably functional areas of the plasma membrane , this approach provides a more functional view than many of the more conventional methods of measuring receptor levels . whereas in multiple studies from our laboratory we have found both increases in gene expression of dynorphin , and increases in kappaopioid receptor densities , and a correlated increase in kappa - opioid receptor mrna levels , with kappa , unlike mu - opioid receptors , which are found to be persistently increased in density following 14 days of withdrawal from binge - pattern escalating - dose cocaine , in this study there was lowering of kappa - opioid receptors in two specific brain regions in animals in long - term withdrawal from cocaine . in our recent studies , we again found that both dopamine d1-like and dopamine d2-like antagonists attenuated the elevation of corticosterone levels by acute , as well as in our earlier studies of chronic , binge - pattern cocaine . thus , these results further support our earlier concept , that both dopamine d1 receptors and dopamine d2 receptors mediate acute as well as chronic cocaine 's stimulatory effects on the hypothalamic - pituitary - adrenal ( hpa ) axis . to further our studies on the relative role of the d1-like and d2-like ( and also d3-like , which are d2-like ) dopamine receptors in the setting of drug abuse , and , specifically the impact of binge - pattern cocaine administration , we have conducted studies using d1-/- or d3-/- selective dopamine receptor gene deletion mice . these findings suggested that both d1 and d3 receptors are involved in mu - opioid receptor gene regulation in the frontal cortex , and also that d1 and d3 receptors may play opposite roles in the effects of cocaine on mu - opioid receptor gene expression in two striatal areas , the caudate - putamen and the nucleus accumbens core . however , after 12 hours of withdrawal from 10-day chronic morphine administration , several indices documenting stress response in the hpa axis were found , including increased pomc mrna levels in the anterior pituitary , coupled with increases in acth levels , and also increased mu - opioid receptor mrna levels in the lateral hypothalamus , the nucleus accumbens core , and the caudate - putamen . the changes in mu - opioid receptor gene expression suggest both a rebound from the abrupt withdrawal from large doses of the exogenous opioid morphine , as well as changes integral to the hpa stress - responsive axis , as well as in the hypothalamus . several studies from other laboratories have demonstrated a role of lateral hypothalamic orexin ( hypocretin ) activation in drug - related positive reward , as well as in withdrawal effects ; therefore gene expression of this peptide was also studied . in these studies , heroin , the most common human opiate of abuse , was used , coupled with a chronic , intermittent escalating - dose administration paradigm and conducted with doses of heroin ranging on day 1 from 7.5 mg / kg up to 60 mg / kg by day 10 ( it should be noted that in this intermittent morphine escalating - dose paradigm , the starting dose was the same for heroin and morphine ( 7.5 mg / kg ) , but after 10 days , the escalation was up to 120 mg / kg when morphine was used , and 60 mg / kg when heroin was used . in this study , it was found that arglnine vasopressin mrna levels were significantly increased during early spontaneous withdrawal , and , of several brain regions examined , only in the amygdala . as in earlier studies , pomc mrna levels in the anterior pituitary were found to be increased , both 30 mm after chronic heroin administration , which probably is a sign of very early withdrawal , as well as at 12 hours of withdrawal from heroin . although the levels of both acth and corticosterone at the end of the chronic heroin administration , and thus 30 min , after the last dose , were somewhat greater than those in the salinetreated controls , these changes were not significant . in much earlier basic clinical research studies , performed in a stress - minimized research unit , documented that plasma levels of acth and cortisol became elevated before any signs and symptoms of opioid abstinence were observed or reported following very - low - dose opioid antagonist administration in opioid - dependent persons , suggesting that hpa axis activation drives , in part , the stress of opioid withdrawal , rather than reflecting a response to that stress . since vasopressin mrna elevations had been observed in animal models of heroin withdrawal , these studies were designed to look at the effects of a vasopressin receptor ( v1b receptor ) antagonist , ssr149415 , in that setting . these findings suggested that opioid receptors may mediate the increase in arginine vasopressin in the amygdala during acute cocaine withdrawal , and suggest a potential role for arginine vasopressin in the amygdala in some of the adversive effects of withdrawal from cocaine as well as in withdrawal from opiates . a recent set of laboratory - based studies in rats affirm , and further suggest a mechanism , for observations which we have made in two separate clinical studies , around 7 years apart , and in two parts of the world . further , we have suggested that this may be through the mechanism of methadone attenuating or preventing the relative endorphin deficiency resulting from the increased mu - opioid receptor density preceded by increased mu - opioid receptor gene expression , but with no concomitant increase in the endogenous opioids that bind to the mu receptor , that is , no increase in beta - endorphin or in the enkephalin peptides . these studies also build upon the early and also much more recent findings that , despite the fact that up to 70% of all persons in the middle atlantic states , as well as currently in tel aviv , israel , have concomitant dependence upon cocaine , when presenting for treatment for longstanding dependence on heroin , after 1 year or more of methadone treatment , as expected , the numbers using heroin dropped precipitously , to less than 20% of patients using heroin at any time ( as contrasted to heroin use by all patients 3 to 6 times a day prior to entry ) . although these beneficial results of methadone maintenance on managing cocaine addiction were always attributed to the counseling and other psychosocial benefits derived from a good methadone maintenance program , we have , over the last decade , hypothesized that a pharmacological mechanism also is in place , a hypothesis based on our findings that binge cocaine increases acutely mu - opioid receptor gene expression and on a chronic basis , mu - opioid receptor density , and further , that a relative endorphin deficiency thus develops in humans , since there is no concomitant increase of beta - endorphin or enkephalins , as may be directly documented by stress - responsive metyrapone testing . other orphanin - nociceptin ( orl-1 ) receptor agonists may be found to have effectiveness in treatment of alcoholism and possibly other specific addictive diseases , which involve interactions between the dopaminergic system and different components of the opioid and opioid - like system . this large peptide is then further processed to yield many biologically active and important neuropeptides , including the major stress - responsive and glucocorticoid - regulated peptide , acth , as well as the longest ( 31 amino acids ) of the endogenous opioids , and a primary ligand of the endogenous mu - opioid receptor , beta - endorphin . a 24-hour circadian rhythm is thus achieved , with the lowest levels of crf , acth , beta - endorphin and thus cortisol in the late afternoon and early evening in humans , and with levels rising again in the early morning hours , the opposite times pertain in rodents , with highest hormone levels at night , at the beginning of the activity period . very recently , in collaboration with the group of bidlack , we have shown that the kappa opioid receptor effect of nalmefene is that of a partial agonist ( that is , with some agonist and some antagonist properties ) , whereas the mu component is pure mu - opioid receptor antagonist . of great interest in this study , however , was the finding that nalmefene causes a significantly greater activation of the hpa axis , with higher resultant peripheral levels of acth and cortisol . our more recent studies , in which we found that the kappa component of nalmefene is a partial agonist , suggest that whereas the mu antagonists act at mu - opioid receptors of the hypothalamic and anterior pituitary sites , and through the mechanism of disinhibition bring about the increased release of crf and acth and beta - endorphin , the kappa partial agonist component of nalmefene may act directly to enhance release of crf and/or of the pomc peptides , acth and beta - endorphin , thus directly activating the hpa stress - responsive axis , which has been suggested by several workers in preclinical studies . in earlier studies , it has been shown that activation of the hpa axis , with increased levels of plasma acth and cortisol , occurs after administration of alcohol or cocaine , and many groups have made similar findings in animal models . therefore , it is not surprising , as we had predicted , that most opiate addicts will not willingly accept chronic daily naltrexone or other opioid antagonist treatment once experienced , whereas alcoholics would accept such treatment , and might be directly benefited . in many of our earlier studies , we have shown that during chronic methadone maintenance treatment , which provides steady perfusion with a synthetic ligand of the mu - opioid receptor , complete normalization of the hpa axis occurs , including normalization of basal levels of hormones , as well as responsivity in various functional tests . to dissect further the relative contribution of the glucocorticoid system contrasted to the mu - opioid receptor endogenous ligands , that is , beta - endorphins and enkephalins , we have conducted further studies using metyrapone . therefore , one can measure the levels of acth ( which also reflect the equimolar release and levels of beta - endorphin ) following metyrapone administration which are elevated because with cortisol synthesis blocked , and the normal negative feedback is transiently cut off . as discussed above , we have found that chronic binge cocaine administration causes an increase in gene expression in the mu - opioid receptor , as well as an increase in density in mu - opioid receptors , in specific brain regions with abundant dopaminergic terminals , and , further , in recent studies , we have found that this increase in mu - opioid receptor density persists for a protracted period of time after last cocaine exposure . thus , a relative endorphin deficiency has been documented both in humans as well as in rodent models , in humans directly shown by testing of the stress - responsive system . as discussed above , an early clinical study from our laboratory , a very recent clinical study from our laboratory , and a recent laboratory - based study have all suggested that increasing the dose of methadone may decrease cocaine addiction in maintenance patients with dual - dependency , and further , in the rodent model , that the addition of steady - state methadone may prevent alterations in mu - opioid receptor gene expression and attenuate or prevent conditioned place preference to cocaine . in another set of studies reported in the last decade we have re - explored the glucocorticoid negative feedback both in methadone - maintained former heroin addicts , as well as those with ongoing cocaine dependence . in all our earlier studies , we found , surprisingly , that all of the methadone - maintained patients had normal suppression to dexamethasone and , in this study , we also used two lower doses than the usual suppression dose , that is , 0.5 and .125 mg and found that all subjects suppressed completely ( as reviewed in refs 5,7,57 ) . although not significant , the glucocorticoid feedback effects in the cocaine - dependent , methadone - maintained patients , and also in the otherwise well - stabilized methadone - maintained patients appeared to be greater than the normal volunteers in the late afternoon , suggesting that there may be a modestly altered , or enhanced , negative feedback by glucocorticolds , in at least some subjects . this , in turn , may contribute to the observed attenuation of both basal and cocaine - induced responsivity of the hpa axis in humans and in rodents in other studies from our laboratory and others . this suggested that in long - term methadone - maintained patients some abnormalities in hpa axis responsivity may pertain , in this case , a greater sensitivity of the anterior pituitary to crf stimulation . further studies to explore this altered sensitivity in other persons with specific addictive diseases , not in treatment , as well as in treatment , are in progress . in another series of studies , we have been able to pursue in humans findings which we and others had made in rodents , that is , that dynorphin , the natural endogenous opioid ligand of the kappa - opioid receptor , may directly act to alter ( lower ) dopaminergic tone . building upon the established biological fact that , in humans , prolactin release is almost exclusively under dopaminergic tone , and thus , that a lowering of dopamine in the tuberoinfundibular dopaminergic region results in a rise in prolactin levels , we conducted studies first in healthy volunteers using two different doses of intravenously - administered dynorphin a(1 - 13 ) ( 120 g / kg and 500 g / kg ) . when we conducted these studies in a stress - minimized environment of our rockefeller hospital clinical research center , we found that peripheral administration of dynorphin a(l13 ) gave a prompt dose - dependent increase in serum prolactin levels , which then returned to normal within 120 minutes . in these initial studies , a further very provocative ( but not completely unexpected given the physiological differences ) observation was made , an unusual finding in our human studies of addictive diseases , specifically that female subjects had modestly higher basal levels of prolactin than males , but when given dynorphin , gave a significantly exaggerated response , with higher levels of prolactin achieved after dynorphin administration and thus the reduction of dopamine in the tuberoinfundibular region . thus , in this study , we were able to observe both the dynorphin- and methadone - induced lowering of tuberoinfundibular dopaminergic tone , resulting in both rises in serum prolactin levels . this suggests , however , that nalmefene or other mu - opioid receptor antagonists , which have kappa - partial agonism ( probably also true for naltrexone ) may have augmented benefit for management of alcoholism , and possibly even for treatment for stimulant , such as cocaine , dependency , since a modest lowering of dopaminergic tone could be helpful in decreasing or attenuating the reward effect , whereas the inhibition of the mu - opioid receptor regulation of the stress - responsive hpa axis could provide modest activation of this axis , which we have directly documented to be sought by alcoholics , and in our animal modeling suggests is also sought by the cocaine self - administering animals . in these basic clinical research studies , we have again found an extremely important role of the mu - opioid receptor system , as well as identifying a previously not - appreciated role of the kappa - opioid receptor system in modulation of the human stress - responsive hpa axis . all these findings have taught us that physiogenetics may occur , that is , difference in our response to our own proteins , peptides , neurotransmitters , or steroids , based on a polymorphism of a receptor or some polymorphism of the ligand or the pathway producing the ligand . further , such studies , in the future , may give us increasing insights into targets for therapeutics , as well as providing a basis for effective primary prevention of specific addictive diseases .

cervical cancer is the third most common cancer worldwide and has an annual mortality rate of 270,000 . recurrence mainly occurs during the first 2 years after treatment ( 60 - 90% of cases ) [ 1 , 2 , 3 ] . the presence of persistent disease after radiotherapy ( rt ) is a significant predictor of patient survival . although surgery has been suggested as an adjuvant therapy for cervical carcinoma patients after rt , it is clear that it does not seem to improve overall survival [ 4 , 5 ] . moreover , the addition of salvage surgery to rt may increase both morbidity and cost . therefore , salvage surgery should be reserved for selected cases with residual tumor [ 6 , 7 ] . to allow for such treatment stratification , however , no standard protocol for evaluating tumor response after rt is available . gynecologic examination combined with biopsies several weeks after rt is often performed routinely . though , it is difficult to recognize small tumors within areas of radiation - induced fibrosis in suspicious cases . furthermore , sampling errors may explain why , even with random biopsies , small microscopic tumor sites are easily missed , especially when suspected lesions are located outside the cervix or vagina . more accurate and ( preferably ) less invasive diagnostic modalities are required to allow a more reliable identification of patients who require salvage surgery to improve survival . magnetic resonance imaging ( mri ) is often used to assess responses of various tumors to rt including pelvic and cervical tumors [ 8 , 9 , 10 , 11 , 12 ] . however , it is unclear which mr criteria are diagnostic for residual tumor tissue after rt . after treatment , the irradiated tumor bed is often replaced by fibrotic tissue . the problem with mri is that differentiating fibrotic tissue from residual tumor tissue is difficult ; for example , mri of rectal cancer results in a false - positive rate of 50% [ 8 , 13 ] . the aims of the present study were to evaluate the ability of pelvic mri to detect residual tumor after rt , to identify and validate objective imaging criteria predictive for residual tumor , and to assess their performance . data for 42 patients with primary cervical cancer ( international federation of gynecology and obstetrics [ figo ] stage ib1 ) who were referred to our gynecologic oncological center ( maastricht university medical centre , maastricht , the netherlands ) for radiation therapy between may 2004 and august 2010 were analyzed retrospectively . the inclusion criteria were as follows : ( a ) histologically - proven primary cervical carcinoma , ( b ) availability of pre- and post - treatment pelvic mris , ( c ) treatment with external beam radiation therapy ( ebrt ) ( 46.0 - 50.4 gy ) , high - dose - rate brachytherapy with chemotherapy ( bct ) or without chemotherapy ( ct ) , or hyperthermia ( ht ) . initially , bct comprised 2 - 3 fractions for prescribing a total dose between 17 - 21 gy . however , this has been replaced with mr - image - guided intra - cavitary plus interstitial bct , which comprises 3 - 4 fractions of 7 gy according to gec - estro guidelines . the bct was delivered over two or three sessions , with a 1-week interval between each session . the first bct session was usually scheduled during the 5 week of ebrt . typically , the overall treatment time was 6 - 7 weeks . the retrospective nature of the study meant that ethics board approval was not required ( not subject to the medical research involving human subjects act ) . baseline characteristics , patients ( n = 42 ) figo international federation of gynecology and obstetric , mri magnetic resonance imaging the majority of the mri examinations ( 64% ) were performed using a 1.5-tesla mri unit ( intera achieve ; philips medical systems , best , the netherlands ; or siemens magnetom avanto , erlangen , germany ) and with a phased array surface coil . the imaging protocol comprised standard 2-dimensional t2w fast spin - echo images in three orthogonal directions ( tr / te ( 3200 - 3427)/(100 - 150 msec ) , 90 - 150 flip angle , 18 - 24 echo train length , 3 - 4 nsa , 0.98 0.98 ( 3.00 - 5.00 ) mm acquisition voxel size , 24 - 48 slices , 3.15 - 5.33 min acquisition time ) . the axial and coronal images were angled perpendicular and parallel to the cervical axis , respectively . the remaining mr examinations ( 36% ; 44% of which comprised examinations performed during bct ) were performed using a 3.0-tesla mri unit using a similar protocol ( t2w fse ; tr / te [ 7000/150 ] , 28 echo train length , 2 nsa , 0.98 0.98 ( 3.004.00 ) mm acquisition voxel size , 40 slices , 3.15 min acquisition time ) . magnetic resonance imaging was performed at different time points : ( a ) before the onset of external beam radiation treatment , ( b ) immediately before the final bct application , and ( c ) at a median 9 weeks ( range 4 - 51 weeks ) after the completion of radiotherapy . the mr images were independently scored by a senior radiologist ( fb , reader 1 ) and a junior radiologist ( sm , reader 2 ) , who have been 7 and 2 years experienced in pelvic mr imaging , respectively . the two readers were only aware of the initial figo stage but were blinded to each other 's interpretation of the results and the follow - up data . first , the readers were asked to assess the presence of residual tumor based on a subjective visual assessment of the t2w images using the following confidence level scores : 0 definitely no residual tumor , 1 probably no residual tumor , 2 possibly residual tumor , 3 probably residual tumor , 4 definitely residual tumor . the readers were not given any instructions or asked to search for certain criteria , and were free to interpreted the scans based on prior experience . subsequently , both readers were asked to evaluate each of the following imaging criteria using a five - point confidence level score : ( a ) the presence of an isointense mass ( isointense compared with the initial tumor , mostly hyperintense lesions at the time of presentation ) , ( b ) the presence of a hypointense ( fibrotic ) mass , ( c ) the shape of the tumor ( nodular or non - nodular ) , ( d ) the aspect of the border of the cervix ( regular or irregular ) , ( e ) signal homogeneity or heterogeneity , and ( f ) the shape of the cervix ( normal or deformed ) . all interpretations were performed using mr images obtained after radiotherapy and , if available ( n = 29/42 ) , on mr images obtained immediately before the final bct application . the pre - treatment images were at the reader 's disposal . if mri was performed immediately before the final bct , these images were also at the disposal of the reader . sagittal ( a - d , g , h ) and axial ( e , f ) t2-weighted images of different patients with a cervical carcinoma after radiotherapy . the patients in a , d , e , g , and h had no residual tumor . to the contrary : patients in b , c , and f showed residual tumor . the images illustrate the imaging criteria used to assess the presence of residual tumour . a , b ) the first criteria was the signal intensity of the cervix after treatment , which was scored as hypointense without any signs of an isointense tumour mass ( a ) or as a persistent isointense signal intensity area , indicative of residual tumour ( white arrow in b ) . c , d ) the second criterion was the homogeneity of the signal intensity in the cervix , which was scored as either homogeneous ( like the homogeneously isointense lesion indicated by the white arrow in c ) or heterogeneous , like the partly isointense and partly hypointense lesion indicated by the arrow in d. e , f ) the third and fourth criterion were the aspect of the cervical border and shape ( nodular vs. nonnodular ) of a ( partly ) isointense laesion . the cervical border was scored as irregular ( e ) or regular ( f ) . the shape was scored as either non - nodular ( e ) or nodular ( f ) . g , h ) the final criterion was the presence or absence of persistent deformation of the cervix after treatment . the cervical shape was scored either as normalized ( g ) or persistently deformed ( h ) the presence or absence of local residual tumor ( in the cervix and/or vagina , parametrium , bladder , and rectum ) was determined by : 1 ) histopathology after surgical resection ( n = 6 ) ; or 2 ) post - treatment gynecologic examination ( under anesthesia ) 3 months after the completion of rt , either with ( n = 21 ) or without a biopsy ( not clinically indicated n = 15 patients ) , combined with at least 12 months documented follow - up . follow - up comprised a gynecologic examination every 3 months , which was conducted by a gynecologic or radiation oncologist . local residual tumor and/or metastasis was defined as the presence of residual tumor ( identified pathologically ) or a growing mass on consecutive images , along with an increase in the level of tumor markers in the serum ( squamous - cell carcinoma [ scc ] or carcinoembryonic antigen [ cea ] ) . statistical analyses were performed using spss statistics , v18.0 ( spss inc , chicago , ill . interobserver variations were assessed by means of weighted kappa statistics , with quadratic kappa weighting . each of the mri criteria were compared and then combined to determine the best combination . receiver operating characteristics ( roc ) curves were constructed to evaluate diagnostic performance for ( a ) the subjective t2w visual response assessment , ( b ) the objective imaging criteria , and ( c ) the combined mri criteria . the areas under the roc curve ( auc ) , sensitivity , specificity , and positive- and negative - predictive value were calculated . at the beginning of the study , the decision was made to dichotomize the confidence level scores between 2 and 3 to yield a relatively high sensitivity for residual tumor . the sensitivity and specificity of the different scoring methods were compared using the mcnemar test ( for paired data ) or the test ( for unpaired data ) . data for 42 patients with primary cervical cancer ( international federation of gynecology and obstetrics [ figo ] stage ib1 ) who were referred to our gynecologic oncological center ( maastricht university medical centre , maastricht , the netherlands ) for radiation therapy between may 2004 and august 2010 were analyzed retrospectively . the inclusion criteria were as follows : ( a ) histologically - proven primary cervical carcinoma , ( b ) availability of pre- and post - treatment pelvic mris , ( c ) treatment with external beam radiation therapy ( ebrt ) ( 46.0 - 50.4 gy ) , high - dose - rate brachytherapy with chemotherapy ( bct ) or without chemotherapy ( ct ) , or hyperthermia ( ht ) . initially , bct comprised 2 - 3 fractions for prescribing a total dose between 17 - 21 gy . however , this has been replaced with mr - image - guided intra - cavitary plus interstitial bct , which comprises 3 - 4 fractions of 7 gy according to gec - estro guidelines . the bct was delivered over two or three sessions , with a 1-week interval between each session . the first bct session was usually scheduled during the 5 week of ebrt . typically , the overall treatment time was 6 - 7 weeks . the retrospective nature of the study meant that ethics board approval was not required ( not subject to the medical research involving human subjects act ) . baseline characteristics , patients ( n = 42 ) figo international federation of gynecology and obstetric , mri magnetic resonance imaging the majority of the mri examinations ( 64% ) were performed using a 1.5-tesla mri unit ( intera achieve ; philips medical systems , best , the netherlands ; or siemens magnetom avanto , erlangen , germany ) and with a phased array surface coil . the imaging protocol comprised standard 2-dimensional t2w fast spin - echo images in three orthogonal directions ( tr / te ( 3200 - 3427)/(100 - 150 msec ) , 90 - 150 flip angle , 18 - 24 echo train length , 3 - 4 nsa , 0.98 0.98 ( 3.00 - 5.00 ) mm acquisition voxel size , 24 - 48 slices , 3.15 - 5.33 min acquisition time ) . the axial and coronal images were angled perpendicular and parallel to the cervical axis , respectively . the remaining mr examinations ( 36% ; 44% of which comprised examinations performed during bct ) were performed using a 3.0-tesla mri unit using a similar protocol ( t2w fse ; tr / te [ 7000/150 ] , 28 echo train length , 2 nsa , 0.98 0.98 ( 3.004.00 ) mm acquisition voxel size , 40 slices , 3.15 min acquisition time ) . magnetic resonance imaging was performed at different time points : ( a ) before the onset of external beam radiation treatment , ( b ) immediately before the final bct application , and ( c ) at a median 9 weeks ( range 4 - 51 weeks ) after the completion of radiotherapy . the mr images were independently scored by a senior radiologist ( fb , reader 1 ) and a junior radiologist ( sm , reader 2 ) , who have been 7 and 2 years experienced in pelvic mr imaging , respectively . the two readers were only aware of the initial figo stage but were blinded to each other 's interpretation of the results and the follow - up data . first , the readers were asked to assess the presence of residual tumor based on a subjective visual assessment of the t2w images using the following confidence level scores : 0 definitely no residual tumor , 1 probably no residual tumor , 2 possibly residual tumor , 3 probably residual tumor , 4 definitely residual tumor . the readers were not given any instructions or asked to search for certain criteria , and were free to interpreted the scans based on prior experience . subsequently , both readers were asked to evaluate each of the following imaging criteria using a five - point confidence level score : ( a ) the presence of an isointense mass ( isointense compared with the initial tumor , mostly hyperintense lesions at the time of presentation ) , ( b ) the presence of a hypointense ( fibrotic ) mass , ( c ) the shape of the tumor ( nodular or non - nodular ) , ( d ) the aspect of the border of the cervix ( regular or irregular ) , ( e ) signal homogeneity or heterogeneity , and ( f ) the shape of the cervix ( normal or deformed ) . all interpretations were performed using mr images obtained after radiotherapy and , if available ( n = 29/42 ) , on mr images obtained immediately before the final bct application . if mri was performed immediately before the final bct , these images were also at the disposal of the reader . sagittal ( a - d , g , h ) and axial ( e , f ) t2-weighted images of different patients with a cervical carcinoma after radiotherapy . the patients in a , d , e , g , and h had no residual tumor . to the contrary : patients in b , c , and f showed residual tumor . the images illustrate the imaging criteria used to assess the presence of residual tumour . a , b ) the first criteria was the signal intensity of the cervix after treatment , which was scored as hypointense without any signs of an isointense tumour mass ( a ) or as a persistent isointense signal intensity area , indicative of residual tumour ( white arrow in b ) . c , d ) the second criterion was the homogeneity of the signal intensity in the cervix , which was scored as either homogeneous ( like the homogeneously isointense lesion indicated by the white arrow in c ) or heterogeneous , like the partly isointense and partly hypointense lesion indicated by the arrow in d. e , f ) the third and fourth criterion were the aspect of the cervical border and shape ( nodular vs. nonnodular ) of a ( partly ) isointense laesion . the cervical border was scored as irregular ( e ) or regular ( f ) . the shape was scored as either non - nodular ( e ) or nodular ( f ) . g , h ) the final criterion was the presence or absence of persistent deformation of the cervix after treatment . the cervical shape was scored either as normalized ( g ) or persistently deformed ( h ) the presence or absence of local residual tumor ( in the cervix and/or vagina , parametrium , bladder , and rectum ) was determined by : 1 ) histopathology after surgical resection ( n = 6 ) ; or 2 ) post - treatment gynecologic examination ( under anesthesia ) 3 months after the completion of rt , either with ( n = 21 ) or without a biopsy ( not clinically indicated n = 15 patients ) , combined with at least 12 months documented follow - up . follow - up comprised a gynecologic examination every 3 months , which was conducted by a gynecologic or radiation oncologist . local residual tumor and/or metastasis was defined as the presence of residual tumor ( identified pathologically ) or a growing mass on consecutive images , along with an increase in the level of tumor markers in the serum ( squamous - cell carcinoma [ scc ] or carcinoembryonic antigen [ cea ] ) . statistical analyses were performed using spss statistics , v18.0 ( spss inc , chicago , ill . interobserver variations were assessed by means of weighted kappa statistics , with quadratic kappa weighting . each of the mri criteria were compared and then combined to determine the best combination . receiver operating characteristics ( roc ) curves were constructed to evaluate diagnostic performance for ( a ) the the areas under the roc curve ( auc ) , sensitivity , specificity , and positive- and negative - predictive value were calculated . at the beginning of the study , the decision was made to dichotomize the confidence level scores between 2 and 3 to yield a relatively high sensitivity for residual tumor . the sensitivity and specificity of the different scoring methods were compared using the mcnemar test ( for paired data ) or the test ( for unpaired data ) . after radiation treatment , seven patients still had local residual tumor ; five of these patients also had concurrent distant metastases . eight patients had distant metastases only , median time to metastases 15 months ( range 3 - 31 months ) . twenty - seven patients had no evidence of disease after a median follow - up of 24 months ( range 13 - 81 months ) . the two patients with local residual tumor but without distant metastasis underwent salvage surgery . figure 2 and tables 2 and 3 show the diagnostic performance of both readers based on the subjective visual response , the objective imaging criteria , and the combined mri criteria . the interobserver agreement for the individual imaging criteria is shown in tables 2 , 3 , and 4 . reciever operating curve for magnetic resonance imaging ( mri ) analysis after radiotherapy , observer 1 and 2 diagnostic performance of subjective and objective magnetic resonance imaging ( mri ) criteria analyses for assessing residual tumor ( n = 7 ) with mri after radiotherapy ( n = 42 ) auc area under the receiver operating characteristics curve , hypointense mass , confidence interval = 0 , no hypointense mass , confidence interval = 4 sensitivity and specificity are given with the corresponding 95% confidence intervals diagnostic performance of subjective and objective magnetic resonance imaging ( mri ) criteria for assessing residual tumor ( n = 4 ) with mri during brachytherapy ( n = 29 ) auc area under the receiver operating characteristics curve , hypointense mass , confidence interval = 0 , no hypointense mass , confidence interval = 4 sensitivity and specificity are given with the corresponding 95% for reader 1 ( the more experienced ) , the subjective visual assessment after radiotherapy resulted in an auc of 0.79 , sensitivity of 71% , and specificity of 86% . for the junior reader ( reader 2 ) , interobserver agreement for the subjective visual response assessment after treatment was moderate ( = 0.41 ) . in 29 patients , additional mr images taken immediately before the final bct application were available and at the disposal of the reader . using this mri , the auc for reader 1 regarding assessment of the tumor response after completion of rt was 0.99 . in the group with 13 patients who did not have this mri , the corresponding aucs for reader 2 were 0.90 and 0.60 ( p = 0.08 ) . a hypointense mass and signal homogeneity showed a low auc 0.49 - 0.63 . an isointense mass , a nodular shape , and an irregular border yielded aucs of 0.79 , 0.79 - 0.83 , and 0.82 - 0.87 , respectively , when each was used as a stand - alone criterion . combining all three criteria yielded an auc of 0.85 - 0.91 . for both readers , using the combined criteria resulted in an auc higher than that obtained when using subjective visual assessment ( auc 0.91 vs. auc 0.79 , p = 0.15 for reader 1 ; and auc 0.85 vs. auc 0.75 , p = 0.02 for reader 2 ) . for the less experienced observer , interobserver agreement regarding the individual imaging criteria ranged from = 0.12 to = 0.91 . the value for the combined criteria set ( isointense mass , a nodular shape , and an irregular border ) was 0.84 . as shown in table 4 there were no major , nor substantial differences between both observers for the mr criteria set . both moderate differences correspond to two cases with no evidence of local residual disease after follow - up , in which the less experienced reader scored an isointense mass in contrast to the experienced reader . case - by - case comparison and interobserver agreement of subjective and objective magnetic resonance imaging ( mri ) for assessing residual tumor after rt ( n = 42 ) perfect match : no difference in agreement between observers ; minor : 1-point difference , moderate : 2-point difference , substantial : 3-point difference , major : 4-point difference magnetic resonance images taken immediately before the final bct were available for 29/42 patients . the subjective ( visual ) assessment of mr images taken immediately before the final bct resulted in an auc of 0.75 , sensitivity of 75% , and specificity of 36% for reader 1 , and 0.43 , 100% , and 8% , respectively , for reader 2 . objective imaging criteria on images taken immediately before the final bct resulted in aucs of 0.51 - 0.82 . only abnormal shape and a hypointense mass showed a higher auc than the subjective assessment when used as stand - alone criteria . after radiation treatment , seven patients still had local residual tumor ; five of these patients also had concurrent distant metastases . eight patients had distant metastases only , median time to metastases 15 months ( range 3 - 31 months ) . twenty - seven patients had no evidence of disease after a median follow - up of 24 months ( range 13 - 81 months ) . figure 2 and tables 2 and 3 show the diagnostic performance of both readers based on the subjective visual response , the objective imaging criteria , and the combined mri criteria . the interobserver agreement for the individual imaging criteria is shown in tables 2 , 3 , and 4 . reciever operating curve for magnetic resonance imaging ( mri ) analysis after radiotherapy , observer 1 and 2 diagnostic performance of subjective and objective magnetic resonance imaging ( mri ) criteria analyses for assessing residual tumor ( n = 7 ) with mri after radiotherapy ( n = 42 ) auc area under the receiver operating characteristics curve , hypointense mass , confidence interval = 0 , no hypointense mass , confidence interval = 4 sensitivity and specificity are given with the corresponding 95% confidence intervals diagnostic performance of subjective and objective magnetic resonance imaging ( mri ) criteria for assessing residual tumor ( n = 4 ) with mri during brachytherapy ( n = 29 ) auc area under the receiver operating characteristics curve , hypointense mass , confidence interval = 0 , no hypointense mass , confidence interval = 4 sensitivity and specificity are given with the corresponding 95% for reader 1 ( the more experienced ) , the subjective visual assessment after radiotherapy resulted in an auc of 0.79 , sensitivity of 71% , and specificity of 86% . for the junior reader ( reader 2 ) , the results were 0.75 , 86% , and 46% , respectively . interobserver agreement for the subjective visual response assessment after treatment was moderate ( = 0.41 ) . in 29 patients , additional mr images taken immediately before the final bct application were available and at the disposal of the reader . using this mri , the auc for reader 1 regarding assessment of the tumor response after completion of rt was 0.99 . in the group with 13 patients who did not have this mri , the corresponding aucs for reader 2 were 0.90 and 0.60 ( p = 0.08 ) . an isointense mass , a nodular shape , and an irregular border yielded aucs of 0.79 , 0.79 - 0.83 , and 0.82 - 0.87 , respectively , when each was used as a stand - alone criterion . combining all three criteria yielded an auc of 0.85 - 0.91 . for both readers , using the combined criteria resulted in an auc higher than that obtained when using subjective visual assessment ( auc 0.91 vs. auc 0.79 , p = 0.15 for reader 1 ; and auc 0.85 vs. auc 0.75 , p = 0.02 for reader 2 ) . for the less experienced observer , the specificity improved from 46% to 80% ( p interobserver agreement regarding the individual imaging criteria ranged from = 0.12 to = 0.91 . the value for the combined criteria set ( isointense mass , a nodular shape , and an irregular border ) was 0.84 . as shown in table 4 there were no major , nor substantial differences between both observers for the mr criteria set . both moderate differences correspond to two cases with no evidence of local residual disease after follow - up , in which the less experienced reader scored an isointense mass in contrast to the experienced reader . objective magnetic resonance imaging ( mri ) for assessing residual tumor after rt ( n = 42 ) perfect match : no difference in agreement between observers ; minor : 1-point difference , moderate : 2-point difference , substantial : 3-point difference , major : 4-point difference the subjective ( visual ) assessment of mr images taken immediately before the final bct resulted in an auc of 0.75 , sensitivity of 75% , and specificity of 36% for reader 1 , and 0.43 , 100% , and 8% , respectively , for reader 2 . objective imaging criteria on images taken immediately before the final bct resulted in aucs of 0.51 - 0.82 . only abnormal shape and a hypointense mass showed a higher auc than the subjective assessment when used as stand - alone criteria . the present study examined the utility of mri performed during and after rt ( either with or without ct or ht ) for detecting residual tumor . we also attempted to identify imaging criteria predictive for residual tumor and assessed their accuracy . subjective visual assessment of residual disease on post - rt mri resulted in a reasonable auc of 0.79 for the more experienced reader and an auc of 0.75 for the junior reader . however , the use of combined objective criteria ( isointense mass , nodular shape , and irregular border ) to assess residual disease after rt led to a much better performance , and increased the specificity parameter for the less experienced reader . moreover , the interobserver agreement improved to excellent when the assessment was performed using three objective criteria . this is the first report focusing on observer performance when interpreting mri scans with the aim of identifying residual disease after rt . this is due to the introduction of a systematic scoring system , in which certain criteria have to be scored separately . experienced readers may unconsciously use similar criteria ; this may explain why the predictive value for the less expert reader improved to a greater extent than that of the more experienced reader when using the objective criteria rather than the subjective assessment method . the mri criteria were derived from the literature in conjunction with an independent expert pelvic radiologist . morphologic visual t2w assessment of cervical tumors is often used to evaluate mr imaging of residual disease after rt . initial studies reported different values for diagnostic accuracy ( sensitivity 80 - 100% and specificity 56 - 100% ) [ 9 , 10 , 11 , 12 ] . reported an equivocal diagnosis in 16/44 cases when iso-/hyperintensity combined with contrast enhancement were used as objective mri criteria ; subjective assessment led to an equivocal diagnosis in only 5/44 cases . however , specificity was still low ( 55% ) for the group with a definite diagnosis , indicating that isointensity is inaccurate when used as a single mri criterion . in our data assessed treatment response at 3 - 8 weeks after the completion of rt , whereas we assessed the response at a median 9 weeks . may have been affected by early radiation effects , with granulomatous tissue formed in the acute post - rt phase mimicking residual tumor isointensity on mri , resulting in many more false - positives . this hypothesis is supported by the fact that we observed a similarly low specificity for isointensity on mri performed immediately before the completion of the final bct . we found it interesting that both readers performed better when visually assessing the post - rt response of patients for whom an additional scan was performed immediately before the last bct . this suggests that the readers found the additional information provided by the bct mr images very useful during their final assessment of the treatment response . this can be explained by the fact that volume regression over time can be observed more accurately on consecutive scans . this phenomenon was suggested by mayr et al . , who found volume regression analyses after ebrt showed a relatively high diagnostic performance . the prevalence of recurrent / persistent local tumor in our dataset ( 17% ) is rather low when compared to that reported in the literature . first , due to our inclusion criteria , patients who did not finish the imaging or treatment protocols due to the presence of early progressive disease were excluded ; 38% of the current study population had tumors at figo stage iia or less . second , almost all patients received concurrent ct or ht treatment in addition to rt , which has a higher cure rate than rt alone [ 18 , 19 ] . mri - based bct is superior to 2d - based radiotherapy with respect to local relapse rates for cervical carcinomas measuring > 5 cm [ 20 , 21 , 22 , 23 ] . however , we believe that the absence of recurrence at long - term follow - up is a good indicator of a lack of residual disease . moreover , when surgery is performed early after rt , microscopic tumors may still be present ( rt may not have exerted its full effect at this time ) . other research groups tried to solve this problem by including patients with microscopic tumors ( < 1 cm ) in the complete response group . however , this could lead to an underestimation of the number of patients with recurrent tumor as these foci can still progress to a recurrence . specifically , the number of patients that underwent mri during brachytherapy was low ; this was because we only started performing mri - based bct at our center in 2008 . a third limitation was the retrospective nature of the study , which meant that different imaging protocols , different mri scanners , and different magnetic fields were used . this may have affected the results . however , because 71% of all post- and pre - treatment scans were performed using a 1.5 tesla mri scanner , and all imaging protocols were routine clinical protocols , we believe that these differences would not have had a significant effect on the final results . the use of predefined objective mri criteria for assessing residual tumor after rt increases diagnostic performance and makes an accurate diagnosis less dependent upon observer experience . the availability of a brachytherapy mri scan during treatment improves the detection of residual cervical cancers on post - treatment mri scans . with the increasing use of mri during brachytherapy , in future prospective studies , this finding should be investigated more accurately in order to tailor treatment for the patient .

purposefor cervical carcinoma , the presence of persistent disease after radiotherapy ( rt ) is a significant predictor for survival . to date , no standard protocol is available to evaluate a response . this study was performed to assess magnetic resonance imaging ( mri ) to evaluate presence of local residual disease during and after rt for federation of gynecology and obstetrics ( figo ) stage ib1-iva cervical cancer.material and methodsforty - two patients were included . patients underwent mri before external beam rt , at final intracavitary brachytherapy ( bct ) and 2 - 3 months after completion of rt . two blinded radiologists ( observer 1 : experienced , observer 2 : less experienced ) scored the likelihood of residual tumor . magnetic resonance imaging was evaluated by means of ( a ) subjective visual evaluation of t2 weighted mri images , and ( b ) objective visual evaluation of t2 weighted mri images according to predefined imaging criteria.resultsseven patients had residual disease . area under the receiver operating characteristics curve ( auc ) for subjective visual assessment was 0.79/0.75 ( observer 1/observer 2 ) after rt and 0.75/0.43 at final bct . the combined objective mri criteria ( isointense , nodular , and irregular ) resulted in improved prediction of residual tumor ( aucs of 0.91/0.85 after rt ) . for the less experienced observer , the mri criteria set significantly improved prediction of residual tumor compared to subjective visual assessment . observer dependency decreased , kappa of 0.41 compared to 0.84 for the mri criteria set after rt.conclusioncompared to subjective visual assessment , predefined objective mri criteria increase diagnostic performance and decrease observer dependency for assessing residual tumor after rt in cervical cancer .

Purpose Material and methods Patients Magnetic resonance imaging Image evaluation Standard of reference Statistical analysis Results Patient and treatment characteristics Diagnostic performance for assessment of response Subjective visual assessment after completion of radiotherapy Objective imaging criteria after completion of radiotherapy Response assessment during brachytherapy Discussion Conclusions Disclosure

the presence of persistent disease after radiotherapy ( rt ) is a significant predictor of patient survival . although surgery has been suggested as an adjuvant therapy for cervical carcinoma patients after rt , it is clear that it does not seem to improve overall survival [ 4 , 5 ] . to allow for such treatment stratification , however , no standard protocol for evaluating tumor response after rt is available . magnetic resonance imaging ( mri ) is often used to assess responses of various tumors to rt including pelvic and cervical tumors [ 8 , 9 , 10 , 11 , 12 ] . the aims of the present study were to evaluate the ability of pelvic mri to detect residual tumor after rt , to identify and validate objective imaging criteria predictive for residual tumor , and to assess their performance . data for 42 patients with primary cervical cancer ( international federation of gynecology and obstetrics [ figo ] stage ib1 ) who were referred to our gynecologic oncological center ( maastricht university medical centre , maastricht , the netherlands ) for radiation therapy between may 2004 and august 2010 were analyzed retrospectively . the inclusion criteria were as follows : ( a ) histologically - proven primary cervical carcinoma , ( b ) availability of pre- and post - treatment pelvic mris , ( c ) treatment with external beam radiation therapy ( ebrt ) ( 46.0 - 50.4 gy ) , high - dose - rate brachytherapy with chemotherapy ( bct ) or without chemotherapy ( ct ) , or hyperthermia ( ht ) . initially , bct comprised 2 - 3 fractions for prescribing a total dose between 17 - 21 gy . baseline characteristics , patients ( n = 42 ) figo international federation of gynecology and obstetric , mri magnetic resonance imaging the majority of the mri examinations ( 64% ) were performed using a 1.5-tesla mri unit ( intera achieve ; philips medical systems , best , the netherlands ; or siemens magnetom avanto , erlangen , germany ) and with a phased array surface coil . magnetic resonance imaging was performed at different time points : ( a ) before the onset of external beam radiation treatment , ( b ) immediately before the final bct application , and ( c ) at a median 9 weeks ( range 4 - 51 weeks ) after the completion of radiotherapy . the mr images were independently scored by a senior radiologist ( fb , reader 1 ) and a junior radiologist ( sm , reader 2 ) , who have been 7 and 2 years experienced in pelvic mr imaging , respectively . first , the readers were asked to assess the presence of residual tumor based on a subjective visual assessment of the t2w images using the following confidence level scores : 0 definitely no residual tumor , 1 probably no residual tumor , 2 possibly residual tumor , 3 probably residual tumor , 4 definitely residual tumor . subsequently , both readers were asked to evaluate each of the following imaging criteria using a five - point confidence level score : ( a ) the presence of an isointense mass ( isointense compared with the initial tumor , mostly hyperintense lesions at the time of presentation ) , ( b ) the presence of a hypointense ( fibrotic ) mass , ( c ) the shape of the tumor ( nodular or non - nodular ) , ( d ) the aspect of the border of the cervix ( regular or irregular ) , ( e ) signal homogeneity or heterogeneity , and ( f ) the shape of the cervix ( normal or deformed ) . if mri was performed immediately before the final bct , these images were also at the disposal of the reader . sagittal ( a - d , g , h ) and axial ( e , f ) t2-weighted images of different patients with a cervical carcinoma after radiotherapy . the images illustrate the imaging criteria used to assess the presence of residual tumour . a , b ) the first criteria was the signal intensity of the cervix after treatment , which was scored as hypointense without any signs of an isointense tumour mass ( a ) or as a persistent isointense signal intensity area , indicative of residual tumour ( white arrow in b ) . g , h ) the final criterion was the presence or absence of persistent deformation of the cervix after treatment . the cervical shape was scored either as normalized ( g ) or persistently deformed ( h ) the presence or absence of local residual tumor ( in the cervix and/or vagina , parametrium , bladder , and rectum ) was determined by : 1 ) histopathology after surgical resection ( n = 6 ) ; or 2 ) post - treatment gynecologic examination ( under anesthesia ) 3 months after the completion of rt , either with ( n = 21 ) or without a biopsy ( not clinically indicated n = 15 patients ) , combined with at least 12 months documented follow - up . local residual tumor and/or metastasis was defined as the presence of residual tumor ( identified pathologically ) or a growing mass on consecutive images , along with an increase in the level of tumor markers in the serum ( squamous - cell carcinoma [ scc ] or carcinoembryonic antigen [ cea ] ) . each of the mri criteria were compared and then combined to determine the best combination . receiver operating characteristics ( roc ) curves were constructed to evaluate diagnostic performance for ( a ) the subjective t2w visual response assessment , ( b ) the objective imaging criteria , and ( c ) the combined mri criteria . the areas under the roc curve ( auc ) , sensitivity , specificity , and positive- and negative - predictive value were calculated . data for 42 patients with primary cervical cancer ( international federation of gynecology and obstetrics [ figo ] stage ib1 ) who were referred to our gynecologic oncological center ( maastricht university medical centre , maastricht , the netherlands ) for radiation therapy between may 2004 and august 2010 were analyzed retrospectively . the inclusion criteria were as follows : ( a ) histologically - proven primary cervical carcinoma , ( b ) availability of pre- and post - treatment pelvic mris , ( c ) treatment with external beam radiation therapy ( ebrt ) ( 46.0 - 50.4 gy ) , high - dose - rate brachytherapy with chemotherapy ( bct ) or without chemotherapy ( ct ) , or hyperthermia ( ht ) . initially , bct comprised 2 - 3 fractions for prescribing a total dose between 17 - 21 gy . baseline characteristics , patients ( n = 42 ) figo international federation of gynecology and obstetric , mri magnetic resonance imaging the majority of the mri examinations ( 64% ) were performed using a 1.5-tesla mri unit ( intera achieve ; philips medical systems , best , the netherlands ; or siemens magnetom avanto , erlangen , germany ) and with a phased array surface coil . magnetic resonance imaging was performed at different time points : ( a ) before the onset of external beam radiation treatment , ( b ) immediately before the final bct application , and ( c ) at a median 9 weeks ( range 4 - 51 weeks ) after the completion of radiotherapy . the mr images were independently scored by a senior radiologist ( fb , reader 1 ) and a junior radiologist ( sm , reader 2 ) , who have been 7 and 2 years experienced in pelvic mr imaging , respectively . first , the readers were asked to assess the presence of residual tumor based on a subjective visual assessment of the t2w images using the following confidence level scores : 0 definitely no residual tumor , 1 probably no residual tumor , 2 possibly residual tumor , 3 probably residual tumor , 4 definitely residual tumor . subsequently , both readers were asked to evaluate each of the following imaging criteria using a five - point confidence level score : ( a ) the presence of an isointense mass ( isointense compared with the initial tumor , mostly hyperintense lesions at the time of presentation ) , ( b ) the presence of a hypointense ( fibrotic ) mass , ( c ) the shape of the tumor ( nodular or non - nodular ) , ( d ) the aspect of the border of the cervix ( regular or irregular ) , ( e ) signal homogeneity or heterogeneity , and ( f ) the shape of the cervix ( normal or deformed ) . sagittal ( a - d , g , h ) and axial ( e , f ) t2-weighted images of different patients with a cervical carcinoma after radiotherapy . the images illustrate the imaging criteria used to assess the presence of residual tumour . a , b ) the first criteria was the signal intensity of the cervix after treatment , which was scored as hypointense without any signs of an isointense tumour mass ( a ) or as a persistent isointense signal intensity area , indicative of residual tumour ( white arrow in b ) . the cervical shape was scored either as normalized ( g ) or persistently deformed ( h ) the presence or absence of local residual tumor ( in the cervix and/or vagina , parametrium , bladder , and rectum ) was determined by : 1 ) histopathology after surgical resection ( n = 6 ) ; or 2 ) post - treatment gynecologic examination ( under anesthesia ) 3 months after the completion of rt , either with ( n = 21 ) or without a biopsy ( not clinically indicated n = 15 patients ) , combined with at least 12 months documented follow - up . local residual tumor and/or metastasis was defined as the presence of residual tumor ( identified pathologically ) or a growing mass on consecutive images , along with an increase in the level of tumor markers in the serum ( squamous - cell carcinoma [ scc ] or carcinoembryonic antigen [ cea ] ) . interobserver variations were assessed by means of weighted kappa statistics , with quadratic kappa weighting . each of the mri criteria were compared and then combined to determine the best combination . receiver operating characteristics ( roc ) curves were constructed to evaluate diagnostic performance for ( a ) the the areas under the roc curve ( auc ) , sensitivity , specificity , and positive- and negative - predictive value were calculated . at the beginning of the study , the decision was made to dichotomize the confidence level scores between 2 and 3 to yield a relatively high sensitivity for residual tumor . the two patients with local residual tumor but without distant metastasis underwent salvage surgery . figure 2 and tables 2 and 3 show the diagnostic performance of both readers based on the subjective visual response , the objective imaging criteria , and the combined mri criteria . the interobserver agreement for the individual imaging criteria is shown in tables 2 , 3 , and 4 . reciever operating curve for magnetic resonance imaging ( mri ) analysis after radiotherapy , observer 1 and 2 diagnostic performance of subjective and objective magnetic resonance imaging ( mri ) criteria analyses for assessing residual tumor ( n = 7 ) with mri after radiotherapy ( n = 42 ) auc area under the receiver operating characteristics curve , hypointense mass , confidence interval = 0 , no hypointense mass , confidence interval = 4 sensitivity and specificity are given with the corresponding 95% confidence intervals diagnostic performance of subjective and objective magnetic resonance imaging ( mri ) criteria for assessing residual tumor ( n = 4 ) with mri during brachytherapy ( n = 29 ) auc area under the receiver operating characteristics curve , hypointense mass , confidence interval = 0 , no hypointense mass , confidence interval = 4 sensitivity and specificity are given with the corresponding 95% for reader 1 ( the more experienced ) , the subjective visual assessment after radiotherapy resulted in an auc of 0.79 , sensitivity of 71% , and specificity of 86% . for the junior reader ( reader 2 ) , interobserver agreement for the subjective visual response assessment after treatment was moderate ( = 0.41 ) . using this mri , the auc for reader 1 regarding assessment of the tumor response after completion of rt was 0.99 . an isointense mass , a nodular shape , and an irregular border yielded aucs of 0.79 , 0.79 - 0.83 , and 0.82 - 0.87 , respectively , when each was used as a stand - alone criterion . for both readers , using the combined criteria resulted in an auc higher than that obtained when using subjective visual assessment ( auc 0.91 vs. auc 0.79 , p = 0.15 for reader 1 ; and auc 0.85 vs. auc 0.75 , p = 0.02 for reader 2 ) . for the less experienced observer , interobserver agreement regarding the individual imaging criteria ranged from = 0.12 to = 0.91 . the value for the combined criteria set ( isointense mass , a nodular shape , and an irregular border ) was 0.84 . both moderate differences correspond to two cases with no evidence of local residual disease after follow - up , in which the less experienced reader scored an isointense mass in contrast to the experienced reader . case - by - case comparison and interobserver agreement of subjective and objective magnetic resonance imaging ( mri ) for assessing residual tumor after rt ( n = 42 ) perfect match : no difference in agreement between observers ; minor : 1-point difference , moderate : 2-point difference , substantial : 3-point difference , major : 4-point difference magnetic resonance images taken immediately before the final bct were available for 29/42 patients . the subjective ( visual ) assessment of mr images taken immediately before the final bct resulted in an auc of 0.75 , sensitivity of 75% , and specificity of 36% for reader 1 , and 0.43 , 100% , and 8% , respectively , for reader 2 . objective imaging criteria on images taken immediately before the final bct resulted in aucs of 0.51 - 0.82 . twenty - seven patients had no evidence of disease after a median follow - up of 24 months ( range 13 - 81 months ) . figure 2 and tables 2 and 3 show the diagnostic performance of both readers based on the subjective visual response , the objective imaging criteria , and the combined mri criteria . the interobserver agreement for the individual imaging criteria is shown in tables 2 , 3 , and 4 . reciever operating curve for magnetic resonance imaging ( mri ) analysis after radiotherapy , observer 1 and 2 diagnostic performance of subjective and objective magnetic resonance imaging ( mri ) criteria analyses for assessing residual tumor ( n = 7 ) with mri after radiotherapy ( n = 42 ) auc area under the receiver operating characteristics curve , hypointense mass , confidence interval = 0 , no hypointense mass , confidence interval = 4 sensitivity and specificity are given with the corresponding 95% confidence intervals diagnostic performance of subjective and objective magnetic resonance imaging ( mri ) criteria for assessing residual tumor ( n = 4 ) with mri during brachytherapy ( n = 29 ) auc area under the receiver operating characteristics curve , hypointense mass , confidence interval = 0 , no hypointense mass , confidence interval = 4 sensitivity and specificity are given with the corresponding 95% for reader 1 ( the more experienced ) , the subjective visual assessment after radiotherapy resulted in an auc of 0.79 , sensitivity of 71% , and specificity of 86% . for the junior reader ( reader 2 ) , the results were 0.75 , 86% , and 46% , respectively . interobserver agreement for the subjective visual response assessment after treatment was moderate ( = 0.41 ) . using this mri , the auc for reader 1 regarding assessment of the tumor response after completion of rt was 0.99 . an isointense mass , a nodular shape , and an irregular border yielded aucs of 0.79 , 0.79 - 0.83 , and 0.82 - 0.87 , respectively , when each was used as a stand - alone criterion . for both readers , using the combined criteria resulted in an auc higher than that obtained when using subjective visual assessment ( auc 0.91 vs. auc 0.79 , p = 0.15 for reader 1 ; and auc 0.85 vs. auc 0.75 , p = 0.02 for reader 2 ) . for the less experienced observer , the specificity improved from 46% to 80% ( p interobserver agreement regarding the individual imaging criteria ranged from = 0.12 to = 0.91 . the value for the combined criteria set ( isointense mass , a nodular shape , and an irregular border ) was 0.84 . both moderate differences correspond to two cases with no evidence of local residual disease after follow - up , in which the less experienced reader scored an isointense mass in contrast to the experienced reader . objective magnetic resonance imaging ( mri ) for assessing residual tumor after rt ( n = 42 ) perfect match : no difference in agreement between observers ; minor : 1-point difference , moderate : 2-point difference , substantial : 3-point difference , major : 4-point difference the subjective ( visual ) assessment of mr images taken immediately before the final bct resulted in an auc of 0.75 , sensitivity of 75% , and specificity of 36% for reader 1 , and 0.43 , 100% , and 8% , respectively , for reader 2 . objective imaging criteria on images taken immediately before the final bct resulted in aucs of 0.51 - 0.82 . the present study examined the utility of mri performed during and after rt ( either with or without ct or ht ) for detecting residual tumor . subjective visual assessment of residual disease on post - rt mri resulted in a reasonable auc of 0.79 for the more experienced reader and an auc of 0.75 for the junior reader . however , the use of combined objective criteria ( isointense mass , nodular shape , and irregular border ) to assess residual disease after rt led to a much better performance , and increased the specificity parameter for the less experienced reader . moreover , the interobserver agreement improved to excellent when the assessment was performed using three objective criteria . this is the first report focusing on observer performance when interpreting mri scans with the aim of identifying residual disease after rt . morphologic visual t2w assessment of cervical tumors is often used to evaluate mr imaging of residual disease after rt . reported an equivocal diagnosis in 16/44 cases when iso-/hyperintensity combined with contrast enhancement were used as objective mri criteria ; subjective assessment led to an equivocal diagnosis in only 5/44 cases . in our data assessed treatment response at 3 - 8 weeks after the completion of rt , whereas we assessed the response at a median 9 weeks . however , we believe that the absence of recurrence at long - term follow - up is a good indicator of a lack of residual disease . moreover , when surgery is performed early after rt , microscopic tumors may still be present ( rt may not have exerted its full effect at this time ) . the use of predefined objective mri criteria for assessing residual tumor after rt increases diagnostic performance and makes an accurate diagnosis less dependent upon observer experience .

adverse blood pressure ( bp ) , prehypertensive and hypertensive , is an established major independent risk factor for epidemic cardiovascular diseases ( cvd ) , afflicting a high proportion of the adult population worldwide . in 20112012 , approximately 29 % of us adults had hypertension ( htn ) , defined as systolic blood pressure ( sbp ) 140 mmhg or higher and/or diastolic blood pressure ( dbp ) 90 mmhg or higher and/or the current use of antihypertensive medication . studies have consistently reported higher prevalence of adverse bp levels in blacks than in whites [ 2 , 47 ] . latest reports show that the prevalence of htn in us adults was about 42 % for blacks and 28 % for whites . compared with whites , blacks develop high bp earlier in life , and their average bps are higher [ 5 , 8 ] . the higher bp levels for blacks are associated with the increased risk of heart disease and stroke [ 5 , 9 , 10 ] . among adults with htn , blacks had significantly ( p < 0.05 ) higher mortality rates than whites for diseases of the circulatory system in the cohorts of the national health and nutrition examination survey ( nhanes ) . although the underlying explanations for these ethnic disparities remain poorly understood , they have been related to differences in the environment and lifestyles , such as education and socioeconomic status , body weight , physical activity , tobacco use , and nutrition [ 6 , 1215 ] . studies have shown that dietary behavior is an important lifestyle factor impacting on the risk of developing htn [ 12 , 13 ] . however , the influences of specific dietary factors on high bp risk for blacks remain uncertain , in part because dietary behaviors and patterns differ over time , across geographical areas , and across demographic subgroups ( e.g. , north and south , urban and rural , lower and higher socioeconomic status ) [ 1618 ] . the nhanes study ( 20092010 ) reported that compared with whites , blacks consumed on average lower amounts of whole grains , fruits , and vegetables ( 0.8 , 1.2 , 1.3 servings / day for black men , respectively , vs. 1.1 , 1.6 , 2.1 servings / day for white men ) and higher amounts of sugar - sweetened beverages ( 11.2 servings / day for black men vs. 8.3 servings / day for white men ) . blacks had a higher average intake of dietary cholesterol ( 311 mg / day for black men vs. 263 mg / day for white men ) and lower average intake of dietary fiber compared with that of whites ( 13.6 g / day for black men vs. 16.3 g / day for white men ) . data from nhanes 20052010 showed that blacks consumed a larger percentage of energy from added sugars than that of whites ( 14.5 % for black men vs. 12.8 % for white men ) . nhanes also reported that lower percentages of blacks met the dietary guidelines for americans compared with that of whites for whole grains ( 3 servings / day ) , fruits ( 2 cups / day ) , vegetables ( 2 cups / day ) , nuts , legumes and seeds ( 4 servings / week ) , and sugar - sweetened beverages ( 36 oz./week ) . about 11 % of whites and 8 % of blacks met guidelines for fruits ; with 100 % fruit juices included , the number of servings increased and the proportions of whites consuming 2 cups / day doubled to 26 % and nearly quadrupled in blacks to 29 % . blacks were 43 % less likely than whites to meet fruit and vegetable guidelines . the 2005 behavioral risk factor surveillance system ( brfss ) reported that only about 21 % of blacks consumed 5 servings / day of fruits and vegetables ; the lowest of any us ethnic group . the reasons for geographic and racial differences in stroke ( regards ) study examined nutrient intakes among 21,334 blacks and whites in the stroke belt ( non - coastal regions of north carolina , south carolina , and georgia , as well as alabama , arkansas , georgia , louisiana , mississippi , and tennessee ; 20 % ) , stroke buckle ( coastal plain regions of north carolina , south carolina , and georgia ; 30 % ) , and elsewhere in the usa ( 50 % ) [ 17 , 18 ] . compared with whites , blacks within each region consumed a higher percentage of energy from carbohydrates and a lower percentage of energy from fats , and less fiber and alcohol ( table 1 ) [ 17 , 18 ] . the daily intakes of na , k , magnesium ( mg ) , and calcium ( ca ) were lower among black men compared with white men , whereas cholesterol intake was higher in blacks ( table 1 ) . black women also had significantly lower intakes of ca , mg , k , iron ( fe ) , and also na compared to that of white women within each region ( table 1 ) .table 1daily intake of macro / micronutrients of 5,105 men and 7,079 women from the reasons for geographic and racial differences in stroke ( regards ) study [ 17 , 18]men women stroke belt stroke buckle stroke belt stroke buckle black ( n = 793)white ( n = 2,456)black ( n = 418)white ( n = 1,438)black ( n = 1,600)white ( n = 2,603)black ( n = 971)white ( n = 1,905)carbohydrates , % energy49.5 ( 12.6)47.1 ( 11.3)**50.0 ( 12.1)46.7 ( 11.2)**50.9 ( 11.9)48.5 ( 11.7)**51.6 ( 12.3)48.1 ( 11.6)**protein , % energy13.6 ( 3.7)14.4 ( 3.6)**13.7 ( 3.9)14.3 ( 3.9)**13.7 ( 4.0)14.5 ( 4.1)**13.8 ( 4.1)14.5 ( 3.8)**total fats , % energy36.2 ( 9.4)37.9 ( 97)**35.8 ( 9.7)37.8 ( 9.9)**36.2 ( 10.1)37.8 ( 10.0)**36.0 ( 9.5)37.9 ( 9.8)**alcohol , % energy0.3 ( 3.1)0.4 ( 4.7)*0.2 ( 2.0)0.5 ( 5.9)**0.0 ( 0.5)0.1 ( 1.1)**0.0 ( 0.4)0.2 ( 1.7)**fiber , g13.5 ( 10.2)15.3 ( 10.3)**12.6 ( 9.6)15.0 ( 9.6)**12.8 ( 9.5)14.3 ( 10)**12.8 ( 9.6)13.7 ( 9.2)**cholesterol , mg222 ( 200)215 ( 159)239 ( 216)207 ( 159)*167 ( 150)156 ( 124)*161 ( 159)155 ( 113)sodium , mg2170 ( 1476)2370 ( 1304)**2149 ( 1478)2321 ( 1286)**1854 ( 1356)1947 ( 1186)*1779 ( 1352)1889 ( 1112)*potassium , mg2306 ( 1453)2706 ( 1393)**2218 ( 1379)2647 ( 1242)**2148 ( 1346)2464 ( 1341)**2093 ( 1362)2361 ( 1276)**calcium , mg559 ( 405)666 ( 455)**523 ( 376)621 ( 411)**506 ( 394)606 ( 433)**460 ( 394)572 ( 419)**magnesium , mg238 ( 149)282 ( 157)**232 ( 140)275 ( 133)**218 ( 138)255 ( 147)**212 ( 137)247 ( 140)**iron , mg11.2 ( 6.9)12.8 ( 7.5)**11.7 ( 7.6)12.7 ( 7.0)**10.1 ( 6.8)11.0 ( 6.8)**9.7 ( 6.9)10.5 ( 6.3 ) * * non - coastal regions of north carolina , south carolina , and georgia , as well as alabama , arkansas , georgia , louisiana , mississippi , and tennessee coastal plain regions of north carolina , south carolina , and georgia*p < 0.05 for the wilcoxon two - sample test within region ( black vs. white ) ; * * p < 0.001 for the wilcoxon two - sample test within region ( black vs. white ) daily intake of macro / micronutrients of 5,105 men and 7,079 women from the reasons for geographic and racial differences in stroke ( regards ) study [ 17 , 18 ] non - coastal regions of north carolina , south carolina , and georgia , as well as alabama , arkansas , georgia , louisiana , mississippi , and tennessee coastal plain regions of north carolina , south carolina , and georgia * p < 0.05 for the wilcoxon two - sample test within region ( black vs. white ) ; * * p < 0.001 for the wilcoxon two - sample test within region ( black vs. white ) data from the continuing survey of food intakes by individuals ( csfii ) 19941998 and nhanes 19992000 showed that blacks in all age groups consumed significantly fewer servings / day of total dairy , milk , cheese , and yogurt than that of non - blacks , and blacks in all age groups did not meet dairy recommendations from the u.s . black women aged 3150 in the csfii had a mean ( sd ) total dairy intake of 0.71 ( 0.07 ) servings / day compared to other women in the same age group of 1.21 ( 0.03 ) servings / day . the total dairy intake of black women in the nhanes ( 0.83 servings / day ) was similar to black women reported in the csfii . data from nhanes reported that compared with whites ( n = 8,302 ) , blacks ( n = 3,458 ) had lower adjusted mean intakes of total dairy ( 138.8 g / day for blacks vs. 273.4 g / day for whites ) , low - fat milk ( 31.8 vs. 88.9 g / day ) , yogurt ( 2.1 vs. 7.1 g / day ) , and lower intakes of nutrients found in dairy foods ( k , ca , mg , phosphorus ( p ) , and vitamin d ) [ 24 , 25 ] . whites were more likely than blacks to use dietary supplements ; nhanes 20032006 reported that 59 % of whites took dietary supplements compared to 36 % blacks [ 26 , 27 ] . the multiethnic cohort study investigated multivitamin / mineral intakes of 159,017 participants and reported a higher rate of supplement use in whites ( 57 % ) than blacks ( 43 % ) . however , median intakes from supplements for most nutrients ( e.g. , folate , vitamin b6 , vitamin d , ca ) were similar for blacks and whites except for vitamin a , where the median intake was 1,473 g retinol activity equivalents for whites and 1,233 g retinol activity equivalents for blacks . data from nhanes 20072010 showed that there was a significant difference between blacks and whites in the percentage of calories consumed from fast foods ( defined as foods usually sold at eating establishments for quick availability or takeout ) . young adults aged 2039 consumed the highest percentage of calories from fast foods ( 21 % in blacks vs. 15 % in whites , p < 0.05 ) . no ethnic differences prevailed in calorie intake from fast foods among adults aged 60 and over . the dietary approaches to stop hypertension ( dash ) and the dash - na trials demonstrated that dietary patterns rich in vegetables , fruits , and low - fat dairy products and reduced in na , total fat , saturated fat , and dietary cholesterol lower bp effectively in prehypertensive and hypertensive adults , blacks and whites [ 3035 ] . in the dash trial , 459 participants ( sbp < 160 mmhg and dbp 8095 mmhg ) were randomly assigned to a control diet ( low in fruits , vegetables , and dairy products , with a fat content typical of the average diet in usa ) , fruits - and - vegetables diet ( rich in fruits and vegetables ) , or combination diet ( rich in fruits , vegetables , and low - fat dairy products and with reduced saturated and total fats ) . with the combination diet , sbp was lower by 5.5 mmhg ( 95 % confidence interval ( ci ) , 3.77.4 mmhg ) than that with the control diet ( p < 0.001 ) . with the fruits - and - vegetables diet , sbp was lower by 2.8 mmhg ( 0.94.7 mmhg ) than that with the control diet ( p < 0.001 ) . compared with the fruits - and - vegetables diet , the combination diet reduced sbp by 2.7 mmhg ( 0.94.6 ) more ( p < 0.001 ) . the dash combination diet lowered bp significantly more in blacks than in whites ( fig . 1 ) despite similar bp levels at baseline ( 131.8/84.8 mmhg for blacks vs. 130.9/84.5 mmhg for whites ) .fig . 1effect of ethnicity and hypertension status on ( a ) systolic blood pressure and ( b ) diastolic blood pressure response to dietary approaches to stop hypertension ( dash ) combination diet , adjusted for site and cohort effect effect of ethnicity and hypertension status on ( a ) systolic blood pressure and ( b ) diastolic blood pressure response to dietary approaches to stop hypertension ( dash ) combination diet , adjusted for site and cohort effect in the dash - na trial , 412 participants ( with sbp 120159 mmhg and dbp 8095 mmhg ) were randomly assigned to a control diet or the dash diet ; within the assigned diet , participants ate foods with three different levels of na ( for a 2100-kcal diet : lower 50 mmol / day , intermediate 100 mmol / day , and higher 150 mmol / day ) . the reduction of na intake from the high to the intermediate level lowered sbp by 2.1 mmhg ( 95 % ci 0.83.4 mmhg , p < 0.001 ) with the control diet and by 1.3 mmhg ( 95 % ci 0.02.6 mmhg , p = 0.03 ) with the dash diet . the reduction of na intake from the intermediate to the low level resulted in additional bp lowering of 4.6 mmhg ( 95 % ci 3.25.9 mmhg ) with the control diet ( p < 0.001 ) and 1.7 mmhg ( 95 % ci 0.43.0 mmhg ) with the dash diet ( p < 0.01 ) . the baseline bp levels were similar for blacks and whites ; average sbp / dbp was 135.3/86.1 mmhg for blacks and 134.1/85.1 mmhg for whites . among participants on the control diet , lower ( vs. higher ) na intake decreased sbp by 8.0 ( 95 % ci 6.59.4 mmhg ) in blacks and by 5.1 mmhg ( 3.46.7 ) in whites ( p < 0.01 ) . among participants on the dash diet , lower ( vs. higher ) na intake decreased sbp by 3.6 mmhg ( 95 % ci 2.25.1 ) in blacks and by 2.2 mmhg ( 0.53.8 ) in whites [ 36 , 37 ] . the dash / dash - na diet bp reduction was more pronounced for blacks compared to whites [ 31 , 36 , 37 ] . although the dash dietary approach has been incorporated into lifestyle changes recommended for patients with htn , data show that few hypertensive americans consume diets even modestly concordant with the dash diet and less so for blacks . only about 19 % of individuals with known htn from nhanes the exercise and nutrition interventions for cardiovascular health study ( encore ) , a 16-week intervention trial of 144 participants with high bp ( sbp 130159 mmhg and/or dbp 8599 mmhg ) , reported that greater adherence to the dash diet was associated with larger bp reductions [ 39 ] . each 2-point increase in dash diet adherence was associated with a 3.4 mmhg ( 95 % ci 2.4 to 4.4 ) reduction in sbp . the dash adherence score ( adopted from folsom and colleagues ) , ranging from 0 to 10 , was calculated from a food frequency questionnaire ( ffq ) . at baseline , black participants in the trial were less likely to consume foods consistent with the dash diet compared with that of whites ; they consumed fewer low - fat dairy products and more sweets compared with that of whites . after intervention , participants increased their consumption of dash - designated foods , and the dash adherence score increased to 4.68 in blacks and 5.83 in whites ( p < 0.001 ) ; compared with whites , blacks continued to consume more meats , sweets , fats , and fewer fruits . these findings indicated lower adherence by black than white participants to the dash diet and , in turn , smaller bp reduction independent of weight loss . the atherosclerosis risk in communities ( aric ) study analyzed ffq data for 8,208 non - hypertensive women and men aged 45 to 64 years . whites consuming 3 daily servings of low - fat milk , compared with those consuming < 1 serving , had a 2.7 mmhg smaller sbp increase with a 9-year follow - up ( p for trend = 0.01 ) . the aric study also reported that p intake was inversely associated with sbp . compared with participants in the lowest quintile of p intake at baseline , those in the highest quintile had significantly lower baseline sbp for both whites and blacks after adjustment for non - dietary confounders ( 2.3 mmhg , 95 % ci 3.4 to 1.2 , p for trend < 0.0001 for whites ; 2.3 mmhg , 5.5 to 0.8 , p for trend = 0.01 for blacks ) , but not after additional adjustment for dietary confounders ( 2.9 mmhg , 95 % ci 4.7 to 1.1 , p for trend = 0.002 for whites ; 0.8 mmhg , 6.5 to 4.9 , p for trend = 0.63 for blacks ) . further analyses showed that p from dairy products ( the main source of p with 31 % contribution)but not from other sources ( fish for 7 % and red meat for 7 % of p)was associated with lower baseline bp and reduced risk of htn . nhanes 19992004 reported that bp was inversely and significantly ( p < 0.05 ) associated with fluid milk , yogurt , and mg , while cheese was positively and significantly associated with sbp and dbp . the adjusted mean sbp was 125.7 mmhg for blacks and 122.6 mmhg for whites ( p < 0.05 ) ; this sbp difference between blacks and whites was partly explained by dairy - related nutrients . the international study of macro / micronutrients and blood pressure ( intermap ) of 2,195 men and women aged 40 to 59 from 8 us population samples reported that less favorable intakes of multiple foods / nutrients by blacks than whites partly accounted for the higher bp of blacks [ 43 ] . the average bp was 124.2/78.4 mmhg for black men and 123.9/75.1 mmhg for black women and 120.0/75.9 mmhg for white men and 114.5/70.6 mmhg for white women . of black women , 47 % were hypertensive compared to 20 % of white women ( p < 0.0001 ) . compared with whites , blacks had lower average intake of fresh fruits , total vegetables , total grains , bread / rolls , and cheese , and higher intake of processed meats , pork , eggs , fruit juice / drinks , sugar - sweetened drinks , fish , and poultry ( table 2 ) . black participants also had lower average intakes of vegetable protein , glutamic acid , starch , fiber , ca , mg , p , and fe , and lower urinary k excretion , along with higher intakes of dietary cholesterol , total sugars , fructose / glucose / sucrose , glycine and higher urinary na / k ratio , related to higher black bp ( table 2 ) . compared with whites , mean sbp of blacks was higher by 4.8 mmhg for men ( p < 0.001 ) and 9.0 mmhg for women adjusted for non - dietary confounders ( p < 0.0001 ) . with additional adjustment for nutrients , the effects on black - white sbp difference reduced to 2.3 mmhg ( 52 % reduction ) in men and 5.3 mmhg ( 21 % reduction ) in women . the additional combinations of foods and urinary metabolites had little further influence on higher bp in blacks.table 2average intake of foods , macro / micronutrients , of 785 men and 774 women from the international study of macro / micronutrients and blood pressure ( intermap ) [ 43]menwomenblack ( n = 165)white ( n = 620)black ( n = 204)white ( n = 570)fresh fruit , g/1,000 kcal42.1 ( 54.0)53.0 ( 64.8)*57.4 ( 73.5)73.2 ( 67.9)**total vegetable , g/1,000 kcal107.2 ( 60.2)127.2 ( 65.6)***133.9 ( 79.6)144.5 ( 74.9)total grains , g/1,000 kcal89.0 ( 40.4)97.3 ( 36.4)*85.0 ( 33.8)106.2 ( 37.0)***bread / rolls / biscuits , g/1,000 kcal30.7 ( 16.3)38.2 ( 21.0)***31.3 ( 19.7)39.4 ( 22.6)***cheese , g/1,000 kcal6.8 ( 6.4)13.1 ( 12.1)***9.2 ( 9.3)15.6 ( 14.8)***processed meats , g/1,000 kcal11.1 ( 12.0)9.9 ( 11.9)10.3 ( 13.5)6.9 ( 9.4)***pork , g/1,000 kcal10.0 ( 13.2)8.5 ( 11.8)10.0 ( 15.5)6.3 ( 9.9)***eggs , g/1,000 kcal12.5 ( 13.1)9.9 ( 9.9)**12.4 ( 12.7)9.4 ( 9.1)***fruit juices / drinks , g/1,000 kcal122.5 ( 122.7)69.9 ( 102.1)***110.1 ( 126.0)66.4 ( 118.6)***sugar - sweetened beverages , g/1,000 kcal210.1 ( 165.6)126.3 ( 150.9)***194.7 ( 166.7)76.7 ( 108.0)***fish / fish roe / shellfish , g/1,000 kcal10.2 ( 16.3)7.5 ( 12.8)*9.9 ( 14.6)8.5 ( 13.7)poultry , g/1,000 kcal27.6 ( 22.6)17.1 ( 16.8)***30.1 ( 23.7)17.8 ( 17.0)***vegetable protein , % kcal4.4 ( 1.7)5.0 ( 1.5)***4.5 ( 1.3)5.4 ( 1.4)***glutamic acid , % kcal2.8 ( 0.5)3.0 ( 0.6)***2.8 ( 0.6)3.1 ( 0.5)***glycine , % kcal0.7 ( 0.2)0.6 ( 0.2)***0.7 ( 0.2)0.6 ( 0.2)***starch , % kcal19.9 ( 5.4)21.9 ( 5.0)***19.8 ( 5.0)23.2 ( 5.0)***total sugars , % kcal28.6 ( 8.7)26.6 ( 8.4)***29.8 ( 8.5)27.7 ( 7.2)***fructose / glucose / sucrose , % kcal24.2 ( 8.8)20.7 ( 8.4)***25.1 ( 8.6)20.8 ( 6.9)***total dietary fiber , g/1,000 kcal7.6 ( 3.4)8.9 ( 3.3)***8.2 ( 3.4)9.9 ( 3.4)***dietary cholesterol , mg/1,000 kcal140.7 ( 62.1)123.6 ( 52.9)***141.9 ( 64.1)117.4 ( 48.6)***urinary potassium , mmol/24 h55.5 ( 17.9)71.7 ( 21.2)***44.2 ( 15.9)56.8 ( 17.9)***urinary sodium / potassium ratio3.55 ( 1.28)2.75 ( 0.98)***3.69 ( 1.53)2.68 ( 0.98)***calcium , mg/1,000 kcal288.9 ( 107.3)382.3 ( 138.4)***308.8 ( 114.4)430.1 ( 147.4)***magnesium , mg/1,000 kcal125.4 ( 37.0)147.6 ( 35.1)***130.4 ( 37.6)159.7 ( 39.0)***iron , mg/1,000 kcal6.9 ( 3.1)7.8 ( 2.9)***7.0 ( 2.3)8.3 ( 2.8)***non - heme iron , mg/1,000 kcal6.3 ( 3.1)7.3 ( 2.9)***6.5 ( 2.2)7.8 ( 2.8)***phosphorus , mg/1,000 kcal523.9 ( 103.3)600.9 ( 125.7)***534.4 ( 117.5)629.9 ( 127.9)****p < 0.05 for the student s t test ; * * p < 0.01 for the student s t test ; * * * p < 0.001 for student s the t test average intake of foods , macro / micronutrients , of 785 men and 774 women from the international study of macro / micronutrients and blood pressure ( intermap ) [ 43 ] * p < 0.05 for the student s t test ; * * p < 0.01 for the student s t test ; * * * p < 0.001 for student s the t test the heart follow - up study ( hfus ) , an investigation of 1,568 men and women in new york city , using 24-h urine collection to assess na and k intake , found that black participants had higher weighted mean sbp ( 126.0 mmhg for blacks vs. 121.3 mmhg for whites , p < 0.01 ) and dbp ( 77.2 vs. 72.8 mmhg , p < 0.001 ) compared with that of whites . black men had significantly higher na and na / k ratio and lower k intakes than that of white men ( p < 0.001 ) ; black women had higher na / k ratio and lower k intake compared with that of white women ( p < 0.001 ) . among black men aged 50 , 1,000 mg greater na intake was associated with 1.9 mmhg higher in sbp ( p < 0.05 ) ; 1,000 mg higher k intake was associated with 4.1 mmhg lower in sbp ( p < 0.05 ) ; 1 unit higher in na / k ratio was associated with 3.4 mmhg higher in sbp ( p < 0.01 ) . no significant relationships between na , k intakes , and sbp were found in white men of the same age group . the jackson heart study ( jhs ) , using a validated ffq to assess nutrition among 1,775 blacks in the southern usa ( jackson , ms ) , identified three major dietary patterns : southern , fast food , and prudent , and reported that higher adjusted southern pattern scores were significantly associated with higher odds ratio ( or ) for htn ( p < 0.05 ) . the southern dietary pattern was characterized by the high consumption of vegetables with high starch content ( e.g. , beans , corn , and potatoes ) , fried meats , poultry , and fish , margarine and butter for cooking , and whole milk and buttermilk for cornbread and rolls . the fast food pattern was characterized by high consumption of sugar - sweetened drinks , salty snacks , and fast foods , and the prudent pattern was characterized by high intakes of fruits and vegetables and cereals and low intakes of white bread and sweets . after adjustment for non - dietary confounders , higher southern pattern scores ( or = 1.42 , 95 % ci 1.11.9 for tertile 2 , p = 0.02 and or = 1.14 , 95 % ci 0.71.8 for tertile 3 , p = 0.6 ) and higher fast food pattern scores ( or = 1.35 , 95 % ci 0.91.8 for tertile 2 , p = 0.06 and or = 1.67 , 95 % ci 1.12.7 for tertile 3 , p = 0.03 ) were significantly associated with htn . the prudent pattern score was inversely associated with htn ( or = 0.75 , 95 % ci 0.60.9 in tertile 2 , p = 0.05 and or = 0.69 , 0.50.9 in tertile 3 , p = 0.02 ) . blood pressure levels have consistently been found to be higher in blacks than whites , with an earlier onset of htn . data showed blacks had significantly lower intakes of fruits , vegetables , and dairy products , and lower intakes of k , mg , ca , and p compared with that of whites . however , dietary differences between blacks and whites , including na and k intakes , do not fully explain the higher prevalence of adverse bp levels in blacks . the dash diet with na reduction resulted in a greater reduction of bp in blacks than whites . lower average incomes for blacks compared to whites and strong cultural influences relating to food preferences , food preparation , and perceptions about eating practices may make it more challenging for blacks to adhere to a dash - type diet . african - americans have a distinctive culinary heritage with diverse flavors ( derived from the african continent , the west indies , and north america ) . more work is needed on the implementation of the dash bp reduction diet for blacks , including ways to identify affordable nutrient - rich foods and reduce consumption of fried , energy - dense , salt - dense , and nutrient - poor foods .

adverse blood pressure ( bp ) is a major independent risk factor for epidemic cardiovascular diseases affecting almost one third of the us adult population . this review synthesizes results from studies published over the past few years on bp differences and prevalent hypertension between us blacks and whites and their different intakes of foods ( e.g. , fruits , vegetables , and dairy products ) and micronutrients ( e.g. , vitamin d , calcium , potassium , and phosphorus ) . studies have consistently reported higher prevalence of adverse bp levels and hypertension and less favorable dietary intakes in blacks than in whites , but the influence of specific dietary factors on high bp risk for blacks remains unclear .

Introduction Dietary Differences Between Blacks and Whites Diet and Blood Pressure Conclusions

adverse blood pressure ( bp ) , prehypertensive and hypertensive , is an established major independent risk factor for epidemic cardiovascular diseases ( cvd ) , afflicting a high proportion of the adult population worldwide . studies have consistently reported higher prevalence of adverse bp levels in blacks than in whites [ 2 , 47 ] . latest reports show that the prevalence of htn in us adults was about 42 % for blacks and 28 % for whites . compared with whites , blacks develop high bp earlier in life , and their average bps are higher [ 5 , 8 ] . however , the influences of specific dietary factors on high bp risk for blacks remain uncertain , in part because dietary behaviors and patterns differ over time , across geographical areas , and across demographic subgroups ( e.g. the nhanes study ( 20092010 ) reported that compared with whites , blacks consumed on average lower amounts of whole grains , fruits , and vegetables ( 0.8 , 1.2 , 1.3 servings / day for black men , respectively , vs. 1.1 , 1.6 , 2.1 servings / day for white men ) and higher amounts of sugar - sweetened beverages ( 11.2 servings / day for black men vs. 8.3 servings / day for white men ) . nhanes also reported that lower percentages of blacks met the dietary guidelines for americans compared with that of whites for whole grains ( 3 servings / day ) , fruits ( 2 cups / day ) , vegetables ( 2 cups / day ) , nuts , legumes and seeds ( 4 servings / week ) , and sugar - sweetened beverages ( 36 oz./week ) . about 11 % of whites and 8 % of blacks met guidelines for fruits ; with 100 % fruit juices included , the number of servings increased and the proportions of whites consuming 2 cups / day doubled to 26 % and nearly quadrupled in blacks to 29 % . the reasons for geographic and racial differences in stroke ( regards ) study examined nutrient intakes among 21,334 blacks and whites in the stroke belt ( non - coastal regions of north carolina , south carolina , and georgia , as well as alabama , arkansas , georgia , louisiana , mississippi , and tennessee ; 20 % ) , stroke buckle ( coastal plain regions of north carolina , south carolina , and georgia ; 30 % ) , and elsewhere in the usa ( 50 % ) [ 17 , 18 ] . compared with whites , blacks within each region consumed a higher percentage of energy from carbohydrates and a lower percentage of energy from fats , and less fiber and alcohol ( table 1 ) [ 17 , 18 ] . the daily intakes of na , k , magnesium ( mg ) , and calcium ( ca ) were lower among black men compared with white men , whereas cholesterol intake was higher in blacks ( table 1 ) . black women also had significantly lower intakes of ca , mg , k , iron ( fe ) , and also na compared to that of white women within each region ( table 1 ) .table 1daily intake of macro / micronutrients of 5,105 men and 7,079 women from the reasons for geographic and racial differences in stroke ( regards ) study [ 17 , 18]men women stroke belt stroke buckle stroke belt stroke buckle black ( n = 793)white ( n = 2,456)black ( n = 418)white ( n = 1,438)black ( n = 1,600)white ( n = 2,603)black ( n = 971)white ( n = 1,905)carbohydrates , % energy49.5 ( 12.6)47.1 ( 11.3)**50.0 ( 12.1)46.7 ( 11.2)**50.9 ( 11.9)48.5 ( 11.7)**51.6 ( 12.3)48.1 ( 11.6)**protein , % energy13.6 ( 3.7)14.4 ( 3.6)**13.7 ( 3.9)14.3 ( 3.9)**13.7 ( 4.0)14.5 ( 4.1)**13.8 ( 4.1)14.5 ( 3.8)**total fats , % energy36.2 ( 9.4)37.9 ( 97)**35.8 ( 9.7)37.8 ( 9.9)**36.2 ( 10.1)37.8 ( 10.0)**36.0 ( 9.5)37.9 ( 9.8)**alcohol , % energy0.3 ( 3.1)0.4 ( 4.7)*0.2 ( 2.0)0.5 ( 5.9)**0.0 ( 0.5)0.1 ( 1.1)**0.0 ( 0.4)0.2 ( 1.7)**fiber , g13.5 ( 10.2)15.3 ( 10.3)**12.6 ( 9.6)15.0 ( 9.6)**12.8 ( 9.5)14.3 ( 10)**12.8 ( 9.6)13.7 ( 9.2)**cholesterol , mg222 ( 200)215 ( 159)239 ( 216)207 ( 159)*167 ( 150)156 ( 124)*161 ( 159)155 ( 113)sodium , mg2170 ( 1476)2370 ( 1304)**2149 ( 1478)2321 ( 1286)**1854 ( 1356)1947 ( 1186)*1779 ( 1352)1889 ( 1112)*potassium , mg2306 ( 1453)2706 ( 1393)**2218 ( 1379)2647 ( 1242)**2148 ( 1346)2464 ( 1341)**2093 ( 1362)2361 ( 1276)**calcium , mg559 ( 405)666 ( 455)**523 ( 376)621 ( 411)**506 ( 394)606 ( 433)**460 ( 394)572 ( 419)**magnesium , mg238 ( 149)282 ( 157)**232 ( 140)275 ( 133)**218 ( 138)255 ( 147)**212 ( 137)247 ( 140)**iron , mg11.2 ( 6.9)12.8 ( 7.5)**11.7 ( 7.6)12.7 ( 7.0)**10.1 ( 6.8)11.0 ( 6.8)**9.7 ( 6.9)10.5 ( 6.3 ) * * non - coastal regions of north carolina , south carolina , and georgia , as well as alabama , arkansas , georgia , louisiana , mississippi , and tennessee coastal plain regions of north carolina , south carolina , and georgia*p < 0.05 for the wilcoxon two - sample test within region ( black vs. white ) ; * * p < 0.001 for the wilcoxon two - sample test within region ( black vs. white ) daily intake of macro / micronutrients of 5,105 men and 7,079 women from the reasons for geographic and racial differences in stroke ( regards ) study [ 17 , 18 ] non - coastal regions of north carolina , south carolina , and georgia , as well as alabama , arkansas , georgia , louisiana , mississippi , and tennessee coastal plain regions of north carolina , south carolina , and georgia * p < 0.05 for the wilcoxon two - sample test within region ( black vs. white ) ; * * p < 0.001 for the wilcoxon two - sample test within region ( black vs. white ) data from the continuing survey of food intakes by individuals ( csfii ) 19941998 and nhanes 19992000 showed that blacks in all age groups consumed significantly fewer servings / day of total dairy , milk , cheese , and yogurt than that of non - blacks , and blacks in all age groups did not meet dairy recommendations from the u.s . data from nhanes reported that compared with whites ( n = 8,302 ) , blacks ( n = 3,458 ) had lower adjusted mean intakes of total dairy ( 138.8 g / day for blacks vs. 273.4 g / day for whites ) , low - fat milk ( 31.8 vs. 88.9 g / day ) , yogurt ( 2.1 vs. 7.1 g / day ) , and lower intakes of nutrients found in dairy foods ( k , ca , mg , phosphorus ( p ) , and vitamin d ) [ 24 , 25 ] . the multiethnic cohort study investigated multivitamin / mineral intakes of 159,017 participants and reported a higher rate of supplement use in whites ( 57 % ) than blacks ( 43 % ) . however , median intakes from supplements for most nutrients ( e.g. , folate , vitamin b6 , vitamin d , ca ) were similar for blacks and whites except for vitamin a , where the median intake was 1,473 g retinol activity equivalents for whites and 1,233 g retinol activity equivalents for blacks . data from nhanes 20072010 showed that there was a significant difference between blacks and whites in the percentage of calories consumed from fast foods ( defined as foods usually sold at eating establishments for quick availability or takeout ) . young adults aged 2039 consumed the highest percentage of calories from fast foods ( 21 % in blacks vs. 15 % in whites , p < 0.05 ) . the dietary approaches to stop hypertension ( dash ) and the dash - na trials demonstrated that dietary patterns rich in vegetables , fruits , and low - fat dairy products and reduced in na , total fat , saturated fat , and dietary cholesterol lower bp effectively in prehypertensive and hypertensive adults , blacks and whites [ 3035 ] . in the dash trial , 459 participants ( sbp < 160 mmhg and dbp 8095 mmhg ) were randomly assigned to a control diet ( low in fruits , vegetables , and dairy products , with a fat content typical of the average diet in usa ) , fruits - and - vegetables diet ( rich in fruits and vegetables ) , or combination diet ( rich in fruits , vegetables , and low - fat dairy products and with reduced saturated and total fats ) . the dash combination diet lowered bp significantly more in blacks than in whites ( fig . 1 ) despite similar bp levels at baseline ( 131.8/84.8 mmhg for blacks vs. 130.9/84.5 mmhg for whites ) .fig . 1effect of ethnicity and hypertension status on ( a ) systolic blood pressure and ( b ) diastolic blood pressure response to dietary approaches to stop hypertension ( dash ) combination diet , adjusted for site and cohort effect effect of ethnicity and hypertension status on ( a ) systolic blood pressure and ( b ) diastolic blood pressure response to dietary approaches to stop hypertension ( dash ) combination diet , adjusted for site and cohort effect in the dash - na trial , 412 participants ( with sbp 120159 mmhg and dbp 8095 mmhg ) were randomly assigned to a control diet or the dash diet ; within the assigned diet , participants ate foods with three different levels of na ( for a 2100-kcal diet : lower 50 mmol / day , intermediate 100 mmol / day , and higher 150 mmol / day ) . the baseline bp levels were similar for blacks and whites ; average sbp / dbp was 135.3/86.1 mmhg for blacks and 134.1/85.1 mmhg for whites . among participants on the control diet , lower ( vs. higher ) na intake decreased sbp by 8.0 ( 95 % ci 6.59.4 mmhg ) in blacks and by 5.1 mmhg ( 3.46.7 ) in whites ( p < 0.01 ) . among participants on the dash diet , lower ( vs. higher ) na intake decreased sbp by 3.6 mmhg ( 95 % ci 2.25.1 ) in blacks and by 2.2 mmhg ( 0.53.8 ) in whites [ 36 , 37 ] . after intervention , participants increased their consumption of dash - designated foods , and the dash adherence score increased to 4.68 in blacks and 5.83 in whites ( p < 0.001 ) ; compared with whites , blacks continued to consume more meats , sweets , fats , and fewer fruits . compared with participants in the lowest quintile of p intake at baseline , those in the highest quintile had significantly lower baseline sbp for both whites and blacks after adjustment for non - dietary confounders ( 2.3 mmhg , 95 % ci 3.4 to 1.2 , p for trend < 0.0001 for whites ; 2.3 mmhg , 5.5 to 0.8 , p for trend = 0.01 for blacks ) , but not after additional adjustment for dietary confounders ( 2.9 mmhg , 95 % ci 4.7 to 1.1 , p for trend = 0.002 for whites ; 0.8 mmhg , 6.5 to 4.9 , p for trend = 0.63 for blacks ) . the adjusted mean sbp was 125.7 mmhg for blacks and 122.6 mmhg for whites ( p < 0.05 ) ; this sbp difference between blacks and whites was partly explained by dairy - related nutrients . the international study of macro / micronutrients and blood pressure ( intermap ) of 2,195 men and women aged 40 to 59 from 8 us population samples reported that less favorable intakes of multiple foods / nutrients by blacks than whites partly accounted for the higher bp of blacks [ 43 ] . compared with whites , blacks had lower average intake of fresh fruits , total vegetables , total grains , bread / rolls , and cheese , and higher intake of processed meats , pork , eggs , fruit juice / drinks , sugar - sweetened drinks , fish , and poultry ( table 2 ) . black participants also had lower average intakes of vegetable protein , glutamic acid , starch , fiber , ca , mg , p , and fe , and lower urinary k excretion , along with higher intakes of dietary cholesterol , total sugars , fructose / glucose / sucrose , glycine and higher urinary na / k ratio , related to higher black bp ( table 2 ) . compared with whites , mean sbp of blacks was higher by 4.8 mmhg for men ( p < 0.001 ) and 9.0 mmhg for women adjusted for non - dietary confounders ( p < 0.0001 ) . the additional combinations of foods and urinary metabolites had little further influence on higher bp in blacks.table 2average intake of foods , macro / micronutrients , of 785 men and 774 women from the international study of macro / micronutrients and blood pressure ( intermap ) [ 43]menwomenblack ( n = 165)white ( n = 620)black ( n = 204)white ( n = 570)fresh fruit , g/1,000 kcal42.1 ( 54.0)53.0 ( 64.8)*57.4 ( 73.5)73.2 ( 67.9)**total vegetable , g/1,000 kcal107.2 ( 60.2)127.2 ( 65.6)***133.9 ( 79.6)144.5 ( 74.9)total grains , g/1,000 kcal89.0 ( 40.4)97.3 ( 36.4)*85.0 ( 33.8)106.2 ( 37.0)***bread / rolls / biscuits , g/1,000 kcal30.7 ( 16.3)38.2 ( 21.0)***31.3 ( 19.7)39.4 ( 22.6)***cheese , g/1,000 kcal6.8 ( 6.4)13.1 ( 12.1)***9.2 ( 9.3)15.6 ( 14.8)***processed meats , g/1,000 kcal11.1 ( 12.0)9.9 ( 11.9)10.3 ( 13.5)6.9 ( 9.4)***pork , g/1,000 kcal10.0 ( 13.2)8.5 ( 11.8)10.0 ( 15.5)6.3 ( 9.9)***eggs , g/1,000 kcal12.5 ( 13.1)9.9 ( 9.9)**12.4 ( 12.7)9.4 ( 9.1)***fruit juices / drinks , g/1,000 kcal122.5 ( 122.7)69.9 ( 102.1)***110.1 ( 126.0)66.4 ( 118.6)***sugar - sweetened beverages , g/1,000 kcal210.1 ( 165.6)126.3 ( 150.9)***194.7 ( 166.7)76.7 ( 108.0)***fish / fish roe / shellfish , g/1,000 kcal10.2 ( 16.3)7.5 ( 12.8)*9.9 ( 14.6)8.5 ( 13.7)poultry , g/1,000 kcal27.6 ( 22.6)17.1 ( 16.8)***30.1 ( 23.7)17.8 ( 17.0)***vegetable protein , % kcal4.4 ( 1.7)5.0 ( 1.5)***4.5 ( 1.3)5.4 ( 1.4)***glutamic acid , % kcal2.8 ( 0.5)3.0 ( 0.6)***2.8 ( 0.6)3.1 ( 0.5)***glycine , % kcal0.7 ( 0.2)0.6 ( 0.2)***0.7 ( 0.2)0.6 ( 0.2)***starch , % kcal19.9 ( 5.4)21.9 ( 5.0)***19.8 ( 5.0)23.2 ( 5.0)***total sugars , % kcal28.6 ( 8.7)26.6 ( 8.4)***29.8 ( 8.5)27.7 ( 7.2)***fructose / glucose / sucrose , % kcal24.2 ( 8.8)20.7 ( 8.4)***25.1 ( 8.6)20.8 ( 6.9)***total dietary fiber , g/1,000 kcal7.6 ( 3.4)8.9 ( 3.3)***8.2 ( 3.4)9.9 ( 3.4)***dietary cholesterol , mg/1,000 kcal140.7 ( 62.1)123.6 ( 52.9)***141.9 ( 64.1)117.4 ( 48.6)***urinary potassium , mmol/24 h55.5 ( 17.9)71.7 ( 21.2)***44.2 ( 15.9)56.8 ( 17.9)***urinary sodium / potassium ratio3.55 ( 1.28)2.75 ( 0.98)***3.69 ( 1.53)2.68 ( 0.98)***calcium , mg/1,000 kcal288.9 ( 107.3)382.3 ( 138.4)***308.8 ( 114.4)430.1 ( 147.4)***magnesium , mg/1,000 kcal125.4 ( 37.0)147.6 ( 35.1)***130.4 ( 37.6)159.7 ( 39.0)***iron , mg/1,000 kcal6.9 ( 3.1)7.8 ( 2.9)***7.0 ( 2.3)8.3 ( 2.8)***non - heme iron , mg/1,000 kcal6.3 ( 3.1)7.3 ( 2.9)***6.5 ( 2.2)7.8 ( 2.8)***phosphorus , mg/1,000 kcal523.9 ( 103.3)600.9 ( 125.7)***534.4 ( 117.5)629.9 ( 127.9)****p < 0.05 for the student s t test ; * * p < 0.01 for the student s t test ; * * * p < 0.001 for student s the t test average intake of foods , macro / micronutrients , of 785 men and 774 women from the international study of macro / micronutrients and blood pressure ( intermap ) [ 43 ] * p < 0.05 for the student s t test ; * * p < 0.01 for the student s t test ; * * * p < 0.001 for student s the t test the heart follow - up study ( hfus ) , an investigation of 1,568 men and women in new york city , using 24-h urine collection to assess na and k intake , found that black participants had higher weighted mean sbp ( 126.0 mmhg for blacks vs. 121.3 mmhg for whites , p < 0.01 ) and dbp ( 77.2 vs. 72.8 mmhg , p < 0.001 ) compared with that of whites . no significant relationships between na , k intakes , and sbp were found in white men of the same age group . the southern dietary pattern was characterized by the high consumption of vegetables with high starch content ( e.g. the fast food pattern was characterized by high consumption of sugar - sweetened drinks , salty snacks , and fast foods , and the prudent pattern was characterized by high intakes of fruits and vegetables and cereals and low intakes of white bread and sweets . blood pressure levels have consistently been found to be higher in blacks than whites , with an earlier onset of htn . data showed blacks had significantly lower intakes of fruits , vegetables , and dairy products , and lower intakes of k , mg , ca , and p compared with that of whites . however , dietary differences between blacks and whites , including na and k intakes , do not fully explain the higher prevalence of adverse bp levels in blacks . the dash diet with na reduction resulted in a greater reduction of bp in blacks than whites . lower average incomes for blacks compared to whites and strong cultural influences relating to food preferences , food preparation , and perceptions about eating practices may make it more challenging for blacks to adhere to a dash - type diet . more work is needed on the implementation of the dash bp reduction diet for blacks , including ways to identify affordable nutrient - rich foods and reduce consumption of fried , energy - dense , salt - dense , and nutrient - poor foods .

gastric specification in the mouse begins during gastrulation with derivation of the endodermal germ layer that eventually will seed the epithelial lining of the digestive , respiratory , and urogenital systems . the endoderm germ layer is formed by the ingression of epiblast cells through the primitive streak . as the cells exit the primitive streak , they arrange into a single - layered epithelial sheet on the outside of the embryo ( embryonic day [ e]6e7.5 ) . this sheet forms pockets at the anterior ( future foregut ) and posterior ( future hindgut ) end of the embryo and progressively zippers into a complete gut tube . zippering of the gut tube , mesodermal growth , and embryonic turning transform the endodermal sheet on the outside of the embryo into an internal tube consisting of 3 major regions : foregut , midgut , and hindgut ( e7.5e9 ) . regional and subsequent organ identity is assembled within the naive , as yet unspecified , gut tube through the integration of signaling inputs from mesodermal tissues located apposed to the endoderm and the endodermal progenitors themselves . one recognizable output of the stage when regional identity is acquired is a pattern of expression of overlapping transcription factor domains that facilitate subsequent organ - specific differentiation programs . stomach epithelial progenitors derive from the foregut region of the endoderm , which also gives rise to liver , pancreas , lungs , and the luminal gastrointestinal organs from the pharynx to the anterior duodenum . signaling pathways and transcription factors that drive specification of pregastric endodermal progenitors from other emerging organs within the foregut have not been well characterized . however , a number of signaling pathways that promote or restrict foregut identity by patterning the anterior / posterior axis of the endoderm are known . retinoic acid ( ra ) , for example , has a complex spatiotemporal role patterning the anterior posterior axis of the endoderm . during late gastrulation , ra signaling promotes the specification of posterior endodermal fates over anterior endodermal fates , particularly at the foregut midgut boundary.12 , 13 subsequently , ra signaling is required to promote the development of a number of foregut tissues . animals with defective ra signaling have abnormal stomach development , but a specific consequence to gastric specification is unclear . wnt and fibroblast growth factor ( fgf ) signals produced by the mesoderm promote expression of posterior endodermal markers such as cdx2 over anterior endodermal markers.15 , 16 , 17 studies in zebrafish also have shown that bone morphogenetic protein ( bmp ) signaling drives posterior over anterior endodermal fates . through the study of other endodermal organs , a number of tissues have been shown to produce important signaling molecules to promote foregut organ specification . for example , the dorsal aorta and notochord produce several key signaling molecules involved in dorsal pancreatic specification.19 , 20 these same tissues also could impact pregastric gene expression given the proximity of gastric and dorsal pancreatic progenitors . ventral tissues , including cardiac mesoderm , also could impact gastric specification from other ventral organs such as the liver and lung . other signaling pathways such as sonic hedgehog ( shh ) have been implicated in gastric growth through epithelial to mesenchyme signaling , although shh does not appear to be involved in gastric specification . during endodermal specification , a highly conserved core transcription network ( including foxa , gata , sox17 , and mixl1 transcription factors ) is activated and guides the growth and survival of endodermal cells before regionalization . expression of these transcription factors in early endoderm is necessary to generate foregut progenitors that give rise to the stomach . as the endoderm regionalizes , a number of transcription factors are expressed either throughout the foregut endoderm or regionally in the pregastric domain . broadly expressed transcription factors such as foxa1/2/3 , gata4/6 , hnf1 , and sox2 all could play an important role in gastric specification ( figure 2 ) . for example , the foxa family is expressed throughout the early endoderm and is important in the development of a number of organs including the liver , pancreas , and intestine.24 , 25 , 26 the specific role of this family in the stomach has yet to be determined , however , foxas are known to be involved in promoting pdx1 expression in the foregut ( figure 2 ) . because pdx1 is expressed only in the gastric antrum and not the more proximal corpus , foxa factors thus could be involved in regionalizing the stomach . gata4 and gata6 are involved in the specification of the extraembryonic endoderm28 , 29 , 30 and are expressed throughout the early definitive endoderm . during endodermal regionalization , both genes are expressed in the foregut . the expression domain of gata4 is particularly interesting because its anterior boundary resides at the future forestomach / glandular stomach boundary . potentially , gata4 may have an important role in specifying the glandular stomach or specifying the forestomach vs the glandular stomach ( figure 2 ) . consistent with the idea that gata4 is important for glandular stomach specification , gata4 null cells do not appear to be able to adopt gastric identity in chimeric embryos when they are competing with wild - type cells . sox2 is expressed broadly throughout the foregut from the most anterior pharyngeal endoderm to the future boundary of gastric antrum and duodenum . studies wherein expression of sox2 is reduced in developing endoderm have shown that it helps govern the development of a number of foregut organs including the stomach , esophagus , trachea , and lung.32 , 33 such experiments involved hypomorphic animals , so it will be interesting to know what the effects of complete loss of sox2 from early endoderm might be . perhaps sox2 has an even more critical role in anterior foregut and stomach specification than currently thought . the border between sox2 and cdx2 expression during development ( figure 2 ) resides at the prospective gastrointestinal junction and suggests that sox2 could define this boundary . misexpressing sox2 in cdx2-positive progenitors in the developing intestine increases expression of gastric - specific differentiation markers . interestingly , loss of cdx2 during early development causes a dramatic transformation of the prospective intestine into sox2-expressing esophageal - like progenitors and not gastric progenitors , indicating that sox2 is not a simple progastric , anti - intestine transcription factor . pdx1 is expressed regionally within the posterior foregut in the areas that give rise to the posterior stomach ( antrum / pylorus ) , anterior duodenum , dorsal and ventral pancreatic buds , and proximal extrahepatic biliary system.27 , 37 pdx1 expression can be used during development to distinguish antral gastric progenitors ( sox2gata4pdx1 ) from corpus progenitors ( sox2gata4pdx1 ) . loss of pdx1 causes aberrant antral stomach progenitors including pyloric defects and loss of gastrin - producing endocrine cells . definitive endoderm - specific knockout of hnf1 alters gene expression within caudal stomach progenitors , including causing loss of pdx1 and indian hedgehog ( ihh ) . the impact on gastric specification in these knockouts remains unclear , but recent in vitro studies intriguingly have implicated hnf1 in promoting antral stomach specification in organoid culture . to date , no specific gene has been shown to have expression restricted only to early gastric progenitors ; thus , it remains difficult to examine directly how the stomach is specified from other organs , the way , for example , cdx1 and cdx2 have been studied in intestinal specification . instead , investigators rely on more broadly expressed genes ( ie , expressed concomitantly in other organs besides the stomach ) such as sox2 , gata4 , and pdx1 to identify the factors defining the prospective gastric regions . further identification of transcripts that may have more restricted or specific expression to gastric progenitors ( particularly to the glandular stomach ) during early development could lead to the generation of new genetic tools to explore and characterize gastric specification or even to perform stomach - specific epithelial cell gene deletion because intestinal epithelial - specific deletion can be driven by villin - cre . however , there could be marked improvement in our understanding of stomach specification simply by manipulating gene expression in early endoderm with tools that already exist . for example , signaling pathways and transcription factors suspected of being involved in gastric development could be deleted via crosses to well - characterized mouse pedigrees that express foxa3- , sox17- , or shh- cre.41 , 42 , 43 summing up all that currently is known and can be inferred from published studies , we have proposed one possible signaling and epistasis model for specification of glandular stomach ( figure 2 ) . mesenchymal cross - talk , and the stomach does not seem to be an exception . for example , foundational experiments in chicks have shown that placing proventricular ( stomach region in chicks similar to the mammalian glandular stomach ) mesenchyme with gizzard ( anterior chicken stomach ) or esophageal endoderm induces proventricular gene expression and causes gland development in these normally nonglandular tissues.44 , 45 similarly , gizzard or esophageal mesenchyme can suppress proventricular gene expression and gland development in proventricular endoderm and promote squamous fates . interestingly , proventricular mesenchyme could not induce proventricular gene expression in intestinal endoderm ; hence , overlying mesoderm can instruct endoderm identity but only within restricted regions . although bmp signaling , principally deriving from the mesenchyme , influences gastric epithelial development , hedgehog signaling derived from the epithelium influences the mesenchyme . for example , in addition to their early role in foregut growth , hedgehog ( shh / ihh ) signals are produced by the gastric endoderm to support mesenchymal growth and differentiation , a pattern that is maintained in the adult.49 , 50 another example of a factor that originates from the mesenchyme and regulates the epithelium is fgf10 , likely via the fgf receptor 2b . fgf10 promotes epithelial proliferation and gland development.51 , 52 although it may not be required for adult homeostasis , it has been shown to inhibit parietal and chief cell differentiation in favor of the mucous neck cell type . in addition to the themes of epithelial hedgehog and mesenchymal bmp signaling that occur throughout the gastrointestinal tract , there have been some descriptions of signals more specific to gastric development vs other regions . for example , barx1 is a transcription factor that is restricted to the prospective esophageal and gastric mesoderm . the stomach intestinal boundary is disturbed such that ectopic cdx2 + intestinal epithelial cells can be found in the posterior stomach.54 , 55 in addition to the disrupted interorgan patterning , the division of intrastomach domains is altered . for example , h / k adenosine triphosphatase expressing cells are seen intermingled with pdx1 + cells , which in mice are normally exclusive to the corpus and antrum , respectively . bapx1 ( nkx3 - 2 ) , nkx2 - 5 , gata3 , six2 , nr2f2 ( chicken ovalbumine upstream promoter - transcription factor ii ) , and sox9 are other known transcription factors expressed in the posterior stomach mesenchyme and involved in specifying the pylorus.56 , 57 , 58 in the absence of those transcription factors , there is aberrant neuromuscular regulation of the pyloric sphincter , which in human beings can manifest as the relatively common condition known as pyloric stenosis.58 , 59 between the stage of endodermal specification and the stage of specific cell lineage commitment in the stomach , the gastric epithelium remains a simple epithelium with no obvious differentiation . at approximately e14.5e16.5 , markers representative of cell types such as endocrine , parietal , and chief cells begin to be expressed , and small glands begin to invaginate into the mesenchyme from the simple epithelium lining the lumen . between e16.5 and 2 weeks of postnatal development , most of the major cell types arise within the stomach , and the glandular stomach mostly becomes organized into its adult form . however , the murine stomach does not reach adult organization with full chief cell and endocrine cell lineage specification until 68 weeks postnatally . for most cell lineages in the stomach we have a poor understanding of pathways and factors involved in their specification and the progenitors from which they directly derive . for example , and this is truly remarkable when contrasted to the state of our understanding in the intestines , there is no specific factor that is known to be necessary or sufficient for specification of chief , parietal , pit , mucous neck , or isthmal cells . the markers used in gastric biology represent the terminal differentiation of those cells ( eg , atp4b , tff1 , pgc , and gif ) . the lack of this basic specification knowledge greatly hinders deciphering the molecular mechanisms underlying how gastric disease causes the loss or increase of any particular cell lineage . the developmental sequence between gastric epithelial progenitors in an adult gastric unit and the differentiated progeny that arise continuously throughout life also is unknown . it is entirely possible that all the mature cell types are specified from a single multipotent progenitor that persists throughout life , or , in turn , there might be numerous long - lived lineage - restricted progenitors62 , 63 ( figures 3 and 4 , and see detailed discussion later ) . the only stomach lineages with known genetic determinants and known progenitor markers are endocrine cells , which are controlled by the master regulators ascl1 and ngn3.65 , 66 ngn3 marks endocrine progenitor cells but not mature forms . ngn3 null embryos lack gastrin , somatostatin , and glucagon endocrine cell types , with largely reduced census of serotonin - positive cells , but enterochromaffin - like ( ecl ) and ghrelin populations still are present.65 , 66 ascl1 null embryos wholly lack gastrin , somatostatin , and glucagon - secreting endocrine cell types ( the former 2 missing from their usual niches in the stomach ) , and gastric serotonin and ghrelin endocrine cells are decreased in number . ascl1 null embryos die before ecl cells emerge developmentally ; however , it was noted that the vast majority of chromogranin a positive cells ( chromogranin a is a general marker of endocrine cells ) are missing in ascl1 null embryos , and ecl cells represent the majority of chromogranin - positive cells in the corpus . thus , if ascl1 is required for all chromogranin a positive cells to emerge , a conditional deletion in the adult also might show that ecl cells are ascl1-dependent , although this only can be speculated with current data . taken together , the data show that ascl1 and ngn3 are each required to specify gastrin , somatostatin , and glucagon - positive endocrine cells . the eventual emergence of ecl cells may be dependent on ascl1 but not ngn3 . serotonin - positive endocrine cells in the antrum also largely are lost in ascl1 and ngn3 mutants . a recent study showed that serotonin - positive cells in the corpus are bone marrow derived , mucosal - associated mast cells and not descendants from endodermal progenitors , an ngn3 lineage , or epithelial cells at all . if corpus serotonin - positive cells are not derived from the endoderm , those cells likely account for the presence of nonantral serotonin - positive cells in ascl1 mutants mentioned earlier . it is unclear how specific mature endocrine cell types arise from endocrine - committed progenitors during embryogenesis and adult homeostasis ; however , endocrine specification in the pancreas and intestine may serve as an illustrative model.68 , 69 endocrine - committed progenitors derived from ascl1- or ngn3-positive populations differentiate into individual endocrine cell types depending on the specific downstream transcription program that is enacted . there is indication that similar programs exist in the stomach because mice null for various transcription factors have defects in specific endocrine lineages . for example , pdx1 , nkx6.3 , pax4/6 , and arx all have been implicated in controlling differentiation of mature endocrine cell types in the stomach.38 , 70 , 71 , 72 although it is mostly not clear what controls the specification of nonendocrine cell lineages within the stomach , some factors have been implicated in maturation of those cell types . the transcription factor spdef has been shown to be crucial for antral deep mucous cell maturation . foxq1 is necessary for the expression of muc5ac in pit cells ( muc5ac is the key mucin protein secreted by these cells ) . xbp1 and mist1 ( bhlha15 ) are important for the ultrastructural maturation of chief cells.75 , 76 specifically , they coordinate the architectural changes necessary for these cells to become regulated secretory factories . in their absence , chief cells fail to generate a dense rough endoplasmic reticulum network and do not make large zymogen - containing vesicles . mucous neck cells , the progenitors for chief cells , emerge in rodents around the time of weaning in a process that depends in part on transforming growth factor and the epidermal growth factor receptor,77 , 78 although whether these play a role in maturation or specification is not known . the isthmus of the corpus epithelium contains a continuously proliferating cell population that lacks any differentiated nuclear and cytoplasmic features ( eg , secretory granules or specialized organelles ) . nucleotide analog labeling studies ( eg , h - thymidine or bromodeoxyuridine ) show that labeled nucleotides are incorporated most frequently in isthmal cells with those morphologically immature characteristics , and this cell lineage has been termed the granule - free stem cell . pulse - chase experiments with such analogs show that the labeled nucleotides spread bidirectionally from the isthmus . karam and leblond hypothesized that the granule - free stem cell directly gave rise to progenitors that were immature versions of each of the mature corpus lineages . some of the earliest cells to incorporate label were cells characterized by ultrastructural features of immature pit cells ( eg , scant but distinctive pit cell mucous granules ) . label spread more slowly in the other direction ( ie , toward the base and away from the gastric lumen ) . the cells that showed early incorporation of label in that direction commonly have early / immature mucous neck cell features . at 12 weeks after injection of labeled nucleotides , label appears in the prezymogenic chief cells at the top of the base , those with features characteristic of both mucous neck cells and zymogenic cells.76 , 80 it appears eventually , also in parietal cells and endocrine cells , first in the isthmus area.81 , 82 in pulse - chase experiments wherein the nucleotides are given only once as opposed to continuously label typically is not retained for longer than a few days in either the immature ( presumptive progenitor ) cells or in the granule - free cells . rather , label is retained long - term only in mature parietal , chief , and endocrine cells . the simplest interpretation of these observations is that the undifferentiated , granule - free isthmal cell is a constitutively active multipotent stem cell that can give rise to and replenish all the mature cell lineages ( figure 3 ) . other than such studies , wherein lineage relationships are inferred from morphology and labeled nucleotide migration patterns , there is little else known about transitions from the stem cell to progenitors and lineage - committed cells in the corpus . the limited state of understanding in the gastric corpus is in marked contrast to that in the small intestine , where numerous markers of crypt - based cells with stem cell potential have been identified over the past 10 years.83 , 84 , 85 , 86 , 87 strikingly , there is still neither a specific molecular marker nor a specific promoter whose expression is restricted to an undifferentiated isthmal cell in the corpus that has yet been identified . several studies using chimeric mice and mosaic silencing of an x - linked transgene in female mice suggest that stomach glands start off polyclonal but become monoclonal over time.62 , 88 , 89 , 90 these results thus support the single gastric unit stem cell hypothesis . however , bjerknes and cheng found patterns of mutant clones that showed that , as mice age , there might be other progenitor - progeny relationships outside the dogma of the single , long - lived , multipotent stem cell proposed by karam and leblond . in adult mice , bjerknes and cheng saw units that seemingly had stable labeling restricted to specific single lineages , suggesting that there also might be long - lived , lineage - committed progenitor cells rather than the transient ones hypothesized by karam and leblond . we have provided a cartoon to distinguish the 2 models ( ie , a multipotent , undifferentiated stem cell giving rise to all lineages vs multiple long - lived , lineage - committed progenitors , each fueling only their specific lineage ) ( figure 4 ) . genetic lineage tracing experiments that have been attempted in the stomach suffer from the caveat that the promoters used to drive lineage tracing also are expressed ( usually much more strongly ) in differentiated cells . for example , long - term lineage tracing in adult animals using the sox2 promoter suggested that some sox2-promoter - expressing cells have stem cell function with the capacity for self - renewal and differentiation into all lineages in the corpus . rare , highly sox2 + cells were suggested to be the most stem - like . interestingly , those cells localized to the base of corpus units , not the isthmus . sox2 protein can be found in many cell lineages throughout the corpus at mid - to - low levels and even can be used as a marker in human beings of gastric differentiation relative to intestinal.92 , 93 other lineage tracing studies have focused on marking mature chief cells using the tumor necrosis factor receptor superfamily member 19 ( tnfrsf19 or troy ) or muscle , intestine and stomach expression 1 ( mist1 ) promoters . long - term lineage tracing using those promoters suggested that chief cells also can act as stem cells and give rise to all the cell lineages within the corpus . mist1 protein and rna are almost exclusive to mature chief cells , and troy is restricted to a handful of chief and parietal cells . these results indicate that differentiated chief cells have the potential to serve as stem cells in some situations , albeit such functional stem cell activity in chief cells seems relatively rare , at least during homeostasis . recent studies have indicated that rare cells labeled with a mist1 knock - in allele also can be found in the isthmus of the corpus . these occasional , isthmus - localized cells that express the mist1 promoter could be another source of stemness in the corpus . however , the molecular / cellular identity of those cells is defined only by this spurious mist1 expression , given that neither the endogenous mist1 transcript nor the mist1 protein has been shown to be expressed outside of chief cells in wild - type mice . definitive lineage tracing studies in the stomach also have been hampered by a technical problem that does not affect other gastrointestinal organs to the same degree , such as small intestine and pancreas , where lineage tracing has been used to great effect . the vast majority of genetic lineage tracing tools use a modified cre recombinase that requires binding tamoxifen to be transported to the nucleus where it can activate reporter genes or other genetic tools ( cre ) . unfortunately , for gastric researchers , tamoxifen induces parietal cell death and chief cell metaplasia when delivered above a threshold dose.97 , 98 , 99 thus , inducible lineage tracing using cre with tamoxifen can be confounding in the stomach because it may induce nonhomeostatic patterns of differentiation with increased cellular plasticity . the stomach is also particularly sensitive to high doses of cre itself . it would be ideal to develop more stomach - lineage specific promoters and induce lineage tracing with methods such as tetracycline - inducible systems or estrogen receptor agonists that do not induce injury furthermore , any lineage tracing in the stomach should be performed with proper controls : mice homozygous for floxed alleles but lacking cre recombinase expression and mice with cre recombinase but with a nonfloxed allele of the gene of interest . to highlight how our understanding of stem cell dynamics in the intestine is more advanced than in the corpus , we point out how cre driven by the lgr5 promoter is an efficient marker of functional stem cell activity in the intestine . lgr5 is expressed at higher levels in the presumptive stem cells in the small intestine than in differentiated progeny . importantly , both the endogenous lgr5 transcript and lgr5 protein also are expressed preferentially in the presumptive stem cell . lgr5 can be used during homeostasis to trace labeled cells , and all cell lineages can be seen eventually to derive from lgr5-promoter expressing , crypt - resident ( presumptive stem ) cells with undifferentiated features . lgr5 promoter - based studies corroborate other studies that , together , make it seem incontrovertible that there is a population of constitutively active , long - lived , multipotent stem cells in the small intestine . although the gastric epithelial stem cell in the adult corpus remains unidentified , there has been some beginning characterization of the patterns of molecular and cellular differentiation of the various mature cell lineages deriving from that stem cell . one strong line of evidence supports an interesting differentiation pattern wherein mucous neck cells give rise to chief cells , a differentiation step that has been termed a transdifferentiation to chief cells.76 , 78 , 101 evidence supporting the lineage relationship between neck and chief cells is circumstantial , but varied and relatively abundant . for one , there are situations in which neck and chief cell markers are co - expressed . during postnatal maturation , after maturation , units in the corpus harbor similar transitional cells with characteristics of both cell populations by ultrastructural and gene expression analysis.76 , 80 , 101 , 102 when the stomach is injured , metaplastic cells arising from the chief cell lineage express both neck and chief cell markers.6 , 95 , 101 furthermore , lineage tracing studies using the tff2 promoter have suggested that parietal cells and mucous neck / chief cells are derived from a common progenitor pool . finally , slowing maturation of chief cells by deleting either mist1 or xbp1 leads to increased cells with neck - chief transitional characteristics.75 , 76 , 101 the surface mucous ( pit / foveolar ) cells clearly arise rapidly from a progenitor in the isthmus . the nature of the progenitor has not been established but must be either a committed pit - specific , long - lived progenitor or the canonical multipotent stem cell ( or both ) . interestingly , we have observed that decreased proliferation in the isthmal progenitor zone tends to have effects on pit cells more than the deeper glandular cells , indicating that much of the proliferation in the isthmus , at least under normal conditions , is directed toward surface mucous cell replenishment . as mentioned earlier , the transcription factor foxq1 is involved in pit cell differentiation because it is required for expression of the key component of the pit cell mucous granules ( muc5ac ) , although it is not required for specification of the lineage itself . a number of signaling pathways have been shown to be active during stomach homeostasis and affect cell behavior . notch signaling is active in the isthmus of the corpus and promotes proliferation within this region . ectopic notch signaling driven by a parietal cell - lineage specific promoter blocked differentiation and maintained progenitor characteristics in differentiating parietal cells . inhibition of the bmp signaling pathway promotes increased cell proliferation in the adult stomach : glands contained fewer parietal cells and more transitional cells ( cells with both neck and zymogenic chief cell characteristics similar to the metaplastic or transitional cells mentioned earlier ) at the base of the unit , indicating that bmp signals regulate progenitor proliferation and cell maturation in the corpus . the primary physiological role of gastrin is to promote acid secretion by activating ecl and parietal cells . absence of gastrin causes decreased cellular proliferation in the corpus and leads to generation of immature parietal and ecl cells,107 , 108 , 109 , 110 whereas overexpression of gastrin causes increased proliferation of those cell populations . parietal cell production of shh is an important regulator of gastrin production . in the absence of this source of shh , excess gastrin is produced by g cells , and pit cells in the corpus have increased proliferation . the antrum is considerably less complex then the corpus ( in organization and number of cell types ) . lee and leblond identified the most actively proliferating cells as also being the least differentiated ultrastructurally ( like the granule - free presumptive stem cell in the isthmus of the corpus ) . in the antrum , the isthmus is much nearer the base than in the corpus , in a pattern more resembling the large intestine . in pulse - chase experiments , labeled dna spreads both upward to the lumen and further down into the base from this isthmus zone ( figure 1 ) . in contrast to the corpus , markers and gene promoters have been shown to efficiently label cells with multipotent progenitor capacity . for example , as in the small intestine , lgr5 shows a pattern of homeostatic expression that is confined to a specific cell population that frequently and efficiently can be traced into progeny that include all the cell lineages in the antrum and cardia , but not the corpus . similarly , cck2r - based lineage tracing labeled as + 4 ( the designation of + 4 is borrowed from the intestine , wherein cells traditionally have been numbered from the most basal cell upward to the lumen ) antral cells that also has stem cell potential and was shown to give rise to lgr5 + antral cells . in addition , villin- and sox2-promoter based lineage tracing also label rare cells in antral glands that show functional stem cell characteristics.91 , 116 it is not yet clear what the relationship among all of these cell populations with stem cell capacity is yet . the lgr5 + cells clearly are not the granule - free , isthmal antral cells because they commonly are located at the very base of the antral unit , not the isthmus , and they show ultrastructural features of differentiation.113 , 114 the cck2r and lgr5 populations seem to be overlapping , at least functionally , but are distinct from each other . sox2 , on the other hand , is expressed in many cells , therefore it likely is not specific to a defined stem cell . it is possible that cells in the antrum are plastic , so that many cells can serve as stem cells even homeostatically . antral glands also undergo relatively frequent fission events , in which one gland gives rise to another,62 , 89 , 117 so perhaps some of the markers label cells that are not constitutive stem cells but that drive budding off of new glands . in sum , sox2 , lgr5 , and villin are not principally expressed in the zone where the least differentiated , most proliferative cells are . that is in contrast to the intestine where the lgr5-expressing ( crypt - base - columnar ) cells are the most proliferative and the least ultrastructurally differentiated cells . perhaps , thus , a marker of isthmal , antral stem cells that is equivalent to lgr5 in the intestine has yet to be identified in the stomach . in the antrum , inhibition of notch signaling promotes mucous and endocrine cell differentiation , whereas activating the pathway stops differentiation . bmp signaling , through bmpr1a , regulates the proliferation and differentiation of mucous cells in the antrum.100 , 119 in the absence of bmpr1a , antral mucous cells hyperproliferate and fail to express the mucin muc5ac . mutations in the bmp family are known to cause juvenile polyposis syndrome , which presents with polyps throughout the gastrointestinal tract , including the antrum . finally , given that lgr5 is expressed and may regulate stem cell activity in the antrum , there may be a role for wnt signaling in regulating antral homeostasis because lgr5 is a co - receptor for canonical wnt signals . although it is clear that tissue interactions between the gastric endoderm and mesoderm are important for gastric development , there is still scant knowledge about how naive endodermal progenitors become specified to the gastric progenitor state . there is equally poor understanding about the factors that control the specification of gastric lineages during development and adult homeostasis , other than some initial inroads into outlining the origins of endocrine lineages . many more studies are needed to determine which cell types have stem cell properties in the adult stomach ( in particular the corpus ) , and their relative contribution during homeostasis and disease / injury conditions . such studies will depend on the development of promoter - based tools that , similar to the intestine , are specific for stem cells and not expressed in differentiated cells . recent reports have described the derivation of mouse and human gastric organoids derived either from adult stomach94 , 122 , 123 , 124 , 125 , 126 or via differentiation from induced pluripotent stem cells . potentially through the manipulation of these cells , the field might have a new approach to better understand the pathways and factors controlling stemness and specification of gastric lineages . the generation of such new tools to study these processes is an important first step to exploring the mechanisms that control gastric specification .

gastric diseases cause considerable worldwide burden . however , the stomach is still poorly understood in terms of the molecular cellular processes that govern its development and homeostasis . in particular , the complex relationship between the differentiated cell types located within the stomach and the stem and progenitor cells that give rise to them is significantly understudied relative to other organs . in this review , we highlight the current state of the literature relating to specification of gastric cell lineages from embryogenesis to adulthood . special emphasis is placed on substantial gaps in knowledge about stomach specification that we think should be tackled to advance the field . for example , it has long been assumed that adult gastric units have a granule - free stem cell that gives rise to all differentiated lineages . here , we point out that there are also other models that fit all extant data , such as long - lived , lineage - committed progenitors that might serve as a source of new cells during homeostasis .

Early Specification Mesoderm Cell Lineage Specification Adult Homeostasis Conclusions

regional and subsequent organ identity is assembled within the naive , as yet unspecified , gut tube through the integration of signaling inputs from mesodermal tissues located apposed to the endoderm and the endodermal progenitors themselves . stomach epithelial progenitors derive from the foregut region of the endoderm , which also gives rise to liver , pancreas , lungs , and the luminal gastrointestinal organs from the pharynx to the anterior duodenum . signaling pathways and transcription factors that drive specification of pregastric endodermal progenitors from other emerging organs within the foregut have not been well characterized . however , a number of signaling pathways that promote or restrict foregut identity by patterning the anterior / posterior axis of the endoderm are known . retinoic acid ( ra ) , for example , has a complex spatiotemporal role patterning the anterior posterior axis of the endoderm . wnt and fibroblast growth factor ( fgf ) signals produced by the mesoderm promote expression of posterior endodermal markers such as cdx2 over anterior endodermal markers.15 , 16 , 17 studies in zebrafish also have shown that bone morphogenetic protein ( bmp ) signaling drives posterior over anterior endodermal fates . for example , the dorsal aorta and notochord produce several key signaling molecules involved in dorsal pancreatic specification.19 , 20 these same tissues also could impact pregastric gene expression given the proximity of gastric and dorsal pancreatic progenitors . expression of these transcription factors in early endoderm is necessary to generate foregut progenitors that give rise to the stomach . for example , the foxa family is expressed throughout the early endoderm and is important in the development of a number of organs including the liver , pancreas , and intestine.24 , 25 , 26 the specific role of this family in the stomach has yet to be determined , however , foxas are known to be involved in promoting pdx1 expression in the foregut ( figure 2 ) . gata4 and gata6 are involved in the specification of the extraembryonic endoderm28 , 29 , 30 and are expressed throughout the early definitive endoderm . pdx1 is expressed regionally within the posterior foregut in the areas that give rise to the posterior stomach ( antrum / pylorus ) , anterior duodenum , dorsal and ventral pancreatic buds , and proximal extrahepatic biliary system.27 , 37 pdx1 expression can be used during development to distinguish antral gastric progenitors ( sox2gata4pdx1 ) from corpus progenitors ( sox2gata4pdx1 ) . to date , no specific gene has been shown to have expression restricted only to early gastric progenitors ; thus , it remains difficult to examine directly how the stomach is specified from other organs , the way , for example , cdx1 and cdx2 have been studied in intestinal specification . instead , investigators rely on more broadly expressed genes ( ie , expressed concomitantly in other organs besides the stomach ) such as sox2 , gata4 , and pdx1 to identify the factors defining the prospective gastric regions . however , there could be marked improvement in our understanding of stomach specification simply by manipulating gene expression in early endoderm with tools that already exist . for example , signaling pathways and transcription factors suspected of being involved in gastric development could be deleted via crosses to well - characterized mouse pedigrees that express foxa3- , sox17- , or shh- cre.41 , 42 , 43 summing up all that currently is known and can be inferred from published studies , we have proposed one possible signaling and epistasis model for specification of glandular stomach ( figure 2 ) . mesenchymal cross - talk , and the stomach does not seem to be an exception . for example , foundational experiments in chicks have shown that placing proventricular ( stomach region in chicks similar to the mammalian glandular stomach ) mesenchyme with gizzard ( anterior chicken stomach ) or esophageal endoderm induces proventricular gene expression and causes gland development in these normally nonglandular tissues.44 , 45 similarly , gizzard or esophageal mesenchyme can suppress proventricular gene expression and gland development in proventricular endoderm and promote squamous fates . for example , in addition to their early role in foregut growth , hedgehog ( shh / ihh ) signals are produced by the gastric endoderm to support mesenchymal growth and differentiation , a pattern that is maintained in the adult.49 , 50 another example of a factor that originates from the mesenchyme and regulates the epithelium is fgf10 , likely via the fgf receptor 2b . fgf10 promotes epithelial proliferation and gland development.51 , 52 although it may not be required for adult homeostasis , it has been shown to inhibit parietal and chief cell differentiation in favor of the mucous neck cell type . for example , barx1 is a transcription factor that is restricted to the prospective esophageal and gastric mesoderm . the stomach intestinal boundary is disturbed such that ectopic cdx2 + intestinal epithelial cells can be found in the posterior stomach.54 , 55 in addition to the disrupted interorgan patterning , the division of intrastomach domains is altered . for example , h / k adenosine triphosphatase expressing cells are seen intermingled with pdx1 + cells , which in mice are normally exclusive to the corpus and antrum , respectively . bapx1 ( nkx3 - 2 ) , nkx2 - 5 , gata3 , six2 , nr2f2 ( chicken ovalbumine upstream promoter - transcription factor ii ) , and sox9 are other known transcription factors expressed in the posterior stomach mesenchyme and involved in specifying the pylorus.56 , 57 , 58 in the absence of those transcription factors , there is aberrant neuromuscular regulation of the pyloric sphincter , which in human beings can manifest as the relatively common condition known as pyloric stenosis.58 , 59 between the stage of endodermal specification and the stage of specific cell lineage commitment in the stomach , the gastric epithelium remains a simple epithelium with no obvious differentiation . at approximately e14.5e16.5 , markers representative of cell types such as endocrine , parietal , and chief cells begin to be expressed , and small glands begin to invaginate into the mesenchyme from the simple epithelium lining the lumen . between e16.5 and 2 weeks of postnatal development , most of the major cell types arise within the stomach , and the glandular stomach mostly becomes organized into its adult form . however , the murine stomach does not reach adult organization with full chief cell and endocrine cell lineage specification until 68 weeks postnatally . for most cell lineages in the stomach we have a poor understanding of pathways and factors involved in their specification and the progenitors from which they directly derive . for example , and this is truly remarkable when contrasted to the state of our understanding in the intestines , there is no specific factor that is known to be necessary or sufficient for specification of chief , parietal , pit , mucous neck , or isthmal cells . the developmental sequence between gastric epithelial progenitors in an adult gastric unit and the differentiated progeny that arise continuously throughout life also is unknown . it is entirely possible that all the mature cell types are specified from a single multipotent progenitor that persists throughout life , or , in turn , there might be numerous long - lived lineage - restricted progenitors62 , 63 ( figures 3 and 4 , and see detailed discussion later ) . ngn3 null embryos lack gastrin , somatostatin , and glucagon endocrine cell types , with largely reduced census of serotonin - positive cells , but enterochromaffin - like ( ecl ) and ghrelin populations still are present.65 , 66 ascl1 null embryos wholly lack gastrin , somatostatin , and glucagon - secreting endocrine cell types ( the former 2 missing from their usual niches in the stomach ) , and gastric serotonin and ghrelin endocrine cells are decreased in number . ascl1 null embryos die before ecl cells emerge developmentally ; however , it was noted that the vast majority of chromogranin a positive cells ( chromogranin a is a general marker of endocrine cells ) are missing in ascl1 null embryos , and ecl cells represent the majority of chromogranin - positive cells in the corpus . it is unclear how specific mature endocrine cell types arise from endocrine - committed progenitors during embryogenesis and adult homeostasis ; however , endocrine specification in the pancreas and intestine may serve as an illustrative model.68 , 69 endocrine - committed progenitors derived from ascl1- or ngn3-positive populations differentiate into individual endocrine cell types depending on the specific downstream transcription program that is enacted . for example , pdx1 , nkx6.3 , pax4/6 , and arx all have been implicated in controlling differentiation of mature endocrine cell types in the stomach.38 , 70 , 71 , 72 although it is mostly not clear what controls the specification of nonendocrine cell lineages within the stomach , some factors have been implicated in maturation of those cell types . mucous neck cells , the progenitors for chief cells , emerge in rodents around the time of weaning in a process that depends in part on transforming growth factor and the epidermal growth factor receptor,77 , 78 although whether these play a role in maturation or specification is not known . nucleotide analog labeling studies ( eg , h - thymidine or bromodeoxyuridine ) show that labeled nucleotides are incorporated most frequently in isthmal cells with those morphologically immature characteristics , and this cell lineage has been termed the granule - free stem cell . karam and leblond hypothesized that the granule - free stem cell directly gave rise to progenitors that were immature versions of each of the mature corpus lineages . at 12 weeks after injection of labeled nucleotides , label appears in the prezymogenic chief cells at the top of the base , those with features characteristic of both mucous neck cells and zymogenic cells.76 , 80 it appears eventually , also in parietal cells and endocrine cells , first in the isthmus area.81 , 82 in pulse - chase experiments wherein the nucleotides are given only once as opposed to continuously label typically is not retained for longer than a few days in either the immature ( presumptive progenitor ) cells or in the granule - free cells . the simplest interpretation of these observations is that the undifferentiated , granule - free isthmal cell is a constitutively active multipotent stem cell that can give rise to and replenish all the mature cell lineages ( figure 3 ) . other than such studies , wherein lineage relationships are inferred from morphology and labeled nucleotide migration patterns , there is little else known about transitions from the stem cell to progenitors and lineage - committed cells in the corpus . the limited state of understanding in the gastric corpus is in marked contrast to that in the small intestine , where numerous markers of crypt - based cells with stem cell potential have been identified over the past 10 years.83 , 84 , 85 , 86 , 87 strikingly , there is still neither a specific molecular marker nor a specific promoter whose expression is restricted to an undifferentiated isthmal cell in the corpus that has yet been identified . however , bjerknes and cheng found patterns of mutant clones that showed that , as mice age , there might be other progenitor - progeny relationships outside the dogma of the single , long - lived , multipotent stem cell proposed by karam and leblond . in adult mice , bjerknes and cheng saw units that seemingly had stable labeling restricted to specific single lineages , suggesting that there also might be long - lived , lineage - committed progenitor cells rather than the transient ones hypothesized by karam and leblond . we have provided a cartoon to distinguish the 2 models ( ie , a multipotent , undifferentiated stem cell giving rise to all lineages vs multiple long - lived , lineage - committed progenitors , each fueling only their specific lineage ) ( figure 4 ) . for example , long - term lineage tracing in adult animals using the sox2 promoter suggested that some sox2-promoter - expressing cells have stem cell function with the capacity for self - renewal and differentiation into all lineages in the corpus . sox2 protein can be found in many cell lineages throughout the corpus at mid - to - low levels and even can be used as a marker in human beings of gastric differentiation relative to intestinal.92 , 93 other lineage tracing studies have focused on marking mature chief cells using the tumor necrosis factor receptor superfamily member 19 ( tnfrsf19 or troy ) or muscle , intestine and stomach expression 1 ( mist1 ) promoters . long - term lineage tracing using those promoters suggested that chief cells also can act as stem cells and give rise to all the cell lineages within the corpus . these results indicate that differentiated chief cells have the potential to serve as stem cells in some situations , albeit such functional stem cell activity in chief cells seems relatively rare , at least during homeostasis . these occasional , isthmus - localized cells that express the mist1 promoter could be another source of stemness in the corpus . however , the molecular / cellular identity of those cells is defined only by this spurious mist1 expression , given that neither the endogenous mist1 transcript nor the mist1 protein has been shown to be expressed outside of chief cells in wild - type mice . definitive lineage tracing studies in the stomach also have been hampered by a technical problem that does not affect other gastrointestinal organs to the same degree , such as small intestine and pancreas , where lineage tracing has been used to great effect . the stomach is also particularly sensitive to high doses of cre itself . it would be ideal to develop more stomach - lineage specific promoters and induce lineage tracing with methods such as tetracycline - inducible systems or estrogen receptor agonists that do not induce injury furthermore , any lineage tracing in the stomach should be performed with proper controls : mice homozygous for floxed alleles but lacking cre recombinase expression and mice with cre recombinase but with a nonfloxed allele of the gene of interest . to highlight how our understanding of stem cell dynamics in the intestine is more advanced than in the corpus , we point out how cre driven by the lgr5 promoter is an efficient marker of functional stem cell activity in the intestine . lgr5 can be used during homeostasis to trace labeled cells , and all cell lineages can be seen eventually to derive from lgr5-promoter expressing , crypt - resident ( presumptive stem ) cells with undifferentiated features . lgr5 promoter - based studies corroborate other studies that , together , make it seem incontrovertible that there is a population of constitutively active , long - lived , multipotent stem cells in the small intestine . although the gastric epithelial stem cell in the adult corpus remains unidentified , there has been some beginning characterization of the patterns of molecular and cellular differentiation of the various mature cell lineages deriving from that stem cell . one strong line of evidence supports an interesting differentiation pattern wherein mucous neck cells give rise to chief cells , a differentiation step that has been termed a transdifferentiation to chief cells.76 , 78 , 101 evidence supporting the lineage relationship between neck and chief cells is circumstantial , but varied and relatively abundant . during postnatal maturation , after maturation , units in the corpus harbor similar transitional cells with characteristics of both cell populations by ultrastructural and gene expression analysis.76 , 80 , 101 , 102 when the stomach is injured , metaplastic cells arising from the chief cell lineage express both neck and chief cell markers.6 , 95 , 101 furthermore , lineage tracing studies using the tff2 promoter have suggested that parietal cells and mucous neck / chief cells are derived from a common progenitor pool . the nature of the progenitor has not been established but must be either a committed pit - specific , long - lived progenitor or the canonical multipotent stem cell ( or both ) . interestingly , we have observed that decreased proliferation in the isthmal progenitor zone tends to have effects on pit cells more than the deeper glandular cells , indicating that much of the proliferation in the isthmus , at least under normal conditions , is directed toward surface mucous cell replenishment . as mentioned earlier , the transcription factor foxq1 is involved in pit cell differentiation because it is required for expression of the key component of the pit cell mucous granules ( muc5ac ) , although it is not required for specification of the lineage itself . lee and leblond identified the most actively proliferating cells as also being the least differentiated ultrastructurally ( like the granule - free presumptive stem cell in the isthmus of the corpus ) . for example , as in the small intestine , lgr5 shows a pattern of homeostatic expression that is confined to a specific cell population that frequently and efficiently can be traced into progeny that include all the cell lineages in the antrum and cardia , but not the corpus . similarly , cck2r - based lineage tracing labeled as + 4 ( the designation of + 4 is borrowed from the intestine , wherein cells traditionally have been numbered from the most basal cell upward to the lumen ) antral cells that also has stem cell potential and was shown to give rise to lgr5 + antral cells . the lgr5 + cells clearly are not the granule - free , isthmal antral cells because they commonly are located at the very base of the antral unit , not the isthmus , and they show ultrastructural features of differentiation.113 , 114 the cck2r and lgr5 populations seem to be overlapping , at least functionally , but are distinct from each other . antral glands also undergo relatively frequent fission events , in which one gland gives rise to another,62 , 89 , 117 so perhaps some of the markers label cells that are not constitutive stem cells but that drive budding off of new glands . perhaps , thus , a marker of isthmal , antral stem cells that is equivalent to lgr5 in the intestine has yet to be identified in the stomach . although it is clear that tissue interactions between the gastric endoderm and mesoderm are important for gastric development , there is still scant knowledge about how naive endodermal progenitors become specified to the gastric progenitor state . there is equally poor understanding about the factors that control the specification of gastric lineages during development and adult homeostasis , other than some initial inroads into outlining the origins of endocrine lineages . many more studies are needed to determine which cell types have stem cell properties in the adult stomach ( in particular the corpus ) , and their relative contribution during homeostasis and disease / injury conditions . potentially through the manipulation of these cells , the field might have a new approach to better understand the pathways and factors controlling stemness and specification of gastric lineages .

prenatal alcohol exposure causes impairment in brain structure and function , leading to cognitive and behavioral deficits that range in severity ( archibald et al . , 2001 ; fetal alcohol syndrome ( fas ) , the most severe of the fetal alcohol spectrum disorders ( fasd ) , is characterized by distinctive craniofacial dysmorphology ( short palpebral fissures , thin vermilion , flat philtrum ) , small head circumference and pre- and/or postnatal growth retardation ( hoyme et al . , 2005 ) . the craniofacial dysmorphology is also seen in partial fas ( pfas ) , together with either small head circumference , retarded growth , or neurobehavioral deficits . heavily exposed ( he ) individuals lacking the distinctive dysmorphology are diagnosed with alcohol - related neurodevelopmental disorder ( arnd ) if they exhibit cognitive and/or behavioral impairment ( stratton et al . , 1996 ) . prenatal alcohol exposure is associated with a broad range of cognitive deficits , including low iq ( streissguth et al . , 1990 ; jacobson et al . , 2004 ) , poor attention and executive function ( kodituwakku et al . , 1995 ; coles et al . , 1997 ; mattson et al . , 1999 ; burden et al . , 2005a ) , and slower cognitive processing speed ( streissguth et al . , 1990 ; jacobson et al . , 1993 ; jacobson et al . , 1994 ; coles et al . , 2002 ) . among the cognitive deficits seen in relation to prenatal alcohol exposure , arithmetic is especially sensitive , and mathematical deficits are seen even after controling for iq ( coles et al . , 1991 ; streissguth et al . , 1990 ; streissguth et al . , 1994 ; chiodo et al . , 2004 ; jacobson et al . , 2004 ; burden et al . , 2005b ) , and impaired numerosity is already seen in infants with fas ( s. jacobson et al . , 2011a ) . when academic achievement tests are administered to exposed individuals , arithmetic is consistently more impaired than reading or spelling ( streissguth et al . , 1991 ; goldschmidt et al . , 1996 ; kerns et al . , 1997 ; howell et al . , although the parietal lobe has been known to be involved in number processing since the beginning of the 20th century ( henschen , 1919 ) , fmri has provided a more extensive understanding of the neuroanatomy of this domain of processing . based on brain lesion and neuroimaging findings , dehaene and associates ( dehaene , 1992 ; dehaene and cohen , 1996 ) have proposed a triple - code model of number processing that incorporates the three different systems of representation that may be used in number processing tasks : the quantity system , the verbal system and the visual system . in addition , they have posited a core quantity system a nonverbal abstract representation of numerical quantity , which has been localized bilaterally in the anterior portion of the horizontal segments of the intraparietal sulcus ( ips ) . this area is hypothesized to support number processing irrespective of the notation used ; that is , whether represented symbolically as arabic numbers or sequences of words or analogically by numbers of dots . the verbal processing of numbers is posited to be based on the left angular gyrus ( close to the language areas ) , while the bilateral posterior superior parietal lobules ( pspls ) are hypothesized to be involved in spatial and non - spatial attentional processes contributing to the visual processing of numbers . ( 2005 ) found age - related increases in activation of the anterior ips during number processing between school age and early adulthood , which they attributed to the development of increasing functional specialization of this region as symbolic number processing becomes increasingly automatic . results from a meta - analysis of studies comparing number processing in children vs. adults generally support dehaene 's model but suggest that the localization of parietal activations is more notation - specific in children and that right ips activations in non - symbolic magnitude comparison are slightly more anterior than those observed in adults ( kaufmann et al . , 2011 ) . data from behavioral studies that we have conducted in two different cohorts suggest that a specific deficit in the ability to represent and manipulate quantity may play a critical role in the poor arithmetic performance seen in fasd ( j. jacobson et al . , 2011 ) . a 224-item , 7-subtest , computer - based number processing test that we developed in collaboration with s. dehaene , was administered to 262 adolescents from the detroit longitudinal prenatal alcohol exposure cohort . a factor analysis of the seven subtests yielded two factors , one reflecting exact and approximate calculation ( calculation ) . the other factor , on which number comparison and proximity judgment loaded most strongly , reflected the ability to represent and manipulate quantity ( magnitude comparison ) , corresponding to dehaene 's core quantity system . in a path analytic model , the relation of prenatal alcohol exposure to calculation was fully mediated by its effects on magnitude comparison , suggesting that magnitude comparison is a core deficit involved in the poor arithmetic performance seen in these children . these findings were subsequently confirmed in a sample of school - age children in cape town , south africa ( s. jacobson et al . , 2011a ) . in the detroit cohort , attention - deficit / hyperactivity disorder ( adhd ) was related to poorer performance on all seven number processing subtests , but , by contrast to the pattern seen in the alcohol - exposed children , the associations were markedly stronger with calculation than magnitude comparison . moreover , iq significantly mediated the effect of adhd on calculation , suggesting that the effects of adhd on aspects of calculation not specific to the representation of number , such as attention and executive function , mediate the poorer number processing seen in that disorder . this behavioral evidence linking prenatal alcohol exposure to impairment in the core quantity system is consistent with evidence from mri studies reporting alcohol - related structural impairment in the parietal region , including disproportionate size reductions in the parietal lobe ( archibald et al . , 2001 a high resolution structural mri , surface - based image analysis indicated that the brains of alcohol - exposed individuals were narrower in the inferior parietal and perisylvian regions ( sowell et al . , 2002 ) , and voxel - based morphometry ( vbm ) analysis revealed gray matter abnormalities that were most prominent in the left perisylvian cortices of the parietal and temporal lobes ( sowell et al . , 2001 ) . in addition , significant cortical thickness excesses were observed in children with fasd in large areas of bilateral temporal , bilateral inferior parietal and right frontal regions ( sowell et al . , , adults with and without prenatal alcohol exposure were administered a task involving subtraction from 11 of a series of numbers that appeared on the screen ( santhanam et al . , 2009 ) . exposed individuals with alcohol - related dysmorphology exhibited poorer task performance and lower activation in regions known to be associated with arithmetic processing , including the right inferior and left superior parietal regions and medial frontal gyrus , compared with controls . however , it was not clear whether the reduced activation in the dysmorphic participants reflected a specific deficit in fronto - parietal function or completion of fewer problems due to the difficulty of the task . using simpler proximity judgment and single - digit addition problems , we found that children activate the same fronto - parietal regions activated in number processing by adults ( meintjes et al . , 2010a ) . although children with fas and pfas performed as well as controls on the simple tasks administered in the scanner , they activated a markedly more diffuse parietal region extending into the angular gyrus , precuneus and posterior cingulate and for , exact addition , also the postcentral gyrus ( meintjes et al . , however , the fas / pfas and control groups did not differ significantly in the degree of activation in the anterior portion of the ips and other regions linked by dehaene and associates to number processing , possibly due to the lack of statistical power in the small sample on which that whole brain voxel - wise analysis was conducted . in this study , we examine the effects of both fasd diagnosis and continuous measures of prenatal alcohol exposure on brain activation in the five parietal structures found by dehaene and associates to mediate number processing in a larger sample that includes not only children with fas / pfas and nonexposed controls , but also nonsyndromal heavily - exposed ( he ) children ( suttie et al . , we have found that continuous measures of prenatal alcohol exposure based on a maternal report obtained during pregnancy are often more sensitive in detecting effects of prenatal alcohol exposure than diagnoses based on dysmorphic features in studies using diverse neuroimaging techniques , including tensor - based morphometry ( meintjes et al . , 2014 ) and magnetic resonance spectroscopy ( du plessis et al . , 2014 ) . based on the behavioral findings from our detroit and cape town studies , our central hypothesis was that prenatal alcohol exposure would be associated with reduced activation of the anterior ips , the region believed to mediate abstract representation of numerical quantity . participants were 65 right - handed , 8- to 12-year - old children from the cape coloured ( mixed ancestry ) community in cape town , south africa , of whom 40 had been heavily exposed to alcohol prenatally and 25 were controls in the same age range ( s. jacobson et al . , 2011b ) . the cape coloured community is composed primarily of descendants of white european settlers , malaysian slaves , khoi - san aboriginals , and black african ancestors . the incidence of fasd in this population is exceptionally high due to poor socioeconomic circumstances and historical practices of compensating farm laborers with wine , which have contributed to a tradition of heavy recreational weekend binge drinking ( may et al . , 2007 ) . thirty - seven children were the older siblings of participants in our cape town longitudinal cohort ( jacobson et al . , 2008 ) . the others were identified by screening all of the 8- to 12-year - old children from an elementary school in a rural section of cape town , where there is a very high incidence of alcohol abuse among local farm workers ( meintjes et al . , 2010b ) . our research nurse and staff driver transported the mother and child from their home to our child development laboratory at the faculty of health sciences campus of the university of cape town ( uct ) for a 3-hour neuropsychological assessment and to groote schuur hospital for a neuroimaging assessment , which was administered on the following day . all examiners were blind with regard to maternal alcohol history and the child 's fasd diagnostic status , except in the most severe cases where it was obvious . written informed consent was obtained from each mother ; written assent , from each child . approval for human research was obtained from the wayne state university human investigation committee and the uct faculty of health sciences human research ethics committee . each mother was interviewed in her primary language , afrikaans or english , regarding her alcohol consumption during pregnancy , using a timeline follow - back approach ( sokol et al . volume was recorded for each type of beverage consumed each day , converted to absolute alcohol ( aa ) using multipliers proposed by bowman et al . ( 1975 ) , and averaged to provide summary measures of alcohol consumption during pregnancy . two groups of women were recruited : ( 1 ) heavy drinkers , who consumed at least 14 standard drinks per week ( 1.0 oz aa / day ) on average or engaged in binge drinking ( 5 or more drinks / occasion ) and ( 2 ) controls whose mothers abstained or drank only minimally during pregnancy92.0% ( all but two ) of the control mothers abstained ; one drank 2 drinks on 11 occasions ; the other , 2 drinks on 1 occasion . data from the alcohol consumption interview were tabulated to provide three continuous measures of drinking during pregnancy : average oz aa consumed per day , aa / drinking day ( dose / occasion ) and frequency ( days / week ) . number of cigarettes smoked on a daily basis was also recorded , as was the use of illicit drugs ( yes / no ) . mothers and children were given breakfast , lunch , and a snack during the morning at each laboratory visit . the mother received a small monetary compensation for each visit and photograph of her child , and the child was given a small gift . in september 2005 , we organized a clinic in which each child was independently examined for growth and fas dysmorphology by two expert u.s .- based fas dysmorphologists ( h.e . ( 2005 ) protocol ; a subset of children who could not attend the clinic was examined by a cape town - based dysmorphologist ( n. khaole ) ( jacobson et al . , 2008 ) . there was substantial agreement among the examiners on the assessment of all dysmorphic features , including the three principal fetal alcohol - related features philtrum and vermilion ( which were measured on the astley and clarren ( 2001 ) rating scales ) and palpebral fissure length ( median r = 0.78 ) . fas and pfas diagnoses were determined at a case conference by the dysmorphologists ( heh and lkr ) , swj , jlj , and cdm . criteria for full fas : at least two of the principal dysmorphic features , small head circumference ( bottom 10th percentile ) , and low weight or short stature ( bottom 10th percentile ) . nine met the criteria for pfas ; that is , two features , confirmed maternal alcohol consumption during pregnancy , and at least one of the following : small head circumference , low weight , short stature , or low iq ( < 70 ) . the fas and pfas groups were combined for the analyses , but the he children were treated as a separate group . the decision not to combine the fas / pfas and he groups was based on our findings in diwadkar et al . ( 2013 ) that for some aspects of cognition , distinctly different brain activation patterns are seen in these two groups . was administered the computer - based number processing test described above , which included two 16-item subtests , one of which assessed exact addition ( ea ) ; the other , proximity judgment ( pj ) ( kopera - frye et al . , 1996 ; jacobson et al . , 2003 ) . in ea , a series of problems involving single and/or double digits are displayed on the screen , and the child enters the solution on the computer keypad . in pj , a double - digit number is displayed on the left side of the screen , and the child presses a button to indicate which of the two double - digit numbers shown on the right side is numerically closer to it . each child was also assessed on 7 of the 10 subtests from the wechsler intelligence scale for children , third edition ( wisc - iii)similarities , arithmetic , digit span , symbol search , coding , block design , and picture completion and matrix reasoning from the wisc - iv . the iq subtests were selected to represent the four dimensions of the wisc - iii : verbal comprehension ( similarities ) , perceptual organization ( block design , picture completion , matrix reasoning ) , freedom from distractibility ( digit span , arithmetic ) , and processing speed ( coding , symbol search ) . only similarities were administered in the verbal domain because the other verbal subtests appeared to be less valid in this cross - cultural context . iq was estimated from these subtests using sattler 's ( 1992 ) formula for computing short form iq ; validity coefficients for short form iq based on 5 or more subtests consistently exceed r = 0.90 . all scans were acquired using a 1.5 t magnetom symphony mri scanner ( siemens medical systems , erlangen , germany ) . high - resolution anatomical images were acquired in the sagittal plane using a three - dimensional inversion recovery gradient echo sequence ( 72 slices , tr = 1900 ms , te = 3.93 ms , ti = 1100 ms , slice thickness 2 mm , 250 mm field of view , resolution 1.4 1.0 2 mm ) . during the fmri protocol , 154 functional volumes sensitive to blood oxygen level dependent contrast were acquired with a t2 * -weighted gradient echo , echo planar imaging sequence ( tr = 2000 ms , te = 50 ms , 20 interleaved slices , 5 mm thick , gap 1 mm , 230 mm field of view , resolution 3.6 3.6 5 mm ) . simplified versions of the ea and pj subtests from the computer - based number processing assessment described above were administered . the tasks were simplified to make it easier for the children in the exposed groups to achieve acceptable scores for task accuracy in the scanner . ea was simplified in that the child selected the correct answer from two choices displayed on the screen and the sum was never greater than 12 . pj was simplified by including fewer difficult problems ( i.e. , problems with response choices that were only 12 units apart ) . ea was selected because it is the easiest of the calculation subtests ; pj was selected to represent magnitude comparison because the range of the neurobehavioral scores for number comparison is truncated . each child practiced these tasks initially in a mock scanner built for this study , which was important in reducing anxiety and facilitating completion of the mri scans . the experimental tasks were programmed using e - prime software ( psychology software tools , inc . , pittsburgh , pa ) and were presented using a data projector and a rear projection screen mounted at the foot of the patient bed . , burnaby , canada ) in his / her right hand and responded using the right index and middle finger . the child was able to talk to the examiner using an intercom that is built into the scanner and could stop the scan at any time by squeezing a ball held in his / her left hand . the ea and pj tasks were administered using a self - paced block design , in which the child completed as many problems as possible during each 40 s block . in the active blocks , a fixation square was displayed for 500 ms prior to each trial ( fig . two numbers were displayed one above the other for 1000 ms , after which two possible solutions appeared horizontally below the two numbers . the display remained on the screen until either the child made a selection or the 40 s time limit for the block expired . sums were selected randomly from a list in which the solution was never greater than 12 and from which tie problems ( e.g. , 2 + 2 ) and sums involving unity had been excluded . the child selected the correct answer from the two choices displayed below by pressing the button on the same side as the correct answer . the control blocks in the ea task ( ea_ctl ) followed the same format but with two identical greek symbols displayed vertically initially for 1000 ms ( fig . two different greek symbols were then displayed horizontally below the vertical symbols and the child selected the one that was identical to the initial two by pressing the button on the side of that symbol . each block was repeated three times in the following order : ea , ea_ctl , rest , ea , ea_ctl , rest , ea_ctl , ea and rest . in the rest blocks , the fixation square was displayed for 20 s , resulting in a total task duration of 5 min . the pj task followed the same format , the only difference being that a single number ( the target ) was now displayed for 1000 ms , and the child was instructed to select from two numbers displayed horizontally below it , the one numerically closer to the target . problems were selected randomly from a list of 1-digit and 2-digit numbers . in the control blocks for the pj task , the target consisted of a single greek symbol , followed by two greek symbols displayed horizontally below it . the child pressed the button on the same side as the symbol that was identical to the target . each block was repeated three times in the order pj , pj control ( pj_ctl ) , rest , pj , pj_ctl , rest , pj_ctl , pj and rest , with the fixation square displayed for 20 s during the rest blocks , for a total task duration of 5 min . responses for the number processing tasks administered in the scanner were recorded on a computer . number of trials attempted , number of trials correct , and accuracy ( % correct ) were computed for each task . functional data were excluded for children with poor task performance ( < 66% correct ) to ensure that all those included in the analysis had been mentally engaged in the task . no imaging data were obtained for 6 children ( 4 fas / pfas , 1 he , 1 control ) . for pj , data of 9 children ( 3 fas / pfas , 3 he , 3 controls ) were excluded from the neuroimaging analyses due to poor performance , and another control child fell asleep during the task . for ea , one he child was not able to perform the task , and data from 19 children ( 6 fas / pfas , 7 he , 6 controls ) were excluded due to poor performance . all fmri analyses were performed using brain voyager qx ( brain innovation , maastricht , the netherlands ) . images were motion corrected relative to the first volume of the functional run with trilinear / sinc interpolation . images were corrected for different slice acquisition times and linear trends , spatially smoothed using a gaussian filter ( fwhm 4 mm ) , and temporally smoothed with a high pass filter of 2 cycles / point . all data exceeding the movement criterion of a 3 mm displacement or 3.0 rotation within a functional run were rejected . in addition to the previously noted exclusions for poor performance , pj data for 2 control children and ea data for 1 control child were discarded due to excessive motion . each child 's functional data sets were co - registered to his / her high - resolution anatomical mri , rotated into the ac pc plane and normalized to talairach space using a linear transform calculated on the anatomical images . the 3.6 3.6 5 mm fmri voxels were interpolated during talairach normalization to 3 3 3 mm . a priori regions of interest ( rois ) were defined for each of the five parietal regions identified in dehaene et al . 's ( 2003 ) meta - analysis , namely bilateral anterior horizontal intraparietal sulcus ( ips ) , bilateral pspl and left angular gyrus . each roi consisted of a sphere , with a radius of 6 mm , centered on the coordinates derived from the meta - analysis . separate subject analyses were performed on the average signal in each roi using the general linear model with predictors based on the known experimental blocks convolved by the standard hemodynamic function . the six motion correction parameters were z - transformed and then added as predictors of no interest . the beta values generated by this analysis , which reflect the mean percent signal change for each condition for each subject , were used to calculate percent signal change during the numeric task compared to the control task . one outlier with percent signal change values > 3 sd beyond the mean for the right and left pspl and left ips regions was excluded from analyses of those regions on the pj task , and one outlier for the left pspl and right ips was excluded from analyses of those regions on ea . aa / occasion were positively skewed and were log transformed ( log x + 1 ) . the following variables with outliers greater than 3 sd beyond the mean were transformed by recoding all outlying values to one point beyond the next most extreme observed value : parity ( n = 1 ) , mother 's grade ( n = 1 ) , lead exposure ( n = 1 ) , number of correct ea trials inside the scanner ( n = 1 ) , and pj accuracy inside the scanner ( seven control variables were assessed for consideration as potential confounders of the relation of prenatal alcohol exposure to number processing : three demographic characteristics ( parity , and mother 's age at delivery and years of education ) , two child characteristics ( child gender and age at assessment ) , and two neurotoxic exposures ( maternal smoking during pregnancy and postnatal lead exposure ) that are known to impact on the child 's academic performance . lead exposure , which was based on a venous blood sample obtained from the child , was included because lead levels in this population are within the range in which subtle but meaningful effects on cognitive function have been consistently reported ( e.g. , lanphear et al . , 2000 ; each control variable that was weakly related to a given outcome measure ( at p < 0.10 ) was considered a potential confounder of the effect of each exposure measure on the outcome in question . the outcome measures were accuracy ( % correct ) inside and outside the scanner , number of trials attempted and number completed inside the scanner , and percent signal change relative to the control task in each of the rois for each of the tasks . the relation of diagnostic group ( fas / pfas ; heavily - exposed ( he ) nonsyndromal ; control ) to each of the outcome measures was examined using analyses of variance ( anova ) . post - hoc comparisons were computed using the least - squares difference ( lsd ) approach . each anova was then rerun as an analysis of covariance including as covariates each of the control variables related to the outcome in question at p < 0.10 to adjust for potential confounding . the relation of two continuous measures of prenatal alcohol exposure aa / day and aa / drinking day to each of the outcome measures was examined using pearson correlation analysis . multiple regression analyses were then run relating each of the continuous exposure measures to each of the outcomes controling for potential confounders . pearson correlation was used to examine the relation of percent signal change in each of the rois to behavioral performance inside and outside the scanner . the mothers of children with fas and pfas reported having consumed an average of 13.2 standard drinks of alcohol per drinking occasion during pregnancy ; the mothers of the he group , 11.8 standard drinks . the groups were generally similar in terms of the control variables , except that the mothers of the he group smoked more during pregnancy than the mothers of the control or fas / pfas children and that on average , the control children 's primary caregivers were more educated than either of the other groups . there was very little drug use , with only one mother of an he child reporting use of marijuana during pregnancy . the fas / pfas and he groups scored more poorly on the wisc iq test than the control children ; and the fas / pfas , more poorly than the he group . on the wisc subtests , the alcohol - exposed children performed more poorly than the controls in both the number processing ( i.e. , arithmetic ) and verbal ( i.e. , similarities ) domains . the low iq scores of all of the children in the sample reflect the highly disadvantaged circumstances and poor quality of education available in this community . fifty - nine children were scanned : 14 with fas or pfas , 21 he nonsyndromal and 24 controls . after exclusions due to excessive motion and/or poor accuracy , 49 children with usable data for at least one task remained ( 47 for pj ; 38 for ea ) . the children with usable scanner data did not differ from those in the initial sample in terms of alcohol exposure or diagnostic group ( all ps > 0.15 ) . the excluded children were 1.1 years younger on average than those included ( t(63 ) = 3.56 , p = 0.001 ) , and their estimated iq scores were lower ( t(63 ) = 2.73 , p = 0.008 ) , but they did not differ from the children whose data were included in maternal education , parity , smoking during pregnancy , and age at delivery , or child sex and blood lead concentrations ( all ps > 0.10 ) . behavioral performance on the number processing tasks is summarized by group and in relation to prenatal alcohol exposure in tables 2 and 3 , respectively . all the children in the sample are included in the analyses of the data collected outside the scanner , but behavioral performance inside the scanner is shown only for those whose neuroimaging data were included in the data analysis . outside the scanner the fas / pfas group performed more poorly than the other two groups on pj and more poorly than the controls on ea ( table 2 ) . both continuous measures of prenatal alcohol exposure were related to poorer pj performance outside the scanner , effects that remained significant after controling for potential confounders ( table 3 ) . by contrast , no group differences in performance accuracy were seen when simplified versions of the tasks were administered inside the scanner ( table 2 ) . the only differences between the groups on either task was that in pj the fas / pfas group attempted fewer trials , reflecting a slower rate of completing the pj problems , and got fewer trials correct than those of the control group . aa / day and aa / occasion were inversely correlated with the number of pj trials completed correctly and accuracy inside the scanner ( table 3 ) . the diagnostic groups showed few differences in terms of activation of the five parietal rois . on the pj task , the groups differed only in the left angular gyrus ( f(2,44 ) = 6.93 , p = 0.002 , means standard deviation ( ms sd ) = 0.17 0.11 , 0.02 0.17 and 0.00 0.11 for the fas / pfas , he and control groups , respectively ) , with more activation in the fas / pfas group than in either the he ( p = 0.001 ) or control groups ( p = 0.003 ; see fig . none of the rois showed significant group differences , but the effect on the right ips fell just short of significance ( f(2,34 ) = 2.96 , p = 0.065 ) , with the controls showing more activation than the he group ( p = 0.020 ) ( ms sd = 0.03 0.10 , 0.03 0.15 , and 0.07 0.08 for the fas / pfas , he and control groups , respectively ) . when combining the fas / pfas and he groups to compare activation in exposed children to unexposed controls , controls showed more activation relative to exposed children in the right ips during ea ( t(35 ) = 2.06 , p = 0.47 ; ms sd = 0.00 0.13 and 0.07 0.08 for the exposed and control groups , respectively ) . by contrast , increasing aa / day and aa / drinking day were both related to lower percent signal change relative to the control task in the right ips on both the pj and ea tasks ( table 4 and fig . since the children in the fas / pfas group performed fewer trials during pj than the he and control children , we also examined whether the effects of alcohol on activation during pj remained significant after controling for the number of trials attempted . all differences and associations remained essentially unchanged ( s relating aa / day and aa / occasion to activation of right ips were 0.35 and 0.36 , respectively ; both ps < 0.02 ) . in addition , after controling for the confounding influences of maternal smoking during pregnancy and age at delivery , greater aa / day was related to reduced activation in the left pspl during ea , suggesting that these control variables were suppressors . the relation of both continuous measures of prenatal alcohol exposure to reduced percent signal change in the left angular gyrus on the ea task fell short of statistical significance ( table 4 ) . greater percent signal change in the right ips was related to better ea performance outside the scanner ( r = 0.35 , p = 0.033 ) . for pj , greater percent signal change in the right pspl was associated with poorer accuracy outside the scanner , r = 0.31 , p = 0.037 , and greater signal change in the left pspl was associated with completion of fewer problems correctly inside the scanner , r = 0.35 , p = 0.016 . as can be seen in fig . 5 , the inverse relation between the left pspl activation and the number of correct pj trials was strongest in the fas / pfas group ( r = 0.76 , p = 0.007 , for fas / pfas , compared with r = 0.40 , p = 0.113 , for he and r = 0.31 , p = 0.214 for controls ) . this study examined the relation of fasd diagnosis and continuous measures of prenatal alcohol exposure to activation of the five parietal regions identified by dehaene et al . ( 2003 ) as most critical for number processing , using tasks involving simple addition and number comparison . despite generally similar behavioral performance between diagnostic groups on the simplified tasks administered inside the scanner , the effects of prenatal exposure were observed in altered patterns of brain activation . greater prenatal alcohol exposure was related to less activation in the right ips during both the pj and ea tasks and to less left pspl activation during ea . in addition , the fas / pfas group activated the left angular gyrus more than the other groups in the pj task . when more challenging versions of the number processing tasks were administered outside the scanner , fasd diagnosis was associated with poorer performance on both ea and pj , and degree of prenatal alcohol exposure was associated with poorer performance on pj . while neither measure of alcohol exposure was related to performance on the simpler ea task administered in the scanner , the children with fas or pfas performed the pj task more slowly than the other groups in the scanner , and the continuous measures of exposure were associated with less accurate in - scanner pj performance . these findings suggest that these heavily exposed children found the magnitude comparison processing required for pj more challenging than simple addition problems that could be solved by rote memory . these findings are also consistent with the higher sensitivity of magnitude comparison to prenatal alcohol exposure seen in our detroit longitudinal cohort ( j. jacobson et al . , 2011 ) . this study is the first to demonstrate a direct effect of prenatal alcohol exposure on activation of the ips during number processing . the bilateral ips have been repeatedly linked to nonverbal representation of quantity ( dehaene et al . , 2003 ) . this region is activated when numbers are read even when no arithmetic manipulation is required ( eger et al . , 2003 ) , but activation is greater when comparing numbers ( chochon et al . , 1999 ) or performing calculations ( burbaud et al . , 1999 ; , 1999 ; pesenti et al . , 2000 ) and often larger on the right ( rosselli and ardila , 1989 ; dehaene and cohen , 1996 ; langdon and warrington , 1997 ; chochon et al . , 1999 ; this area is more active when manipulating large numbers ( kiefer and dehaene , 1997 ; stanescu - cosson et al . , 2000 ) , performing more complex arithmetic manipulations ( e.g. , with 3 rather than 2 operands ; rivera et al . , 2002 ) , or comparing numbers separated by a small numerical distance ( dehaene and cohen , 1996 ; pinel et al . it is well - established that numbers closer together ( e.g. , 2 and 3 ) are more difficult ( i.e. , take longer ) to compare than numbers further apart ( e.g. , 2 and 9 ) ( moyer and landauer , 1967 ) . thus , the observed effect of prenatal alcohol exposure on poorer recruitment of the ips provides evidence of a fetal alcohol - related deficit in mental representation and manipulation of quantity , which is consistent with the behavioral evidence from our detroit study suggesting a specific effect of prenatal alcohol on magnitude comparison ( j. jacobson et al . , 2011 ) . although our continuous measure of prenatal alcohol exposure showed a dose - dependent decrease in activation of the right ips during both tasks , diagnostic group differences were only seen in this region when all alcohol exposed children were combined into one group and compared to unexposed controls , a result that seems to be driven by the reduced activation in this region during ea in the he children specifically . this finding is consistent with our previous study , in which children with fas or pfas from this cohort were compared with healthy controls using a whole brain , voxelwise approach ( meintjes et al . although only the control children showed significant activation of the ips in that study , the between - group difference was not significant . the only other previous study to examine number processing in relation to fetal alcohol exposure also found an alcohol exposure - dependent response in a right inferior parietal region that included the ips , with controls showing the most activity , during a subtraction task ( santhanam et al . , 2009 ) . the finding that our continuous measures of prenatal alcohol exposure were more sensitive than diagnosis in detecting effects on brain function is consistent with our findings in several other neuroimaging studies ( de guio et al . , 2014 ; du plessis et al . the poorer activation of the right ips seen in the alcohol - exposed children in this study during number processing is also seen in children with developmental dyscalculia ( dd ) ( kucian et al . , 2006 ; price et al . , 2007 ; kaufmann et al . , 2009a ; kaufmann et al . , 2009b ; rubinsten and henik , 2009 ; mussolin et al . , 2010 ; 2012 ) and poor arithmetical fluency ( de smedt et al . , 2011 ) . dd is a specific learning disability believed to be genetic in origin , which is characterized by impairment in the processing of numerical and arithmetical information in individuals with normal intelligence . in dd , activations of the bilateral ips also fail to exhibit the increased response to differences in numerical distance seen in normal control children ( mussolin et al . , 2010 ) . a voxel - based morphometry study found less gray matter density in the left ips in low birthweight children with dd , compared with healthy controls ( isaacs et al . , 2001 ) . impaired recruitment of the ips during tasks involving number processing has also been found in turner syndrome ( ts ) , a genetic disorder involving a chromosomal defect , in which math is an area where deficits are commonly noted ( molko et al . similarly , in a study of children with fragile x syndrome , another form of mental retardation , rivera et al . ( 2002 ) found that , although the right ips was activated by healthy controls during 3-operand arithmetic problems , it was not activated by fragile x patients presented with those problems . in the pj task , we found greater activation in the left angular gyrus in the fas / pfas group than either the he or control groups . the angular gyrus is adjacent to the perisylvian language processing network and is associated with the verbal processing of numbers ( dehaene et al . , 2003 ) . in typically developing children , it is activated more during addition and multiplication than during subtraction , presumably because addition and multiplication facts are more likely to be retrieved from long - term memory ( stanescu - cosson et al . , 2000 ; simon et al . , 2002 ; delazer et al . , 2005 ) . the increased activation of the angular gyrus suggests that children with fas and pfas may rely on verbal recitation of the numbers and/or verbally mediated subtraction operations to solve the pj problems , instead of the type of nonverbal quantity processing that has been shown to be mediated by the right anterior ips ( chochon et al . , 1999 ) . verbal mediation may provide a compensatory strategy for these children this finding is consistent with results from our previous whole brain voxelwise analysis , in which there was a significant group difference in the same region of the left angular gyrus [ 42,65,36 ] , with greater activation in the fas / pfas group than the controls ( meintjes et al . , 2010b ) . we added the he group to the analyses in the present study and found that , although this group was exposed at similar levels to the fas / pfas group , the he children did not appear to rely on the angular gyrus to perform the pj task . by contrast , on the ea task , in which verbal recall of number facts is presumed to be the most efficient strategy , our data suggest less activation of the angular gyrus by the more heavily exposed children ( table 4 ) . lower levels of activation in the right ips was related to poorer ea performance outside the scanner . by contrast , price et al . ( 2013 ) reported greater activation in the right ips during single digit arithmetic calculation in adolescents with lower math scores . the authors note that their findings suggest that , while ips - mediated quantity processing mechanisms appear to play an important role in the development of elementary arithmetic skills , individuals who continue to rely on them in adolescence may achieve poorer mathematical competence than their peers who do not . a large body of behavioral research has shown that children typically undergo a process of development in arithmetic skill whereby simple calculations are initially computed through procedural strategies , but then gradually come to be solved by memory retrieval ( geary et al . development beyond quantity - based calculation strategies may be essential for the development of more advanced mathematical competence . in the pj task , greater activation of the right pspl was associated with worse performance outside the scanner , and greater activation of the left pspl was associated with fewer trials correct inside the scanner . the pspl , which is activated during counting ( piazza et al . , 2002 ) and a variety of visual spatial tasks , is believed to support the engagement of attention during number processing ( pinel et al . the greater activation of these regions by children with less optimal pj performance suggests engagement of increased attention to compensate for their poorer facility in magnitude comparison . 5 , the inverse relation between the left pspl activation and pj performance was strongest in the fas / pfas group , which appeared to find the pj task particularly challenging . by contrast , in the ea task prenatal alcohol exposure was related to less activation of the left pspl , a region that may facilitate attentional engagement required for simple addition at this age but may no longer be needed by children who have mastered simple proximity judgment . since working memory and attention , which are also mediated , in part , by the parietal lobes ( e.g. , ravizza et al . , 2004 ; diwadkar et al . , 2013 ) , contribute substantially to number processing performance , impairments in these domains may play a role in the number processing deficits observed in fasd . moreover , two studies have reported reduced activation in working memory tasks in individuals with fasd compared to controls in posterior parietal regions close to the pspl roi used in our study ( astley et al . , 2009 ; gautam et al . , the specific section of the ips found to mediate the effects of prenatal alcohol exposure on number processing in this study has , however , not been implicated in working memory or attention per se . one limitation of this study was that the maternal report of drinking during pregnancy was obtained retrospectively several years after the child 's birth . nevertheless , the validity of these reports is supported by our findings that they are predictive of neuroimaging and neurobehavioral outcomes ( meintjes et al . predictive validity for childhood iq was r = 0.36 and 0.40 , for aa / day and aa / occasion , respectively , both ps < 0.001 . a second limitation was the relatively small size of the fas / pfas and he groups and the greater variability in performance accuracy outside the scanner in the fas / pfas group . not surprisingly , the children who were excluded due to poor performance were younger and had lower iq scores . presumably , performance on this test will improve with age , and we assume that the fetal alcohol - related patterns of brain activation seen in this study will also be manifest once these younger children acquire sufficient math skills to perform these tasks . with regard to iq , although proportionately more low iq children were unable to complete the neuroimaging tasks , the sample on whom the neuroimaging data were obtained did include a substantial number of children with low iqs ( < 70 ) . thus , the results can be generalized to all but the most severely retarded children . because the children were socioeconomically and educationally deprived , we can not determine the degree to which the results would hold for children from an educationally less deprived background . moreover , due to the poor educational background of the children , we had to use very easy problems to ensure that they would be able to perform them . if more difficult problems could have been used , a more pronounced group difference might have been seen . the need to use easy problems also precluded the use of a potentially more powerful parametric design to examine the effect of increasing difficulty on the children 's brain activations . the age range of the children7.9 to 13.4 years was relatively large , particularly in light of the changes in number processing strategy that may emerge during this period . a smaller age range might have yielded stronger associations . nevertheless , it is interesting , that , although all the behavioral performance measures were related to age , suggesting an age - dependent effect of education , none of the brain activations were even weakly related to age but instead showed associations with maternal factors ( age , education , smoking , and parity ) . we did not control for multiple comparisons , due to the fact that we examined only a limited number of regions rather than the whole brain . region - of - interest analyses are advantageous in that multiple comparisons are much less of an issue and snr is increased by averaging across the voxels in a region . this study demonstrates poor recruitment of the right ips during number processing tasks by children with heavy prenatal alcohol exposure , confirming our previous report based on behavioral data of a fetal alcohol - related deficit in mental representation and manipulation of quantity ( j. jacobson et al . , 2011 ) . our neuroimaging data also indicated increased activation of the angular gyrus in the fas / pfas group during the proximity judgment task , suggesting that these children may use a verbal mediation strategy to compensate for impairment in magnitude comparison . by contrast , prenatal alcohol exposure was related to lower levels of activation of the angular gyrus during the exact addition task , for which verbal mediation to retrieve addition facts is presumed to be an efficient strategy . although activation of the left pspl , which is believed to support attentional engagement during number processing , was increased in children with less optimal pj performance , particularly in the fas / pfas group , prenatal alcohol was associated with lower levels of activation of this region during the ea task , suggesting less capacity to devote attentional resources to the verbal retrieval of addition facts required for simple addition at this age . in summary , these data demonstrate that prenatal alcohol exposure alters activation of each of the elements of the parietal network known to be critical for number processing , providing additional evidence for a specific fetal alcohol - related deficit in the core quantity system for representation and manipulation of quantity identified by dehaene et al . these findings suggest that remediation focused on the core quantity concepts might be particularly effective in children with fasd who exhibit difficulty with mathematical processing .

number processing deficits are frequently seen in children prenatally exposed to alcohol . although the parietal lobe , which is known to mediate several key aspects of number processing , has been shown to be structurally impaired in fetal alcohol spectrum disorders ( fasd ) , effects on functional activity in this region during number processing have not previously been investigated . this fmri study of 49 children examined differences in activation associated with prenatal alcohol exposure in five key parietal regions involved in number processing , using tasks involving simple addition and magnitude comparison . despite generally similar behavioral performance , in both tasks greater prenatal alcohol exposure was related to less activation in an anterior section of the right horizontal intraparietal sulcus known to mediate mental representation and manipulation of quantity . children with fetal alcohol syndrome and partial fetal alcohol syndrome appeared to compensate for this deficit by increased activation of the angular gyrus during the magnitude comparison task .

Introduction Methods Results Discussion Conclusions

, 2001 ; fetal alcohol syndrome ( fas ) , the most severe of the fetal alcohol spectrum disorders ( fasd ) , is characterized by distinctive craniofacial dysmorphology ( short palpebral fissures , thin vermilion , flat philtrum ) , small head circumference and pre- and/or postnatal growth retardation ( hoyme et al . prenatal alcohol exposure is associated with a broad range of cognitive deficits , including low iq ( streissguth et al . among the cognitive deficits seen in relation to prenatal alcohol exposure , arithmetic is especially sensitive , and mathematical deficits are seen even after controling for iq ( coles et al . , although the parietal lobe has been known to be involved in number processing since the beginning of the 20th century ( henschen , 1919 ) , fmri has provided a more extensive understanding of the neuroanatomy of this domain of processing . based on brain lesion and neuroimaging findings , dehaene and associates ( dehaene , 1992 ; dehaene and cohen , 1996 ) have proposed a triple - code model of number processing that incorporates the three different systems of representation that may be used in number processing tasks : the quantity system , the verbal system and the visual system . in addition , they have posited a core quantity system a nonverbal abstract representation of numerical quantity , which has been localized bilaterally in the anterior portion of the horizontal segments of the intraparietal sulcus ( ips ) . the verbal processing of numbers is posited to be based on the left angular gyrus ( close to the language areas ) , while the bilateral posterior superior parietal lobules ( pspls ) are hypothesized to be involved in spatial and non - spatial attentional processes contributing to the visual processing of numbers . ( 2005 ) found age - related increases in activation of the anterior ips during number processing between school age and early adulthood , which they attributed to the development of increasing functional specialization of this region as symbolic number processing becomes increasingly automatic . results from a meta - analysis of studies comparing number processing in children vs. adults generally support dehaene 's model but suggest that the localization of parietal activations is more notation - specific in children and that right ips activations in non - symbolic magnitude comparison are slightly more anterior than those observed in adults ( kaufmann et al . a 224-item , 7-subtest , computer - based number processing test that we developed in collaboration with s. dehaene , was administered to 262 adolescents from the detroit longitudinal prenatal alcohol exposure cohort . in a path analytic model , the relation of prenatal alcohol exposure to calculation was fully mediated by its effects on magnitude comparison , suggesting that magnitude comparison is a core deficit involved in the poor arithmetic performance seen in these children . in the detroit cohort , attention - deficit / hyperactivity disorder ( adhd ) was related to poorer performance on all seven number processing subtests , but , by contrast to the pattern seen in the alcohol - exposed children , the associations were markedly stronger with calculation than magnitude comparison . moreover , iq significantly mediated the effect of adhd on calculation , suggesting that the effects of adhd on aspects of calculation not specific to the representation of number , such as attention and executive function , mediate the poorer number processing seen in that disorder . this behavioral evidence linking prenatal alcohol exposure to impairment in the core quantity system is consistent with evidence from mri studies reporting alcohol - related structural impairment in the parietal region , including disproportionate size reductions in the parietal lobe ( archibald et al . , 2002 ) , and voxel - based morphometry ( vbm ) analysis revealed gray matter abnormalities that were most prominent in the left perisylvian cortices of the parietal and temporal lobes ( sowell et al . in addition , significant cortical thickness excesses were observed in children with fasd in large areas of bilateral temporal , bilateral inferior parietal and right frontal regions ( sowell et al . exposed individuals with alcohol - related dysmorphology exhibited poorer task performance and lower activation in regions known to be associated with arithmetic processing , including the right inferior and left superior parietal regions and medial frontal gyrus , compared with controls . however , it was not clear whether the reduced activation in the dysmorphic participants reflected a specific deficit in fronto - parietal function or completion of fewer problems due to the difficulty of the task . using simpler proximity judgment and single - digit addition problems , we found that children activate the same fronto - parietal regions activated in number processing by adults ( meintjes et al . although children with fas and pfas performed as well as controls on the simple tasks administered in the scanner , they activated a markedly more diffuse parietal region extending into the angular gyrus , precuneus and posterior cingulate and for , exact addition , also the postcentral gyrus ( meintjes et al . , however , the fas / pfas and control groups did not differ significantly in the degree of activation in the anterior portion of the ips and other regions linked by dehaene and associates to number processing , possibly due to the lack of statistical power in the small sample on which that whole brain voxel - wise analysis was conducted . in this study , we examine the effects of both fasd diagnosis and continuous measures of prenatal alcohol exposure on brain activation in the five parietal structures found by dehaene and associates to mediate number processing in a larger sample that includes not only children with fas / pfas and nonexposed controls , but also nonsyndromal heavily - exposed ( he ) children ( suttie et al . based on the behavioral findings from our detroit and cape town studies , our central hypothesis was that prenatal alcohol exposure would be associated with reduced activation of the anterior ips , the region believed to mediate abstract representation of numerical quantity . the others were identified by screening all of the 8- to 12-year - old children from an elementary school in a rural section of cape town , where there is a very high incidence of alcohol abuse among local farm workers ( meintjes et al . was administered the computer - based number processing test described above , which included two 16-item subtests , one of which assessed exact addition ( ea ) ; the other , proximity judgment ( pj ) ( kopera - frye et al . only similarities were administered in the verbal domain because the other verbal subtests appeared to be less valid in this cross - cultural context . simplified versions of the ea and pj subtests from the computer - based number processing assessment described above were administered . ea was selected because it is the easiest of the calculation subtests ; pj was selected to represent magnitude comparison because the range of the neurobehavioral scores for number comparison is truncated . each child practiced these tasks initially in a mock scanner built for this study , which was important in reducing anxiety and facilitating completion of the mri scans . 's ( 2003 ) meta - analysis , namely bilateral anterior horizontal intraparietal sulcus ( ips ) , bilateral pspl and left angular gyrus . the beta values generated by this analysis , which reflect the mean percent signal change for each condition for each subject , were used to calculate percent signal change during the numeric task compared to the control task . the following variables with outliers greater than 3 sd beyond the mean were transformed by recoding all outlying values to one point beyond the next most extreme observed value : parity ( n = 1 ) , mother 's grade ( n = 1 ) , lead exposure ( n = 1 ) , number of correct ea trials inside the scanner ( n = 1 ) , and pj accuracy inside the scanner ( seven control variables were assessed for consideration as potential confounders of the relation of prenatal alcohol exposure to number processing : three demographic characteristics ( parity , and mother 's age at delivery and years of education ) , two child characteristics ( child gender and age at assessment ) , and two neurotoxic exposures ( maternal smoking during pregnancy and postnatal lead exposure ) that are known to impact on the child 's academic performance . lead exposure , which was based on a venous blood sample obtained from the child , was included because lead levels in this population are within the range in which subtle but meaningful effects on cognitive function have been consistently reported ( e.g. the relation of two continuous measures of prenatal alcohol exposure aa / day and aa / drinking day to each of the outcome measures was examined using pearson correlation analysis . the mothers of children with fas and pfas reported having consumed an average of 13.2 standard drinks of alcohol per drinking occasion during pregnancy ; the mothers of the he group , 11.8 standard drinks . the groups were generally similar in terms of the control variables , except that the mothers of the he group smoked more during pregnancy than the mothers of the control or fas / pfas children and that on average , the control children 's primary caregivers were more educated than either of the other groups . on the wisc subtests , the alcohol - exposed children performed more poorly than the controls in both the number processing ( i.e. behavioral performance on the number processing tasks is summarized by group and in relation to prenatal alcohol exposure in tables 2 and 3 , respectively . both continuous measures of prenatal alcohol exposure were related to poorer pj performance outside the scanner , effects that remained significant after controling for potential confounders ( table 3 ) . the diagnostic groups showed few differences in terms of activation of the five parietal rois . on the pj task , the groups differed only in the left angular gyrus ( f(2,44 ) = 6.93 , p = 0.002 , means standard deviation ( ms sd ) = 0.17 0.11 , 0.02 0.17 and 0.00 0.11 for the fas / pfas , he and control groups , respectively ) , with more activation in the fas / pfas group than in either the he ( p = 0.001 ) or control groups ( p = 0.003 ; see fig . none of the rois showed significant group differences , but the effect on the right ips fell just short of significance ( f(2,34 ) = 2.96 , p = 0.065 ) , with the controls showing more activation than the he group ( p = 0.020 ) ( ms sd = 0.03 0.10 , 0.03 0.15 , and 0.07 0.08 for the fas / pfas , he and control groups , respectively ) . in addition , after controling for the confounding influences of maternal smoking during pregnancy and age at delivery , greater aa / day was related to reduced activation in the left pspl during ea , suggesting that these control variables were suppressors . the relation of both continuous measures of prenatal alcohol exposure to reduced percent signal change in the left angular gyrus on the ea task fell short of statistical significance ( table 4 ) . greater percent signal change in the right ips was related to better ea performance outside the scanner ( r = 0.35 , p = 0.033 ) . this study examined the relation of fasd diagnosis and continuous measures of prenatal alcohol exposure to activation of the five parietal regions identified by dehaene et al . ( 2003 ) as most critical for number processing , using tasks involving simple addition and number comparison . despite generally similar behavioral performance between diagnostic groups on the simplified tasks administered inside the scanner , the effects of prenatal exposure were observed in altered patterns of brain activation . greater prenatal alcohol exposure was related to less activation in the right ips during both the pj and ea tasks and to less left pspl activation during ea . when more challenging versions of the number processing tasks were administered outside the scanner , fasd diagnosis was associated with poorer performance on both ea and pj , and degree of prenatal alcohol exposure was associated with poorer performance on pj . while neither measure of alcohol exposure was related to performance on the simpler ea task administered in the scanner , the children with fas or pfas performed the pj task more slowly than the other groups in the scanner , and the continuous measures of exposure were associated with less accurate in - scanner pj performance . these findings suggest that these heavily exposed children found the magnitude comparison processing required for pj more challenging than simple addition problems that could be solved by rote memory . these findings are also consistent with the higher sensitivity of magnitude comparison to prenatal alcohol exposure seen in our detroit longitudinal cohort ( j. jacobson et al . this study is the first to demonstrate a direct effect of prenatal alcohol exposure on activation of the ips during number processing . thus , the observed effect of prenatal alcohol exposure on poorer recruitment of the ips provides evidence of a fetal alcohol - related deficit in mental representation and manipulation of quantity , which is consistent with the behavioral evidence from our detroit study suggesting a specific effect of prenatal alcohol on magnitude comparison ( j. jacobson et al . although our continuous measure of prenatal alcohol exposure showed a dose - dependent decrease in activation of the right ips during both tasks , diagnostic group differences were only seen in this region when all alcohol exposed children were combined into one group and compared to unexposed controls , a result that seems to be driven by the reduced activation in this region during ea in the he children specifically . the only other previous study to examine number processing in relation to fetal alcohol exposure also found an alcohol exposure - dependent response in a right inferior parietal region that included the ips , with controls showing the most activity , during a subtraction task ( santhanam et al . the finding that our continuous measures of prenatal alcohol exposure were more sensitive than diagnosis in detecting effects on brain function is consistent with our findings in several other neuroimaging studies ( de guio et al . the poorer activation of the right ips seen in the alcohol - exposed children in this study during number processing is also seen in children with developmental dyscalculia ( dd ) ( kucian et al . dd is a specific learning disability believed to be genetic in origin , which is characterized by impairment in the processing of numerical and arithmetical information in individuals with normal intelligence . in dd , activations of the bilateral ips also fail to exhibit the increased response to differences in numerical distance seen in normal control children ( mussolin et al . impaired recruitment of the ips during tasks involving number processing has also been found in turner syndrome ( ts ) , a genetic disorder involving a chromosomal defect , in which math is an area where deficits are commonly noted ( molko et al . similarly , in a study of children with fragile x syndrome , another form of mental retardation , rivera et al . the angular gyrus is adjacent to the perisylvian language processing network and is associated with the verbal processing of numbers ( dehaene et al . in typically developing children , it is activated more during addition and multiplication than during subtraction , presumably because addition and multiplication facts are more likely to be retrieved from long - term memory ( stanescu - cosson et al . the increased activation of the angular gyrus suggests that children with fas and pfas may rely on verbal recitation of the numbers and/or verbally mediated subtraction operations to solve the pj problems , instead of the type of nonverbal quantity processing that has been shown to be mediated by the right anterior ips ( chochon et al . verbal mediation may provide a compensatory strategy for these children this finding is consistent with results from our previous whole brain voxelwise analysis , in which there was a significant group difference in the same region of the left angular gyrus [ 42,65,36 ] , with greater activation in the fas / pfas group than the controls ( meintjes et al . by contrast , on the ea task , in which verbal recall of number facts is presumed to be the most efficient strategy , our data suggest less activation of the angular gyrus by the more heavily exposed children ( table 4 ) . lower levels of activation in the right ips was related to poorer ea performance outside the scanner . ( 2013 ) reported greater activation in the right ips during single digit arithmetic calculation in adolescents with lower math scores . in the pj task , greater activation of the right pspl was associated with worse performance outside the scanner , and greater activation of the left pspl was associated with fewer trials correct inside the scanner . the greater activation of these regions by children with less optimal pj performance suggests engagement of increased attention to compensate for their poorer facility in magnitude comparison . 5 , the inverse relation between the left pspl activation and pj performance was strongest in the fas / pfas group , which appeared to find the pj task particularly challenging . by contrast , in the ea task prenatal alcohol exposure was related to less activation of the left pspl , a region that may facilitate attentional engagement required for simple addition at this age but may no longer be needed by children who have mastered simple proximity judgment . since working memory and attention , which are also mediated , in part , by the parietal lobes ( e.g. , 2013 ) , contribute substantially to number processing performance , impairments in these domains may play a role in the number processing deficits observed in fasd . , the specific section of the ips found to mediate the effects of prenatal alcohol exposure on number processing in this study has , however , not been implicated in working memory or attention per se . presumably , performance on this test will improve with age , and we assume that the fetal alcohol - related patterns of brain activation seen in this study will also be manifest once these younger children acquire sufficient math skills to perform these tasks . the age range of the children7.9 to 13.4 years was relatively large , particularly in light of the changes in number processing strategy that may emerge during this period . nevertheless , it is interesting , that , although all the behavioral performance measures were related to age , suggesting an age - dependent effect of education , none of the brain activations were even weakly related to age but instead showed associations with maternal factors ( age , education , smoking , and parity ) . this study demonstrates poor recruitment of the right ips during number processing tasks by children with heavy prenatal alcohol exposure , confirming our previous report based on behavioral data of a fetal alcohol - related deficit in mental representation and manipulation of quantity ( j. jacobson et al . our neuroimaging data also indicated increased activation of the angular gyrus in the fas / pfas group during the proximity judgment task , suggesting that these children may use a verbal mediation strategy to compensate for impairment in magnitude comparison . by contrast , prenatal alcohol exposure was related to lower levels of activation of the angular gyrus during the exact addition task , for which verbal mediation to retrieve addition facts is presumed to be an efficient strategy . although activation of the left pspl , which is believed to support attentional engagement during number processing , was increased in children with less optimal pj performance , particularly in the fas / pfas group , prenatal alcohol was associated with lower levels of activation of this region during the ea task , suggesting less capacity to devote attentional resources to the verbal retrieval of addition facts required for simple addition at this age . in summary , these data demonstrate that prenatal alcohol exposure alters activation of each of the elements of the parietal network known to be critical for number processing , providing additional evidence for a specific fetal alcohol - related deficit in the core quantity system for representation and manipulation of quantity identified by dehaene et al .

infertility is one of the major health problems in life , and approximately about 30% of this problem is due to male factors . several factors can interfere with the process of spermatogenesis , reduce sperm quantity and quality and decrease male fertility . many diseases such as coronary heart diseases , diabetes mellitus and chronic liver diseases as well as insufficient vitamins intake have deleterious effects on spermatogenesis and production of normal sperm . oxidative stress is a major predisposing risk factor for many chronic diseases , including male infertility problem . on the other side , intake of natural anti - oxidant agents from plants together with vitamins e and c can protect sperm dna from oxidative stress in the testis of rats , and antagonize testicular toxicity in rabbits . medicinal plants are a promising source for safe , natural anti - oxidant agents as they contain many bioactive constituents , especially anti - oxidant flavonoids and polyphenol compounds . tribulus terrestris plant , popularly known as puncture vine , is a perennial creeping herb with a worldwide distribution . since ancient times , t. terrestris has a long history as a powerful aphrodisiac . in traditional medicine , moreover , t. terrestris roots extract and total saponins extracted from the roots have beneficial effects on various ailments such as urinary tract infections , inflammations , leucorrhea , edema , and ascites . t. terrestris fruits extract has been successfully used in europe and asia to treat sexual dysfunction in males . the different pharmacological effects of t. terrestris plant were reviewed by chhatre et al . . the present study was designed to evaluate the protective and anti - oxidant activities of the methanolic extract of t. terrestris fruits ( mett ) against testicular toxicity and oxidative stress induced sodium valproate ( svp ) in rats , and to examine the possible mechanisms . the fruits of t. terrestris ( family zygophyllaceae ) plant were obtained from the company of medicinal herbs , seeds , agricultural products , cairo , egypt . these fruits were botanically authenticated by dr . abdelhalim mohamed , flora and taxonomy department , agricultural research centre , giza , egypt . herbal specimen was deposited at the department of pharmacology , faculty of veterinary medicine , cairo university , egypt . two hundred grams of powdered dried fruits were extracted with 2 l of 90% methanol ( sigma aldrich for chemicals , usa ) by percolation for 72 h. the solvent was evaporated by vacuum distillation at 45c using a rotatory evaporator ( west germany ) . the liquid extract yielded 10 g of gummy residue that used for preparing the different doses of the extract using tween 80 as a suspending agent . it was obtained as an oral solution packed in dark brown bottles each containing 40 ml . a total of 50 mature male sprague dawley rats with average body weight of 200 - 250 g and age of 10 - 13 weeks were used in this study . rats were procured from laboratory animal colony , ministry of public health , helwan , egypt . the animals were housed in clean cages , kept under controlled hygienic conditions and maintained at room temperature at 25c 2c , relative humidity of 50% 5% and photoperiod of 12 h dark/12 h light cycles . rats were fed on rat pellets , and free access of tap water was supplied . the experiment on animals was carried out according to the national regulations on animal welfare and institutional animal ethical committee . group 1 was normal ( negative ) control and administered diluted tween 80 ( 0.1 ml / rat ) . the other four groups were intoxicated by oral administration of svp ( 500 mg / kg ) during the last week of the experiment period for induction of testicular toxicity . group 2 was kept intoxicated positive ( control ) and groups 3 , 4 , and 5 were pre - treated with the mett in daily doses of 2.5 , 5.0 and 10.0 mg / kg , respectively . the extract was orally given once daily for 60 days to cover the period of the spermatogenic cycle in the rat . blood samples were withdrawn by puncture of retro - orbital plexus of veins in the eye using microcapillary tubes . the samples were kept standing for 15 min to clot then centrifuged at 10,000 rpm for 10 min to separate the serum which kept frozen at 70c . the serum was used for estimation of testosterone , follicle stimulating hormone ( fsh ) and luteinizing hormone ( lh ) levels . rats were anesthetized by prolonged exposure to ether , and a longitudinal incision was made in the skin of scrotum and both testes were exposed . semen samples were collected from cauda epididymis by cutting the tail of epididymis and squeezed it into a clean watch glass . the semen samples were used for semen analysis . the testes and accessory sexual organs were dissected out and weighed , and relative weights of sexual organs were calculated . the right testes were immediately taken on ice cooled bags and kept frozen at 70c until the assessment of the activity anti - oxidant enzymes superoxide dismutase ( sod ) , glutathione peroxidase ( gpx ) and catalase ( cat ) . the left testes were preserved in 10% neutral formalin solution till processed for histological examination . serum testosterone concentration was assayed according to the method described in the manufacturer s directions . the method is based on the enzyme - linked immune absorbent assay ( elisa ) as described by tietz . serum testosterone concentrations were obtained by correlating the absorbance of the test sample at 550 nm with the corresponding absorbance on the standard curve . serum levels of fsh and lh hormones were determined using elisa kits ( amersham , buckinghamshire , uk ) according to ballester et al . . calabasas , usa . the seminal content of epididymis was obtained by cutting of cauda epididymis using surgical blades and squeezed into a sterile clean watch glass . this content was diluted 10 times with 2.9% sodium citrate solution and thoroughly mixed to estimate the percentage of sperm progressive motility and sperm count as described by bearden and fluquary . thereafter , one drop of sperm suspension was withdrawn , smeared on a glass slide and stained by eosin - nigrosin stain . the stained seminal smears were examined microscopically to determine the percentage of sperm viability ( alive / dead ) and morphological abnormalities . tissue specimens of the right testes after thawing was homogenized in nine volumes of ice cooled buffered 0.9% saline solution . the homogenate was then centrifuged at 4000 rpm for 15 min at 4c and the supernatant was used for anti - oxidant enzymes assay . the activity of sod was determined as described by nishikimi et al . and expressed as unit / mg protein . gpx activity was determined as described by paglia and valentine and expressed as nmol of glutathione utilized / min / mg protein . the activity of cat was expressed as nmol of h2o2 utilized / min / mg protein . the fixed specimens were trimmed , washed and dehydrated in ascending grades of alcohol , then cleared in xylene , embedded in paraffin , sectioned at 4 - 6 microns thickness and stained with hematoxylen and eosin stain , then examined microscopically . differences between means in the experimental groups were tested for significance using student s t - test . the fruits of t. terrestris ( family zygophyllaceae ) plant were obtained from the company of medicinal herbs , seeds , agricultural products , cairo , egypt . these fruits were botanically authenticated by dr . abdelhalim mohamed , flora and taxonomy department , agricultural research centre , giza , egypt . herbal specimen was deposited at the department of pharmacology , faculty of veterinary medicine , cairo university , egypt . two hundred grams of powdered dried fruits were extracted with 2 l of 90% methanol ( sigma aldrich for chemicals , usa ) by percolation for 72 h. the solvent was evaporated by vacuum distillation at 45c using a rotatory evaporator ( west germany ) . the liquid extract yielded 10 g of gummy residue that used for preparing the different doses of the extract using tween 80 as a suspending agent . it was obtained as an oral solution packed in dark brown bottles each containing 40 ml . a total of 50 mature male sprague dawley rats with average body weight of 200 - 250 g and age of 10 - 13 weeks were used in this study . rats were procured from laboratory animal colony , ministry of public health , helwan , egypt . the animals were housed in clean cages , kept under controlled hygienic conditions and maintained at room temperature at 25c 2c , relative humidity of 50% 5% and photoperiod of 12 h dark/12 h light cycles . rats were fed on rat pellets , and free access of tap water was supplied . the experiment on animals was carried out according to the national regulations on animal welfare and institutional animal ethical committee . group 1 was normal ( negative ) control and administered diluted tween 80 ( 0.1 ml / rat ) . the other four groups were intoxicated by oral administration of svp ( 500 mg / kg ) during the last week of the experiment period for induction of testicular toxicity . group 2 was kept intoxicated positive ( control ) and groups 3 , 4 , and 5 were pre - treated with the mett in daily doses of 2.5 , 5.0 and 10.0 mg / kg , respectively . the extract was orally given once daily for 60 days to cover the period of the spermatogenic cycle in the rat . blood samples were withdrawn by puncture of retro - orbital plexus of veins in the eye using microcapillary tubes . the samples were kept standing for 15 min to clot then centrifuged at 10,000 rpm for 10 min to separate the serum which kept frozen at 70c . the serum was used for estimation of testosterone , follicle stimulating hormone ( fsh ) and luteinizing hormone ( lh ) levels . rats were anesthetized by prolonged exposure to ether , and a longitudinal incision was made in the skin of scrotum and both testes were exposed . semen samples were collected from cauda epididymis by cutting the tail of epididymis and squeezed it into a clean watch glass . the semen samples were used for semen analysis . the testes and accessory sexual organs were dissected out and weighed , and relative weights of sexual organs were calculated . the right testes were immediately taken on ice cooled bags and kept frozen at 70c until the assessment of the activity anti - oxidant enzymes superoxide dismutase ( sod ) , glutathione peroxidase ( gpx ) and catalase ( cat ) . the left testes were preserved in 10% neutral formalin solution till processed for histological examination . serum testosterone concentration was assayed according to the method described in the manufacturer s directions . the method is based on the enzyme - linked immune absorbent assay ( elisa ) as described by tietz . serum testosterone concentrations were obtained by correlating the absorbance of the test sample at 550 nm with the corresponding absorbance on the standard curve . serum levels of fsh and lh hormones were determined using elisa kits ( amersham , buckinghamshire , uk ) according to ballester et al . . the seminal content of epididymis was obtained by cutting of cauda epididymis using surgical blades and squeezed into a sterile clean watch glass . this content was diluted 10 times with 2.9% sodium citrate solution and thoroughly mixed to estimate the percentage of sperm progressive motility and sperm count as described by bearden and fluquary . thereafter , one drop of sperm suspension was withdrawn , smeared on a glass slide and stained by eosin - nigrosin stain . the stained seminal smears were examined microscopically to determine the percentage of sperm viability ( alive / dead ) and morphological abnormalities . tissue specimens of the right testes after thawing was homogenized in nine volumes of ice cooled buffered 0.9% saline solution . the homogenate was then centrifuged at 4000 rpm for 15 min at 4c and the supernatant was used for anti - oxidant enzymes assay . the activity of sod was determined as described by nishikimi et al . and expressed as unit / mg protein . gpx activity was determined as described by paglia and valentine and expressed as nmol of glutathione utilized / min / mg protein . the activity of cat was expressed as nmol of h2o2 utilized / min / mg protein . the fixed specimens were trimmed , washed and dehydrated in ascending grades of alcohol , then cleared in xylene , embedded in paraffin , sectioned at 4 - 6 microns thickness and stained with hematoxylen and eosin stain , then examined microscopically . differences between means in the experimental groups were tested for significance using student s t - test . oral administration of svp to male rats in a dose of 500 mg / kg during the last week of the experiment induced significant ( p < 0.05 ) decreases in weights of testes and seminal vesicles when compared to the normal control group . rats pre - treated with oral administration of mett significantly normalized weights of testes and seminal vesicles , in a dose - dependent manner , when compared to svp - intoxicated control group [ table 1 ] . effect of methanolic extract of tribulus terrestris fruits on weights of sexual organs in sodium valproate - intoxicated rats ( n=10 ) oral administration of svp ( 500 mg / kg ) to rats during the last week of the experimental period significantly decreased serum testosterone , fsh and lh levels when compared with the normal control group . pre - treatments of svp - intoxicated rats with mett significantly increased serum testosterone , fsh and lh levels when compared with the intoxicated control group , in a dose dependent fashion [ table 2 ] . effect of mett on serum testosterone , fsh and lh levels in svp - intoxicated rats ( n=10 ) svp when given orally to male rats ( 500 mg / kg ) during the last week of the experiment induced significant decreases in sperm motility , viability and count and increased percentages of sperm abnormalities when compared with the normal control group . pre - treatment with mett significantly increased sperm motility , viability and count and decreased percentages of sperm abnormalities , in a dose dependent manner , as recorded in table 3 . the most frequently seen sperm abnormalities were detached , double and circular heads ; and bent and coiled tails and a detached neck as demonstrated in figure 1 . seminal smears from cauda epididymis of the rat s testes : ( a ) normal mature sperm ( n ) and detached head of sperm ( arrow ) , ( b ) double head of sperm ( arrow ) , ( c ) circular head ( c ) and deformed head ( d ) , ( d ) bent tail ( arrow ) , ( e ) coiled tail of sperm ( arrow ) and detached neck ( arrow ) effect of mett fruits on sperm motility , viability , abnormality and count in svp - intoxicated rats ( n=10 ) data are shown in table 4 revealed that intoxication of rats by svp induced significant decreases in activities of testicular anti - oxidant enzymes sod , gpx and cat compared with normal control rats . the pre - treatment with mett with the three tested doses significantly increased the activity of testicular sod , gpx and cat , in a dose dependent fashion , when compared with positive intoxicated rats . effect of mett on activity of testicular sod , gpx and cat in svp - intoxicated rats ( n=10 ) histopathological examination of testes of normal control rats revealed normal architecture with normal germinal epithelium and fully mature sperms filled the lumen of seminiferous tubules as shown in figure 2a . the testes of rats orally given svp in a dose of 500 mg / kg during the last week of the experiment period showed histopathological lesions characterized by edema and necrosis of the germinal epithelium with severe atrophy of seminiferous tubules [ figure 2b ] . some testicular sections showed complete absence of mature sperms ( azoospermia ) as demonstrated in figure 2c . the testes of rats given the large dose ( 10 mg / kg ) of mett showed partial improvement in the germinal epithelium of seminiferous tubules , and some mature sperms appeared in them , but the size of seminiferous tubules still atrophied [ figure 2d ] . photomicrographs of testes of : ( a ) normal control rat showing normal histological structure of seminiferous tubules filled with mature sperms . ( h and e 400 ) , ( b ) intoxicated rat by sodium valproate ( svp ) showing atrophied seminiferous tubules and edema with absence of sperms ( arrows ) . ( h and e 100 ) , ( c ) intoxicated rat by svp showing necrosis of the germinal epithelium of seminiferous tubules ( black arrows ) with absence of sperms ( red arrow ) ( h and e 400 ) , ( d ) pre - treated rat by the large dose ( 10 mg / kg ) of tribulus terrestris fruits extract showing partial improvement of the germinal epithelium of seminiferous tubules and mature sperm were found in most of seminiferous tubules , but their sizes still atrophied . histopathological examination of testes of normal control rats revealed normal architecture with normal germinal epithelium and fully mature sperms filled the lumen of seminiferous tubules as shown in figure 2a . the testes of rats orally given svp in a dose of 500 mg / kg during the last week of the experiment period showed histopathological lesions characterized by edema and necrosis of the germinal epithelium with severe atrophy of seminiferous tubules [ figure 2b ] . some testicular sections showed complete absence of mature sperms ( azoospermia ) as demonstrated in figure 2c . the testes of rats given the large dose ( 10 mg / kg ) of mett showed partial improvement in the germinal epithelium of seminiferous tubules , and some mature sperms appeared in them , but the size of seminiferous tubules still atrophied [ figure 2d ] . photomicrographs of testes of : ( a ) normal control rat showing normal histological structure of seminiferous tubules filled with mature sperms . ( h and e 400 ) , ( b ) intoxicated rat by sodium valproate ( svp ) showing atrophied seminiferous tubules and edema with absence of sperms ( arrows ) . ( h and e 100 ) , ( c ) intoxicated rat by svp showing necrosis of the germinal epithelium of seminiferous tubules ( black arrows ) with absence of sperms ( red arrow ) ( h and e 400 ) , ( d ) pre - treated rat by the large dose ( 10 mg / kg ) of tribulus terrestris fruits extract showing partial improvement of the germinal epithelium of seminiferous tubules and mature sperm were found in most of seminiferous tubules , but their sizes still atrophied . the purposes of this study were to evaluate the protective and anti - oxidant activities of the mett against testicular toxicity induced by svp in rats , and to examine its possible protective mechanisms . the toxic effect of spv characterized by decreased weights of the testes and seminal vesicles , low serum testosterone , fsh and lh levels , low semen quantity and quality , as well as incidence of testicular edema and necrosis with markedly atrophied seminiferous tubules . these authors found that the weights of testes and epididymis as well as sperm count and viability were all decreased following spv administration to rats and mice . in addition , serum levels of testosterone were dropped , and degeneration , edema necrosis and atrophy of most seminiferous tubules were seen upon histopathological examination of the testis . the previous authors attributed the toxic effect of spv due to its direct cytotoxic effect on the testis and/or indirectly via decreasing serum testosterone level . moreover , svp is commonly used to induce male reproductive toxicity in rats and mice , and its toxic effect was found to a dose - dependent and of a reversible manner . in this study , the oral pre - treatment with the mett at three dosage levels in svp - intoxicated rats produced a protective effect against testicular toxicity . this protective effect characterized by increased weights of testes and seminal vesicles , improved semen quality and quantity , increased serum testosterone , fsh and lh levels as well as partial amelioration of testicular histopathological lesions seen . these findings were partially in agreement with those previously reported [ 8 - 11,14,29,30 ] . the previous authors concluded that t. terrestris plant acts as a powerful aphrodisiac in rats , mice and humans ; improves semen quality and quantity and increases weights of the testis and epididymis . the reported increase in serum testosterone levels following administration of mett in this study was previously recorded by el - tantawy et al . using both chloroformic and ethanolic extracts of tribulus alatus fruits in rats . however , a limited number of animal studies displayed a significant increase in serum testosterone levels after administration of mett , but this effect was only noted in humans . moreover , qureshi et al . concluded that the release of nitric oxide after administration of t. terrestris extract to rats may offer a possible explanation for its protective activity on male reproductive dysfunction , independent of the serum testosterone level . the authors concluded that t. terrestris plant is successfully used in the management of sexual dysfunction , including erectile dysfunction in patients . in addition , the reported anti - oxidant activity of mett in this study was evident from the enhancement of activities of testicular anti - oxidant enzymes . the anti - oxidant effect of t. terrestris extract was similar to that previously demonstrated . the anti - oxidant activity of t. terrestris fruits was attributed to the presence of active derivatives of 4,5-di - p - coumaroylquinic acid , which were isolated from the fruits and reported to have potent anti - oxidant activity . the mechanism(s ) underlying the protective effect of mett against testicular toxicity induced by svp in rats could be attributed to the increased release of testosterone , fsh and lh serum levels . it was previously reported that abnormalities in the synthesis and release of androgens or their depletion by medical or surgical castration may suppress libido and decline erectile and ejaculatory functions . the other possible mechanism of the protective effect of mett could be due to its potent anti - oxidant property , so reducing oxidative stress in the testis and improving reproductive function . the anti - oxidant activity of mett was previously attributed to the presence of 4,5-di - p - coumaroylquinic acid that previously isolated from t. terrestris fruits and proved to exhibit a potent anti - oxidant effect . the mett has protective and anti - oxidant effects against svp - induced testicular toxicity . the protective effect of mett against svp toxicity might be due to the increased release of testosterone , fsh and lh and the enhancement of activity anti - oxidant enzymes in testicular tissue by mett . these results affirm the traditional use of t. terrestris fruits as a potent aphrodisiac for treating male sexual impotency and erectile dysfunction in patients . therefore , t. terrestris fruits may be beneficial for male patients suffering from infertility due to oxidative stress . moreover , isolation of bioactive constituents of t. terrestris fruits is necessary to search for safe , natural anti - oxidant agents to be developed for the prevention of infertility in males .

aims : this study was carried out to assess the protective and anti - oxidant activities of the methanolic extract of tribulus terrestris fruits ( mett ) against sodium valproate ( svp)-induced testicular toxicity in rats.materials and methods : fifty mature male rats were randomly divided into five equal groups ( n = 10 ) . group 1 was used normal ( negative ) control , and the other four groups were intoxicated with svp ( 500 mg / kg1 , orally ) during the last week of the experiment . group 2 was kept intoxicated ( positive ) control , and groups 3 , 4 and 5 were orally pre - treated with mett in daily doses 2.5 , 5.0 , and 10.0 mg / kg1 for 60 days , respectively . weights of sexual organs , serum testosterone , follicle stimulating hormone ( fsh ) and luteinizing hormone ( lh ) levels , semen picture , testicular anti - oxidant capacity and histopathology of testes were the parameters used in this study.results:oral pre - treatment with mett significantly increased weights of testes and seminal vesicles ; serum testosterone , fsh and lh levels and sperm motility , count and viability in svp - intoxicated rats . mett enhanced the activity of testicular anti - oxidant enzymes and partially alleviated degenerative changes induced by svp in testes.conclusion:the pre - treatment with mett has protective and anti - oxidant effects in svp - intoxicated rats . mechanisms of this protective effect against testicular toxicity may be due to the increased release of testosterone , fsh and lh and the enhanced tissue anti - oxidant capacity . these results affirm the traditional use of t. terrestris fruits as an aphrodisiac for treating male sexual impotency and erectile dysfunction in patients . the study recommends that t. terrestris fruits may be beneficial for male patients suffering from infertility .

INTRODUCTION MATERIALS AND METHODS Plant Material Extraction of Plant Material Sodium Valproate (Depakin Rats and Husbandry Experimental Design Hormone Assay Semen Analysis Anti-oxidant Enzymes Assay Histological Procedure Statistical Analysis RESULTS Histopathological Examination DISSCUSION CONCLUSION

infertility is one of the major health problems in life , and approximately about 30% of this problem is due to male factors . on the other side , intake of natural anti - oxidant agents from plants together with vitamins e and c can protect sperm dna from oxidative stress in the testis of rats , and antagonize testicular toxicity in rabbits . medicinal plants are a promising source for safe , natural anti - oxidant agents as they contain many bioactive constituents , especially anti - oxidant flavonoids and polyphenol compounds . tribulus terrestris plant , popularly known as puncture vine , is a perennial creeping herb with a worldwide distribution . since ancient times , t. terrestris has a long history as a powerful aphrodisiac . in traditional medicine , moreover , t. terrestris roots extract and total saponins extracted from the roots have beneficial effects on various ailments such as urinary tract infections , inflammations , leucorrhea , edema , and ascites . t. terrestris fruits extract has been successfully used in europe and asia to treat sexual dysfunction in males . the different pharmacological effects of t. terrestris plant were reviewed by chhatre et al . the present study was designed to evaluate the protective and anti - oxidant activities of the methanolic extract of t. terrestris fruits ( mett ) against testicular toxicity and oxidative stress induced sodium valproate ( svp ) in rats , and to examine the possible mechanisms . the fruits of t. terrestris ( family zygophyllaceae ) plant were obtained from the company of medicinal herbs , seeds , agricultural products , cairo , egypt . the liquid extract yielded 10 g of gummy residue that used for preparing the different doses of the extract using tween 80 as a suspending agent . it was obtained as an oral solution packed in dark brown bottles each containing 40 ml . a total of 50 mature male sprague dawley rats with average body weight of 200 - 250 g and age of 10 - 13 weeks were used in this study . rats were procured from laboratory animal colony , ministry of public health , helwan , egypt . rats were fed on rat pellets , and free access of tap water was supplied . the experiment on animals was carried out according to the national regulations on animal welfare and institutional animal ethical committee . group 1 was normal ( negative ) control and administered diluted tween 80 ( 0.1 ml / rat ) . the other four groups were intoxicated by oral administration of svp ( 500 mg / kg ) during the last week of the experiment period for induction of testicular toxicity . group 2 was kept intoxicated positive ( control ) and groups 3 , 4 , and 5 were pre - treated with the mett in daily doses of 2.5 , 5.0 and 10.0 mg / kg , respectively . the extract was orally given once daily for 60 days to cover the period of the spermatogenic cycle in the rat . blood samples were withdrawn by puncture of retro - orbital plexus of veins in the eye using microcapillary tubes . the serum was used for estimation of testosterone , follicle stimulating hormone ( fsh ) and luteinizing hormone ( lh ) levels . rats were anesthetized by prolonged exposure to ether , and a longitudinal incision was made in the skin of scrotum and both testes were exposed . the testes and accessory sexual organs were dissected out and weighed , and relative weights of sexual organs were calculated . the right testes were immediately taken on ice cooled bags and kept frozen at 70c until the assessment of the activity anti - oxidant enzymes superoxide dismutase ( sod ) , glutathione peroxidase ( gpx ) and catalase ( cat ) . the left testes were preserved in 10% neutral formalin solution till processed for histological examination . serum testosterone concentration was assayed according to the method described in the manufacturer s directions . serum testosterone concentrations were obtained by correlating the absorbance of the test sample at 550 nm with the corresponding absorbance on the standard curve . serum levels of fsh and lh hormones were determined using elisa kits ( amersham , buckinghamshire , uk ) according to ballester et al . this content was diluted 10 times with 2.9% sodium citrate solution and thoroughly mixed to estimate the percentage of sperm progressive motility and sperm count as described by bearden and fluquary . the stained seminal smears were examined microscopically to determine the percentage of sperm viability ( alive / dead ) and morphological abnormalities . tissue specimens of the right testes after thawing was homogenized in nine volumes of ice cooled buffered 0.9% saline solution . the homogenate was then centrifuged at 4000 rpm for 15 min at 4c and the supernatant was used for anti - oxidant enzymes assay . the activity of sod was determined as described by nishikimi et al . the activity of cat was expressed as nmol of h2o2 utilized / min / mg protein . differences between means in the experimental groups were tested for significance using student s t - test . the fruits of t. terrestris ( family zygophyllaceae ) plant were obtained from the company of medicinal herbs , seeds , agricultural products , cairo , egypt . the liquid extract yielded 10 g of gummy residue that used for preparing the different doses of the extract using tween 80 as a suspending agent . it was obtained as an oral solution packed in dark brown bottles each containing 40 ml . a total of 50 mature male sprague dawley rats with average body weight of 200 - 250 g and age of 10 - 13 weeks were used in this study . rats were procured from laboratory animal colony , ministry of public health , helwan , egypt . the animals were housed in clean cages , kept under controlled hygienic conditions and maintained at room temperature at 25c 2c , relative humidity of 50% 5% and photoperiod of 12 h dark/12 h light cycles . rats were fed on rat pellets , and free access of tap water was supplied . the experiment on animals was carried out according to the national regulations on animal welfare and institutional animal ethical committee . group 1 was normal ( negative ) control and administered diluted tween 80 ( 0.1 ml / rat ) . the other four groups were intoxicated by oral administration of svp ( 500 mg / kg ) during the last week of the experiment period for induction of testicular toxicity . group 2 was kept intoxicated positive ( control ) and groups 3 , 4 , and 5 were pre - treated with the mett in daily doses of 2.5 , 5.0 and 10.0 mg / kg , respectively . the extract was orally given once daily for 60 days to cover the period of the spermatogenic cycle in the rat . the serum was used for estimation of testosterone , follicle stimulating hormone ( fsh ) and luteinizing hormone ( lh ) levels . rats were anesthetized by prolonged exposure to ether , and a longitudinal incision was made in the skin of scrotum and both testes were exposed . the semen samples were used for semen analysis . the testes and accessory sexual organs were dissected out and weighed , and relative weights of sexual organs were calculated . the right testes were immediately taken on ice cooled bags and kept frozen at 70c until the assessment of the activity anti - oxidant enzymes superoxide dismutase ( sod ) , glutathione peroxidase ( gpx ) and catalase ( cat ) . the left testes were preserved in 10% neutral formalin solution till processed for histological examination . serum testosterone concentration was assayed according to the method described in the manufacturer s directions . serum testosterone concentrations were obtained by correlating the absorbance of the test sample at 550 nm with the corresponding absorbance on the standard curve . serum levels of fsh and lh hormones were determined using elisa kits ( amersham , buckinghamshire , uk ) according to ballester et al . the seminal content of epididymis was obtained by cutting of cauda epididymis using surgical blades and squeezed into a sterile clean watch glass . this content was diluted 10 times with 2.9% sodium citrate solution and thoroughly mixed to estimate the percentage of sperm progressive motility and sperm count as described by bearden and fluquary . the stained seminal smears were examined microscopically to determine the percentage of sperm viability ( alive / dead ) and morphological abnormalities . tissue specimens of the right testes after thawing was homogenized in nine volumes of ice cooled buffered 0.9% saline solution . the homogenate was then centrifuged at 4000 rpm for 15 min at 4c and the supernatant was used for anti - oxidant enzymes assay . the activity of sod was determined as described by nishikimi et al . the activity of cat was expressed as nmol of h2o2 utilized / min / mg protein . differences between means in the experimental groups were tested for significance using student s t - test . oral administration of svp to male rats in a dose of 500 mg / kg during the last week of the experiment induced significant ( p < 0.05 ) decreases in weights of testes and seminal vesicles when compared to the normal control group . rats pre - treated with oral administration of mett significantly normalized weights of testes and seminal vesicles , in a dose - dependent manner , when compared to svp - intoxicated control group [ table 1 ] . effect of methanolic extract of tribulus terrestris fruits on weights of sexual organs in sodium valproate - intoxicated rats ( n=10 ) oral administration of svp ( 500 mg / kg ) to rats during the last week of the experimental period significantly decreased serum testosterone , fsh and lh levels when compared with the normal control group . pre - treatments of svp - intoxicated rats with mett significantly increased serum testosterone , fsh and lh levels when compared with the intoxicated control group , in a dose dependent fashion [ table 2 ] . effect of mett on serum testosterone , fsh and lh levels in svp - intoxicated rats ( n=10 ) svp when given orally to male rats ( 500 mg / kg ) during the last week of the experiment induced significant decreases in sperm motility , viability and count and increased percentages of sperm abnormalities when compared with the normal control group . pre - treatment with mett significantly increased sperm motility , viability and count and decreased percentages of sperm abnormalities , in a dose dependent manner , as recorded in table 3 . seminal smears from cauda epididymis of the rat s testes : ( a ) normal mature sperm ( n ) and detached head of sperm ( arrow ) , ( b ) double head of sperm ( arrow ) , ( c ) circular head ( c ) and deformed head ( d ) , ( d ) bent tail ( arrow ) , ( e ) coiled tail of sperm ( arrow ) and detached neck ( arrow ) effect of mett fruits on sperm motility , viability , abnormality and count in svp - intoxicated rats ( n=10 ) data are shown in table 4 revealed that intoxication of rats by svp induced significant decreases in activities of testicular anti - oxidant enzymes sod , gpx and cat compared with normal control rats . the pre - treatment with mett with the three tested doses significantly increased the activity of testicular sod , gpx and cat , in a dose dependent fashion , when compared with positive intoxicated rats . effect of mett on activity of testicular sod , gpx and cat in svp - intoxicated rats ( n=10 ) histopathological examination of testes of normal control rats revealed normal architecture with normal germinal epithelium and fully mature sperms filled the lumen of seminiferous tubules as shown in figure 2a . the testes of rats orally given svp in a dose of 500 mg / kg during the last week of the experiment period showed histopathological lesions characterized by edema and necrosis of the germinal epithelium with severe atrophy of seminiferous tubules [ figure 2b ] . the testes of rats given the large dose ( 10 mg / kg ) of mett showed partial improvement in the germinal epithelium of seminiferous tubules , and some mature sperms appeared in them , but the size of seminiferous tubules still atrophied [ figure 2d ] . photomicrographs of testes of : ( a ) normal control rat showing normal histological structure of seminiferous tubules filled with mature sperms . ( h and e 400 ) , ( b ) intoxicated rat by sodium valproate ( svp ) showing atrophied seminiferous tubules and edema with absence of sperms ( arrows ) . ( h and e 100 ) , ( c ) intoxicated rat by svp showing necrosis of the germinal epithelium of seminiferous tubules ( black arrows ) with absence of sperms ( red arrow ) ( h and e 400 ) , ( d ) pre - treated rat by the large dose ( 10 mg / kg ) of tribulus terrestris fruits extract showing partial improvement of the germinal epithelium of seminiferous tubules and mature sperm were found in most of seminiferous tubules , but their sizes still atrophied . histopathological examination of testes of normal control rats revealed normal architecture with normal germinal epithelium and fully mature sperms filled the lumen of seminiferous tubules as shown in figure 2a . the testes of rats orally given svp in a dose of 500 mg / kg during the last week of the experiment period showed histopathological lesions characterized by edema and necrosis of the germinal epithelium with severe atrophy of seminiferous tubules [ figure 2b ] . the testes of rats given the large dose ( 10 mg / kg ) of mett showed partial improvement in the germinal epithelium of seminiferous tubules , and some mature sperms appeared in them , but the size of seminiferous tubules still atrophied [ figure 2d ] . photomicrographs of testes of : ( a ) normal control rat showing normal histological structure of seminiferous tubules filled with mature sperms . ( h and e 400 ) , ( b ) intoxicated rat by sodium valproate ( svp ) showing atrophied seminiferous tubules and edema with absence of sperms ( arrows ) . ( h and e 100 ) , ( c ) intoxicated rat by svp showing necrosis of the germinal epithelium of seminiferous tubules ( black arrows ) with absence of sperms ( red arrow ) ( h and e 400 ) , ( d ) pre - treated rat by the large dose ( 10 mg / kg ) of tribulus terrestris fruits extract showing partial improvement of the germinal epithelium of seminiferous tubules and mature sperm were found in most of seminiferous tubules , but their sizes still atrophied . the purposes of this study were to evaluate the protective and anti - oxidant activities of the mett against testicular toxicity induced by svp in rats , and to examine its possible protective mechanisms . the toxic effect of spv characterized by decreased weights of the testes and seminal vesicles , low serum testosterone , fsh and lh levels , low semen quantity and quality , as well as incidence of testicular edema and necrosis with markedly atrophied seminiferous tubules . these authors found that the weights of testes and epididymis as well as sperm count and viability were all decreased following spv administration to rats and mice . in addition , serum levels of testosterone were dropped , and degeneration , edema necrosis and atrophy of most seminiferous tubules were seen upon histopathological examination of the testis . the previous authors attributed the toxic effect of spv due to its direct cytotoxic effect on the testis and/or indirectly via decreasing serum testosterone level . moreover , svp is commonly used to induce male reproductive toxicity in rats and mice , and its toxic effect was found to a dose - dependent and of a reversible manner . in this study , the oral pre - treatment with the mett at three dosage levels in svp - intoxicated rats produced a protective effect against testicular toxicity . this protective effect characterized by increased weights of testes and seminal vesicles , improved semen quality and quantity , increased serum testosterone , fsh and lh levels as well as partial amelioration of testicular histopathological lesions seen . the previous authors concluded that t. terrestris plant acts as a powerful aphrodisiac in rats , mice and humans ; improves semen quality and quantity and increases weights of the testis and epididymis . the reported increase in serum testosterone levels following administration of mett in this study was previously recorded by el - tantawy et al . using both chloroformic and ethanolic extracts of tribulus alatus fruits in rats . however , a limited number of animal studies displayed a significant increase in serum testosterone levels after administration of mett , but this effect was only noted in humans . concluded that the release of nitric oxide after administration of t. terrestris extract to rats may offer a possible explanation for its protective activity on male reproductive dysfunction , independent of the serum testosterone level . the authors concluded that t. terrestris plant is successfully used in the management of sexual dysfunction , including erectile dysfunction in patients . in addition , the reported anti - oxidant activity of mett in this study was evident from the enhancement of activities of testicular anti - oxidant enzymes . the anti - oxidant effect of t. terrestris extract was similar to that previously demonstrated . the anti - oxidant activity of t. terrestris fruits was attributed to the presence of active derivatives of 4,5-di - p - coumaroylquinic acid , which were isolated from the fruits and reported to have potent anti - oxidant activity . the mechanism(s ) underlying the protective effect of mett against testicular toxicity induced by svp in rats could be attributed to the increased release of testosterone , fsh and lh serum levels . it was previously reported that abnormalities in the synthesis and release of androgens or their depletion by medical or surgical castration may suppress libido and decline erectile and ejaculatory functions . the other possible mechanism of the protective effect of mett could be due to its potent anti - oxidant property , so reducing oxidative stress in the testis and improving reproductive function . the anti - oxidant activity of mett was previously attributed to the presence of 4,5-di - p - coumaroylquinic acid that previously isolated from t. terrestris fruits and proved to exhibit a potent anti - oxidant effect . the mett has protective and anti - oxidant effects against svp - induced testicular toxicity . the protective effect of mett against svp toxicity might be due to the increased release of testosterone , fsh and lh and the enhancement of activity anti - oxidant enzymes in testicular tissue by mett . these results affirm the traditional use of t. terrestris fruits as a potent aphrodisiac for treating male sexual impotency and erectile dysfunction in patients . therefore , t. terrestris fruits may be beneficial for male patients suffering from infertility due to oxidative stress . moreover , isolation of bioactive constituents of t. terrestris fruits is necessary to search for safe , natural anti - oxidant agents to be developed for the prevention of infertility in males .

neoadjuvant systemic therapy ( nst ) has been used in locally advanced breast cancer in order to convert a previously unresectable cancer into an operable one ( hortobagyi et al . more recently , it has been widely administered in primarily operable breast cancer to reduce tumor volume and allow conservative surgery ( fisher et al . the downstaging of the primary tumor and the increase in breast conservation rates seems to be the only clinical benefit of nst , given that several studies failed to demonstrate an improvement of overall survival compared with postoperative adjuvant chemotherapy ( fisher et al . breast cancer is a heterogeneous disease that varies widely in response to standard therapies and outcomes ( rouzier et al . 2005 ) . in this perspective , nst represents an opportunity to determine the intrinsic resistance / sensitiveness of breast cancer to chemotherapy . moreover , the extent of residual disease in the breast and axillary surgical specimens after nst , classified according to the revised 2003 american joint committee on cancer ( ajcc ) tumor - node - metastasis ( tnm ) staging system , has been reported to be associated with relapse and survival ( carey et al . 2005 ) . pathologic complete response ( pcr ) achieved after nst , when defined as non - invasive and non - in situ cancer in breast and nodes , is predictive of good prognosis and might be used as surrogate of survival ( kuerer et al . however , patients with hormone receptors ( hr)-positive tumors usually have low rates of pcr and maintain a good long - term outcome even in the presence of residual disease ( no pcr ) ( colleoni et al . 2009 ; huober et al . 2010 ; precht et al . 2010 ; straver et al . , pcr fails to predict survival , emphasizing the importance of tumor biology rather than response to neoadjuvant chemotherapy as a prognostic marker in some subtypes of breast cancers ( von minckwitz et al . the aim of this study is to evaluate the long - term outcome in a series of patients with locally advanced breast cancer consecutively treated with nst in our institution . all patients came from the routine clinical practice and were not included in clinical trials . the charts of all patients with locally advanced breast cancer , performance status 02 ( ecog scale ) , consecutively treated with nst at the medical oncology division , university of chieti hospital between january 1999 and december 2011 , were reviewed for this retrospective study . diagnosis of invasive breast cancer was established by tru - cut biopsy of the primary tumor . all patients received preoperative chemotherapy and those with hr - positive tumor received adjuvant hormonal therapy for 5 years . chemotherapy regimens administered included : cmf ( fluorouracil , methotrexate and cyclophosphamide ) ; single - agent epirubicin ; ec ( epirubicin and cyclophosphamide ) ; fec ( fluorouracil , epirubicin and cyclophosphamide ) ; e - cmf ( single - agent epirubicin followed by cmf ) ; single - agent taxanes ; et ( epirubicin and taxol ) ; ec - t ( ec followed by docetaxel ) ; ec - taxel ( ec followed by docetaxel and capecitabine ) and other combinations including platinum compounds , vinorelbine and pegylated doxorubicin . ninety - nine patients received adjuvant tamoxifen , 125 postmenopausal patients received aromatase inhibitors ( anastrozole or letrozole ) and 71 patients tamoxifen followed by exemestane . ninety - four patients treated after 2005 and carrying her2-positive tumors received trastuzumab simultaneously with neoadjuvant chemotherapy and/or postoperatively to complete 1 year of treatment . sentinel node biopsy after nst was performed in 54 patients ; axillary lymph node dissection was performed in 371 ( 90.1 % ) patients , including 16 having positive sentinel nodes . adjuvant breast radiotherapy was delivered to patients who underwent bcs as well as to patients who underwent mastectomy but had initial stage ct3 , cn2 or cn3 disease . estrogen ( er)/progesterone receptors ( pr ) and human epidermal growth factor type 2 receptor ( her2 ) were determined on pretreatment biopsy and on surgical specimens by immunohistochemistry . hr status was considered positive if 10 % of tumor cells stained for er and/or pr . samples were scored as follows : score 0 , membrane staining in 10 % of tumor cells ; score 1 + , partial and/or faint membrane staining in > 10 % of tumor cells ; score 2 + , weak to moderate , complete membrane staining in > 10 % tumor cells and score 3 + , strong , complete membrane staining in > 10 % of tumor cells . fish or cish was carried out on all tumors with herceptest 2 + . tumors with a score of 3 + by immunohistochemistry ( ihc ) or gene amplification by fish were considered as her2 positive . nuclear grade was assessed according to the nottingham grading system ( elston and ellis 1991 ) . we could not exactly define breast cancer intrinsic subtypes with immunohistochemistry in all tumors ( goldhirsch et al . 2011 ) , since ki-67 assessment was not available in 172 ( 42.0 % ) samples . so we classified tumors as follows : ( 1 ) luminal a and luminal b / her2 negative ; ( 2 ) luminal b / her2 positive ; ( 3 ) her2 enriched ; ( 4 ) triple negative ( houssami et al . 2012 ) . pcr was defined as non - invasive cancer within the breast ( ypt0/is ) and lymph node ( ypn0 ) , also classified as stage 0 ( kuerer et al . pathological stages were categorized according to the american joint committee on cancer staging manual , 7th ed . locoregional recurrence ( lrr ) was defined as any chest wall recurrence in those who underwent mastectomy , any ipsilateral in - breast recurrence in those achieving breast conservation and any recurrence in the axillary , supraclavicular or internal mammary nodes . medical records for all patients were reviewed retrospectively and the cut off date for follow - up set on december 31 , 2011 . clinical and pathological characteristics for each patient were entered on an anonymized database . since patients enrollment began in 1999 , complete information was not available for all 409 patients ; thus , denominators may vary throughout the article . the follow - up contacts were carried out at 6-month intervals over the first 5 years , and at 12-month intervals thereafter . the primary endpoint of this study was overall survival ( os ) , defined as the interval between the time of surgery and the date of death from any cause or censoring . the secondary endpoints were rate of pcr and distant relapse free survival ( drfs ) , defined as the time from breast surgery to the first occurrence of distant metastasis or intercurrent deaths without distant recurrence . a two - sided level of significance of 0.05 was applied to all statistical tests . in univariate analysis , the relationships between patients / tumor characteristics and pcr were assessed by pearson s or fisher s exact test , as appropriate . a stepwise multivariate logistic regression was used to identify independent predictors of pcr among baseline patients / tumor characteristics . survival curves were derived from kaplan meier estimates and compared by log - rank test and hazard ratio ( hr ) ( massarweh et al . a multivariate cox proportional hazard model was carried out to assess the relative influence of prognostic factors on survival ( de placido et al . all statistical analyses were performed using the spss statistic software version 19 ( ibm , armonk , new york ) . the charts of all patients with locally advanced breast cancer , performance status 02 ( ecog scale ) , consecutively treated with nst at the medical oncology division , university of chieti hospital between january 1999 and december 2011 , were reviewed for this retrospective study . diagnosis of invasive breast cancer was established by tru - cut biopsy of the primary tumor . all patients received preoperative chemotherapy and those with hr - positive tumor received adjuvant hormonal therapy for 5 years . chemotherapy regimens administered included : cmf ( fluorouracil , methotrexate and cyclophosphamide ) ; single - agent epirubicin ; ec ( epirubicin and cyclophosphamide ) ; fec ( fluorouracil , epirubicin and cyclophosphamide ) ; e - cmf ( single - agent epirubicin followed by cmf ) ; single - agent taxanes ; et ( epirubicin and taxol ) ; ec - t ( ec followed by docetaxel ) ; ec - taxel ( ec followed by docetaxel and capecitabine ) and other combinations including platinum compounds , vinorelbine and pegylated doxorubicin . ninety - nine patients received adjuvant tamoxifen , 125 postmenopausal patients received aromatase inhibitors ( anastrozole or letrozole ) and 71 patients tamoxifen followed by exemestane . ninety - four patients treated after 2005 and carrying her2-positive tumors received trastuzumab simultaneously with neoadjuvant chemotherapy and/or postoperatively to complete 1 year of treatment . sentinel node biopsy after nst was performed in 54 patients ; axillary lymph node dissection was performed in 371 ( 90.1 % ) patients , including 16 having positive sentinel nodes . adjuvant breast radiotherapy was delivered to patients who underwent bcs as well as to patients who underwent mastectomy but had initial stage ct3 , cn2 or cn3 disease . estrogen ( er)/progesterone receptors ( pr ) and human epidermal growth factor type 2 receptor ( her2 ) were determined on pretreatment biopsy and on surgical specimens by immunohistochemistry . hr status was considered positive if 10 % of tumor cells stained for er and/or pr . samples were scored as follows : score 0 , membrane staining in 10 % of tumor cells ; score 1 + , partial and/or faint membrane staining in > 10 % of tumor cells ; score 2 + , weak to moderate , complete membrane staining in > 10 % tumor cells and score 3 + , strong , complete membrane staining in > 10 % of tumor cells . fish or cish was carried out on all tumors with herceptest 2 + . tumors with a score of 3 + by immunohistochemistry ( ihc ) or gene amplification by fish were considered as her2 positive . nuclear grade was assessed according to the nottingham grading system ( elston and ellis 1991 ) . we could not exactly define breast cancer intrinsic subtypes with immunohistochemistry in all tumors ( goldhirsch et al . 2011 ) , since ki-67 assessment was not available in 172 ( 42.0 % ) samples . so we classified tumors as follows : ( 1 ) luminal a and luminal b / her2 negative ; ( 2 ) luminal b / her2 positive ; ( 3 ) her2 enriched ; ( 4 ) triple negative ( houssami et al . 2012 ) . pcr was defined as non - invasive cancer within the breast ( ypt0/is ) and lymph node ( ypn0 ) , also classified as stage 0 ( kuerer et al . pathological stages were categorized according to the american joint committee on cancer staging manual , 7th ed . locoregional recurrence ( lrr ) was defined as any chest wall recurrence in those who underwent mastectomy , any ipsilateral in - breast recurrence in those achieving breast conservation and any recurrence in the axillary , supraclavicular or internal mammary nodes . medical records for all patients were reviewed retrospectively and the cut off date for follow - up set on december 31 , 2011 . clinical and pathological characteristics for each patient were entered on an anonymized database . since patients enrollment began in 1999 , complete information was not available for all 409 patients ; thus , denominators may vary throughout the article . the follow - up contacts were carried out at 6-month intervals over the first 5 years , and at 12-month intervals thereafter . the primary endpoint of this study was overall survival ( os ) , defined as the interval between the time of surgery and the date of death from any cause or censoring . the secondary endpoints were rate of pcr and distant relapse free survival ( drfs ) , defined as the time from breast surgery to the first occurrence of distant metastasis or intercurrent deaths without distant recurrence . a two - sided level of significance of 0.05 was applied to all statistical tests . in univariate analysis , the relationships between patients / tumor characteristics and pcr were assessed by pearson s or fisher s exact test , as appropriate . a stepwise multivariate logistic regression was used to identify independent predictors of pcr among baseline patients / tumor characteristics . survival curves were derived from kaplan meier estimates and compared by log - rank test and hazard ratio ( hr ) ( massarweh et al . a multivariate cox proportional hazard model was carried out to assess the relative influence of prognostic factors on survival ( de placido et al . all statistical analyses were performed using the spss statistic software version 19 ( ibm , armonk , new york ) . the median age was 48.8 years ( range 2580 ) , with 20 ( 4.9 % ) patients being younger than 35 years and 15 ( 3.7 % ) older than 70 years . clinical tumor size was 3 cm in 175 ( 42.8 % ) patients ; 346 ( 84.6 % ) patients had ductal carcinoma . tumor grade was 12 in 289 ( 70.7 % ) and grade 3 in 87 ( 21.2 % ) patients . ki-67 was available in 237 ( 58 % ) cases and was > 14 % in 135 ( 56.9 % ) . tumors were classified in four molecular subtypes according to the tumor staining for er / pr and her2 status : 211 ( 51.9 % ) were her2 negative , luminal a or luminal b ( er and/or pr positive ) ; 84 ( 20.6 % ) were her2 positive , luminal b ( er and/or pr positive ) ; 53 ( 13.0 % ) were her2 enriched ( er and pr negative , her2 positive ) ; 59 ( 14.5 % ) were triple negative ( er and pr negatives , her2 negative ) . most patients , 237 ( 58.0 % ) , received chemotherapy based on anthracycline and taxanes . among 137 women with her2-positive tumor , 43 ( 31.4 % ) , diagnosed before 2005 , were not treated with trastuzumab , 29 ( 21.2 % ) received adjuvant trastuzumab and 65 ( 47.4 % ) received neoadjuvant and adjuvant trastuzumab . a total of 300 ( 73.3 % ) patients received more than four cycles of chemotherapy.table 1association of baseline factors and pcr in univariate analysisno . ( % ) p valueagemedian age 48.8 years ( range 2580 years ) 35 years20 ( 4.9)5 ( 25.0 ) > 35 years389 ( 95.1)56 ( 14.4)n.s.clinical t3 cm213 ( 52.1)30 ( 14.0)3 cm175 ( 42.8)30 ( 17.1)unknown21 ( 5.1)1 ( 4.8)n.s.histologic typeductal346 ( 84.6)51 ( 14.7)lobular57 ( 14.0)10 ( 17.5)others6 ( 1.4)0n.s.grade12289 ( 70.7)25 ( 8.6)387 ( 21.2)19 ( 21.8)unknown33 ( 8.1)17 ( 51.5)0.001ki-6714 % 102 ( 43.1)4 ( 3.9)>14 % 135 ( 56.9)33 ( 22.2)unknown172 ( 50.0)24 ( 14.0)0.000molecular subtypeluminal a & b / her2 negative211 ( 51.9)12 ( 5.7)luminal b / her2 positive84 ( 20.6)14 ( 16.6)her2 enriched53 ( 13.0)18 ( 33.9)triple negative59 ( 14.5)17 ( 28.8)unknown2 ( 0.04)0.000type of nstvarious107 ( 26.1)5 ( 4.5)*anthracycline and taxane237 ( 58.0)30 ( 12.6)*chemotherapy + trastuzumab65 ( 15.9)26 ( 40.0)0.000no . of chemotherapy cycles4109 ( 26.7)7 ( 6.4)>4300 ( 73.3)54 ( 18.0)0.004 * 10.2 % pcr in patients treated with chemotherapy onlyunknown were not included in univariate analysis association of baseline factors and pcr in univariate analysis * 10.2 % pcr in patients treated with chemotherapy only unknown were not included in univariate analysis in the univariate analysis , pcr was significantly associated with tumor grade , proliferative activity , molecular subtype , type of nst and number of chemotherapy cycles ( table 1 ) , patients with the worst prognostic factors having the best pcr rates . in the multivariate analysis , only hr - negative tumors , independently from her2 status ( her2 enriched : p = 0.043 ; triple negative : p = 0.002 ) and the use of neoadjuvant trastuzumab ( p = 0.035 ) were significantly associated with higher pcr rates ( table 2).table 2association of baseline factors and pcr in multivariate analysisodds ratio95 % cip valueage>35 yearsreference35 years2.0810.42610.175n.s.clinical t3 cmreference3 cm1.0400.4142.611n.s.histologic typeotherreferenceductal2.9650.3276.424n.s.lobular5.9950.21310.785n.s.grade12reference31.2750.4183.885n.s.ki-6714 % reference>14 % 3.6890.89915.132n.s.molecular subtypeluminal a and b / her2 negativereferenceluminal b / her2 positive1.5640.2529.716n.s.her2 enriched6.0901.06234.9210.043triple negative10.6462.30740.1250.002type of nstvarious2.290referencen.s.anthracycline and taxane11.3340.30017.4190.035chemotherapy + trastuzumab1.182108.719no . of chemotherapy cycles42.158referencen.s.>40.30815.143 association of baseline factors and pcr in multivariate analysis bcs was performed in 241 ( 58.9 % ) patients and mastectomy in the remaining 168 ( 41.1 % ) . absence of cancer in the breast ( ypt0 ) was found in 75 ( 18.3 % ) patients ; absence of cancer in lymph nodes ( ypn0 ) in 181 ( 44.2 % ) patients and a total of 61 ( 14.9 % ) patients had a pcr , that is , absence of invasive cancer both in breast and nodes . most patients received adjuvant treatments : 125 ( 30.6 % ) patients had only hormonal therapy , 54 ( 13.2 % ) only chemotherapy , 105 ( 25.6 % ) chemotherapy followed by hormonal therapy and 94 ( 23.0 % ) received adjuvant trastuzumab either alone ( 33 patients ) , with hormonal therapy ( 39 patients ) , with chemotherapy ( 11 patients ) or with chemotherapy and hormonal therapy ( 11 patients ) . adjuvant radiotherapy was delivered to 310 ( 75.8 % ) patients , including 101 patients who underwent mastectomy.table 3clinical characteristics of patients after nst therapyno . ( % ) type of surgerybcs241 ( 58.9)mastectomy168 ( 41.1)residual tumor sizeypt075 ( 18.3)ypt1180 ( 44.0)ypt2116 ( 28.3)ypt338 ( 9.2)no . of metastatic nodesnone181 ( 44.2)13109 ( 26.6)4970 ( 17.1)1049 ( 11.9)posttherapy stage061 ( 14.9)i92 ( 22.5)ii129 ( 31.5)iii127 ( 31.9)adjuvant treatmentnil31 ( 7.6)hormonal therapy125 ( 30.6)chemotherapy54 ( 13.2)chemotherapy followed by hormonal therapy105 ( 25.6)trastuzumab94 ( 23.0)radiotherapyyes310 ( 75.8)no99 ( 24.2)mastectomywith radiation101 ( 60.1)without radiation67 ( 39.8 ) clinical characteristics of patients after nst therapy median follow - up was 42.1 months ( range 0.8147.3 months ) . during follow - up , 25 ( 6.1 % ) patients had local relapse , 84 ( 20.5 % ) had distant metastases and 53 ( 13.0 % ) died . we evaluated patients outcome in relation to different variables such as pcr , stage at surgery , tumor molecular subtype , use of trastuzumab for her2-positive tumors , type of surgery and breast radiotherapy . the occurrence of local relapse was not correlated with stage of disease at surgery , type of surgery and radiation therapy , while it was more frequent among patients with hr - negative tumors ( p = 0.007 by pearson s ) . at kaplan meier analysis of the whole population , pcr was not found to be a prognostic factor for drfs and os ( not shown ) . however , excluding from the analyses patients with luminal a or luminal b / her2-negative tumors ( a group of patients with favorable outcome , representing 51.9 % of the entire study population ) , pcr resulted predictive of better drfs ( p = 0.028 : hr = 0.37 , 95 % ci = 0.190.72 ) with a trend toward significance for os ( p = 0.06 ; hr = 0.34 , 95 % ci = 0.160.77 ) ( fig . patients with higher stage of disease after nst were more likely to develop distant metastasis and die from breast cancer ( fig . patients who achieved pcr ( stage 0 ) had drfs and os rates of 87.1 % ( 95 % ci : 77.396.9 % ) and 92.0 % ( 95 % ci : 84.899.2 % ) , respectively , similar to those of patients with surgical stage i ( drfs : 85.9 % ; 95 % ci : 77.194.7 % and os : 80.4 % ; 95 % ci : 66.993.9 % ) . stage ii patients had drfs rates of 61.3 % ( 95 % ci : 39.782.9 % ) and os rates of 76.8 % ( 95 % ci : 55.298.4 % ) , while stage iii patients had drfs rates of 48.5 % ( 95 % ci : 35.461.6 % ) and os rates of 44.6 % ( 95 % ci : 17.771.5 % ) .fig . 1a distant relapse free survival ( drfs ) and b overall survival ( os ) stratified by pathological complete response ( pcr ) for the whole population , excluding patients with hr - positive / her2-negative tumorsfig . 2a distant relapse free survival ( drfs ) and b overall survival ( os ) stratified by pathological stage after systemic neoadjuvant chemotherapy a distant relapse free survival ( drfs ) and b overall survival ( os ) stratified by pathological complete response ( pcr ) for the whole population , excluding patients with hr - positive / her2-negative tumors a distant relapse free survival ( drfs ) and b overall survival ( os ) stratified by pathological stage after systemic neoadjuvant chemotherapy significant differences in drfs ( p = 0.006 ) and os ( p = 0.006 ) were observed among patients with different tumor molecular subtypes ( fig . 3a , b ) , the group of patients with luminal a or luminal b / her2-negative tumors showing a better prognosis . moreover , patients with hr - positive tumors had a significant longer drfs ( p < 0.005 : hr = 0.54 , 95 % ci = 0.350.85 ) and os ( p < 0.0001 : hr = 0.34 , 95 % ci = 0.190.63 ) compared with patients with hr - negative tumors ( fig . when survival analysis was stratified according to her2 status , the drfs and os advantage for hr - positive tumors was limited to the her2-negative population ( p = 0.016 : hr = 0.50 , 95 % ci = 0.250.97 and p < 0.0001 : hr = 0.25 , 95 % ci = 0.110.60 , respectively ) ( fig . 3e , f ) , while among her2-positive group , hr positivity was predictive of a longer drfs ( p = 0.044 : hr = 0.50 , 95 % ci = 0.241.0 ) , but not os ( not shown ) . in our population , neither her2 status nor the use of trastuzumab in the her2-positive patients was statistically associated with clinical outcome ( not shown).fig . 3a , c , e distant relapse free survival ( drfs ) and b , d , f overall survival ( os ) stratified by molecular subtypes for the whole population ( a , b ) , by hormone receptor ( hr ) status ( hr+ and hr ) for the whole population ( c , d ) and by hr status for patients with her2-negative tumors ( e , f ) a , c , e distant relapse free survival ( drfs ) and b , d , f overall survival ( os ) stratified by molecular subtypes for the whole population ( a , b ) , by hormone receptor ( hr ) status ( hr+ and hr ) for the whole population ( c , d ) and by hr status for patients with her2-negative tumors ( e , f ) patients who underwent bcs were more likely to have a better drfs ( p < 0.0001 : hr = 0.36 , 95 % ci = 0.230.55 ) and os ( p = 0.0014 : hr = 0.42 , 95 % ci = 0.240.72 ) compared with those who required mastectomy ( fig . no differences in survival were observed in patients treated with or without radiotherapy after surgery ( not shown).fig . 4a distant relapse free survival ( drfs ) and b overall survival ( os ) stratified by type of surgery . bcs breast conservative surgery a distant relapse free survival ( drfs ) and b overall survival ( os ) stratified by type of surgery . bcs breast conservative surgery in multivariate analysis , the variables independently associated with shorter drfs and os were absence of hr expression ( drfs and os : p < 0.001 ) , mastectomy ( drfs : p = 0.009 ; os : p = 0.05 ) and stage iii disease ( drfs : p < 0.001 ; os : p = 0.003 ) , whereas the molecular subtype luminal b / her2-positive tumors reached statistical significance for os ( p = 0.035 ) , but not for drfs ( tables 4 , 5).table 4multivariable proportional hazard regression model predicting drfsparameter estimatehazard ratio95 % cip valueage>35 yearsreference35 years0.1581.1710.4573.003n.s.histologic typelobularreferencen.s.ductal0.0651.0670.5682.006n.s.others0.1871.2060.2475.874molecular subtype prenstluminal a and b / her2 negativereferenceluminal b / her2 positive0.5911.8070.9703.3650.062her2 enriched1.6785.3542.74710.4350.000triple negative1.1413.1301.7035.7520.000type of surgerybcsreferencemastectomy0.6431.9031.1703.0930.009stage0referencei0.1851.2030.4193.454n.s.ii0.7512.1190.8255.443n.s.iii1.8106.1082.37615.7070.000table 5multivariable proportional hazard regression model predicting osparameter estimatehazard ratio95 % cip valueage>35 yearsreferencen.s.35 years0.1781.1950.4043.533histologic typelobularreferenceductal0.4241.5280.5903.955n.s.others0.5091.6640.17016.266n.s.molecular subtype prenstluminal a and b / her2 negativereferenceluminal b / her2 positive0.8462.3301.0625.1110.035her2 enriched1.7515.7612.43813.6100.000triple negative1.6965.4532.62011.3500.000type of surgerybcsreferencemastectomy0.6061.8341.1703.0930.056stage0referencei0.7262.0660.5477.808n.s.ii0.4661.5930.4096.205n.s.iii1.9006.6831.87723.8010.003 multivariable proportional hazard regression model predicting drfs multivariable proportional hazard regression model predicting os the median age was 48.8 years ( range 2580 ) , with 20 ( 4.9 % ) patients being younger than 35 years and 15 ( 3.7 % ) older than 70 years . clinical tumor size was 3 cm in 175 ( 42.8 % ) patients ; 346 ( 84.6 % ) patients had ductal carcinoma . tumor grade was 12 in 289 ( 70.7 % ) and grade 3 in 87 ( 21.2 % ) patients . ki-67 was available in 237 ( 58 % ) cases and was > 14 % in 135 ( 56.9 % ) . tumors were classified in four molecular subtypes according to the tumor staining for er / pr and her2 status : 211 ( 51.9 % ) were her2 negative , luminal a or luminal b ( er and/or pr positive ) ; 84 ( 20.6 % ) were her2 positive , luminal b ( er and/or pr positive ) ; 53 ( 13.0 % ) were her2 enriched ( er and pr negative , her2 positive ) ; 59 ( 14.5 % ) were triple negative ( er and pr negatives , her2 negative ) . most patients , 237 ( 58.0 % ) , received chemotherapy based on anthracycline and taxanes . among 137 women with her2-positive tumor , 43 ( 31.4 % ) , diagnosed before 2005 , were not treated with trastuzumab , 29 ( 21.2 % ) received adjuvant trastuzumab and 65 ( 47.4 % ) received neoadjuvant and adjuvant trastuzumab . a total of 300 ( 73.3 % ) patients received more than four cycles of chemotherapy.table 1association of baseline factors and pcr in univariate analysisno . ( % ) p valueagemedian age 48.8 years ( range 2580 years ) 35 years20 ( 4.9)5 ( 25.0 ) > 35 years389 ( 95.1)56 ( 14.4)n.s.clinical t3 cm213 ( 52.1)30 ( 14.0)3 cm175 ( 42.8)30 ( 17.1)unknown21 ( 5.1)1 ( 4.8)n.s.histologic typeductal346 ( 84.6)51 ( 14.7)lobular57 ( 14.0)10 ( 17.5)others6 ( 1.4)0n.s.grade12289 ( 70.7)25 ( 8.6)387 ( 21.2)19 ( 21.8)unknown33 ( 8.1)17 ( 51.5)0.001ki-6714 % 102 ( 43.1)4 ( 3.9)>14 % 135 ( 56.9)33 ( 22.2)unknown172 ( 50.0)24 ( 14.0)0.000molecular subtypeluminal a & b / her2 negative211 ( 51.9)12 ( 5.7)luminal b / her2 positive84 ( 20.6)14 ( 16.6)her2 enriched53 ( 13.0)18 ( 33.9)triple negative59 ( 14.5)17 ( 28.8)unknown2 ( 0.04)0.000type of nstvarious107 ( 26.1)5 ( 4.5)*anthracycline and taxane237 ( 58.0)30 ( 12.6)*chemotherapy + trastuzumab65 ( 15.9)26 ( 40.0)0.000no . of chemotherapy cycles4109 ( 26.7)7 ( 6.4)>4300 ( 73.3)54 ( 18.0)0.004 * 10.2 % pcr in patients treated with chemotherapy onlyunknown were not included in univariate analysis association of baseline factors and pcr in univariate analysis * 10.2 % pcr in patients treated with chemotherapy only unknown were not included in univariate analysis in the univariate analysis , pcr was significantly associated with tumor grade , proliferative activity , molecular subtype , type of nst and number of chemotherapy cycles ( table 1 ) , patients with the worst prognostic factors having the best pcr rates . in the multivariate analysis , only hr - negative tumors , independently from her2 status ( her2 enriched : p = 0.043 ; triple negative : p = 0.002 ) and the use of neoadjuvant trastuzumab ( p = 0.035 ) were significantly associated with higher pcr rates ( table 2).table 2association of baseline factors and pcr in multivariate analysisodds ratio95 % cip valueage>35 yearsreference35 years2.0810.42610.175n.s.clinical t3 cmreference3 cm1.0400.4142.611n.s.histologic typeotherreferenceductal2.9650.3276.424n.s.lobular5.9950.21310.785n.s.grade12reference31.2750.4183.885n.s.ki-6714 % reference>14 % 3.6890.89915.132n.s.molecular subtypeluminal a and b / her2 negativereferenceluminal b / her2 positive1.5640.2529.716n.s.her2 enriched6.0901.06234.9210.043triple negative10.6462.30740.1250.002type of nstvarious2.290referencen.s.anthracycline and taxane11.3340.30017.4190.035chemotherapy + trastuzumab1.182108.719no . of chemotherapy cycles42.158referencen.s.>40.30815.143 association of baseline factors and pcr in multivariate analysis bcs was performed in 241 ( 58.9 % ) patients and mastectomy in the remaining 168 ( 41.1 % ) . absence of cancer in the breast ( ypt0 ) was found in 75 ( 18.3 % ) patients ; absence of cancer in lymph nodes ( ypn0 ) in 181 ( 44.2 % ) patients and a total of 61 ( 14.9 % ) patients had a pcr , that is , absence of invasive cancer both in breast and nodes . most patients received adjuvant treatments : 125 ( 30.6 % ) patients had only hormonal therapy , 54 ( 13.2 % ) only chemotherapy , 105 ( 25.6 % ) chemotherapy followed by hormonal therapy and 94 ( 23.0 % ) received adjuvant trastuzumab either alone ( 33 patients ) , with hormonal therapy ( 39 patients ) , with chemotherapy ( 11 patients ) or with chemotherapy and hormonal therapy ( 11 patients ) . adjuvant radiotherapy was delivered to 310 ( 75.8 % ) patients , including 101 patients who underwent mastectomy.table 3clinical characteristics of patients after nst therapyno . ( % ) type of surgerybcs241 ( 58.9)mastectomy168 ( 41.1)residual tumor sizeypt075 ( 18.3)ypt1180 ( 44.0)ypt2116 ( 28.3)ypt338 ( 9.2)no . of metastatic nodesnone181 ( 44.2)13109 ( 26.6)4970 ( 17.1)1049 ( 11.9)posttherapy stage061 ( 14.9)i92 ( 22.5)ii129 ( 31.5)iii127 ( 31.9)adjuvant treatmentnil31 ( 7.6)hormonal therapy125 ( 30.6)chemotherapy54 ( 13.2)chemotherapy followed by hormonal therapy105 ( 25.6)trastuzumab94 ( 23.0)radiotherapyyes310 ( 75.8)no99 ( 24.2)mastectomywith radiation101 ( 60.1)without radiation67 ( 39.8 ) clinical characteristics of patients after nst therapy median follow - up was 42.1 months ( range 0.8147.3 months ) . during follow - up , 25 ( 6.1 % ) patients had local relapse , 84 ( 20.5 % ) had distant metastases and 53 ( 13.0 % ) died . we evaluated patients outcome in relation to different variables such as pcr , stage at surgery , tumor molecular subtype , use of trastuzumab for her2-positive tumors , type of surgery and breast radiotherapy . the occurrence of local relapse was not correlated with stage of disease at surgery , type of surgery and radiation therapy , while it was more frequent among patients with hr - negative tumors ( p = 0.007 by pearson s ) . at kaplan meier analysis of the whole population , pcr was not found to be a prognostic factor for drfs and os ( not shown ) . however , excluding from the analyses patients with luminal a or luminal b / her2-negative tumors ( a group of patients with favorable outcome , representing 51.9 % of the entire study population ) , pcr resulted predictive of better drfs ( p = 0.028 : hr = 0.37 , 95 % ci = 0.190.72 ) with a trend toward significance for os ( p = 0.06 ; hr = 0.34 , 95 % ci = 0.160.77 ) ( fig . patients with higher stage of disease after nst were more likely to develop distant metastasis and die from breast cancer ( fig . . patients who achieved pcr ( stage 0 ) had drfs and os rates of 87.1 % ( 95 % ci : 77.396.9 % ) and 92.0 % ( 95 % ci : 84.899.2 % ) , respectively , similar to those of patients with surgical stage i ( drfs : 85.9 % ; 95 % ci : 77.194.7 % and os : 80.4 % ; 95 % ci : 66.993.9 % ) . stage ii patients had drfs rates of 61.3 % ( 95 % ci : 39.782.9 % ) and os rates of 76.8 % ( 95 % ci : 55.298.4 % ) , while stage iii patients had drfs rates of 48.5 % ( 95 % ci : 35.461.6 % ) and os rates of 44.6 % ( 95 % ci : 17.771.5 % ) .fig . 1a distant relapse free survival ( drfs ) and b overall survival ( os ) stratified by pathological complete response ( pcr ) for the whole population , excluding patients with hr - positive / her2-negative tumorsfig . 2a distant relapse free survival ( drfs ) and b overall survival ( os ) stratified by pathological stage after systemic neoadjuvant chemotherapy a distant relapse free survival ( drfs ) and b overall survival ( os ) stratified by pathological complete response ( pcr ) for the whole population , excluding patients with hr - positive / her2-negative tumors a distant relapse free survival ( drfs ) and b overall survival ( os ) stratified by pathological stage after systemic neoadjuvant chemotherapy significant differences in drfs ( p = 0.006 ) and os ( p = 0.006 ) were observed among patients with different tumor molecular subtypes ( fig . 3a , b ) , the group of patients with luminal a or luminal b / her2-negative tumors showing a better prognosis . moreover , patients with hr - positive tumors had a significant longer drfs ( p < 0.005 : hr = 0.54 , 95 % ci = 0.350.85 ) and os ( p < 0.0001 : hr = 0.34 , 95 % ci = 0.190.63 ) compared with patients with hr - negative tumors ( fig . 3c , d ) . when survival analysis was stratified according to her2 status , the drfs and os advantage for hr - positive tumors was limited to the her2-negative population ( p = 0.016 : hr = 0.50 , 95 % ci = 0.250.97 and p < 0.0001 : hr = 0.25 , 95 % ci = 0.110.60 , respectively ) ( fig . 3e , f ) , while among her2-positive group , hr positivity was predictive of a longer drfs ( p = 0.044 : hr = 0.50 , 95 % ci = 0.241.0 ) , but not os ( not shown ) . in our population , neither her2 status nor the use of trastuzumab in the her2-positive patients was statistically associated with clinical outcome ( not shown).fig . 3a , c , e distant relapse free survival ( drfs ) and b , d , f overall survival ( os ) stratified by molecular subtypes for the whole population ( a , b ) , by hormone receptor ( hr ) status ( hr+ and hr ) for the whole population ( c , d ) and by hr status for patients with her2-negative tumors ( e , f ) a , c , e distant relapse free survival ( drfs ) and b , d , f overall survival ( os ) stratified by molecular subtypes for the whole population ( a , b ) , by hormone receptor ( hr ) status ( hr+ and hr ) for the whole population ( c , d ) and by hr status for patients with her2-negative tumors ( e , f ) patients who underwent bcs were more likely to have a better drfs ( p < 0.0001 : hr = 0.36 , 95 % ci = 0.230.55 ) and os ( p = 0.0014 : hr = 0.42 , 95 % ci = 0.240.72 ) compared with those who required mastectomy ( fig . no differences in survival were observed in patients treated with or without radiotherapy after surgery ( not shown).fig . 4a distant relapse free survival ( drfs ) and b overall survival ( os ) stratified by type of surgery . bcs breast conservative surgery a distant relapse free survival ( drfs ) and b overall survival ( os ) stratified by type of surgery . bcs breast conservative surgery in multivariate analysis , the variables independently associated with shorter drfs and os were absence of hr expression ( drfs and os : p < 0.001 ) , mastectomy ( drfs : p = 0.009 ; os : p = 0.05 ) and stage iii disease ( drfs : p < 0.001 ; os : p = 0.003 ) , whereas the molecular subtype luminal b / her2-positive tumors reached statistical significance for os ( p = 0.035 ) , but not for drfs ( tables 4 , 5).table 4multivariable proportional hazard regression model predicting drfsparameter estimatehazard ratio95 % cip valueage>35 yearsreference35 years0.1581.1710.4573.003n.s.histologic typelobularreferencen.s.ductal0.0651.0670.5682.006n.s.others0.1871.2060.2475.874molecular subtype prenstluminal a and b / her2 negativereferenceluminal b / her2 positive0.5911.8070.9703.3650.062her2 enriched1.6785.3542.74710.4350.000triple negative1.1413.1301.7035.7520.000type of surgerybcsreferencemastectomy0.6431.9031.1703.0930.009stage0referencei0.1851.2030.4193.454n.s.ii0.7512.1190.8255.443n.s.iii1.8106.1082.37615.7070.000table 5multivariable proportional hazard regression model predicting osparameter estimatehazard ratio95 % cip valueage>35 yearsreferencen.s.35 years0.1781.1950.4043.533histologic typelobularreferenceductal0.4241.5280.5903.955n.s.others0.5091.6640.17016.266n.s.molecular subtype prenstluminal a and b / her2 negativereferenceluminal b / her2 positive0.8462.3301.0625.1110.035her2 enriched1.7515.7612.43813.6100.000triple negative1.6965.4532.62011.3500.000type of surgerybcsreferencemastectomy0.6061.8341.1703.0930.056stage0referencei0.7262.0660.5477.808n.s.ii0.4661.5930.4096.205n.s.iii1.9006.6831.87723.8010.003 multivariable proportional hazard regression model predicting drfs multivariable proportional hazard regression model predicting os in this retrospective study , we show that hr negativity , requirement for mastectomy and pathological stage iii disease are independently associated with a worse outcome in breast cancer patients treated with nst in clinical practice . these data are of high interest since they derive from a very heterogeneous group of patients , treated with different neoadjuvant / adjuvant regimens outside of clinical trials and with a long follow - up period . during the course of the last 12 years , the adjuvant treatment of patients affected by early breast cancer is profoundly changed , going from first generation regimens like cmf and epirubicin cmf , second generation regimens like fec to third generation regimens , like ec followed by docetaxel ( sachelarie et al . 2012 ) . also , we delivered trastuzumab in the adjuvant treatment of her2-positive tumors from 2005 and in the neoadjuvant setting from 2006 ( romond et al . finally , surrogate definitions of intrinsic subtypes with immunohistochemistry have only recently proven to be effective in defining prognosis and selecting adjuvant therapy in early stage breast cancer patients ( cheang et al . 2011 ) . in our study , high tumor grade , high proliferative activity , hr - negativity expression in tumor biopsy , the use of neoadjuvant trastuzumab and an increase in number of chemotherapy cycles resulted significantly associated with higher rates of pcr at univariate analysis , consistent with current literature ( colleoni et al . 2009 ; huober et al . ; straver et al . 2010 ; untch et al . 2011 ; von minckwitz et al . 2011 ) , but only hr negativity and neoadjuvant trastuzumab were confirmed at multivariate analysis . the association between hr negativity and pcr has been observed also in a recently published meta - analysis based on 20 studies providing data with classification of her2 positivity according to hr status ( houssami et al . 2012 ) . their estimates of pcr were 8.3 % in the luminal a and luminal b / her2-negative subtype ; 18.7 % in the luminal b / her2-positive subtype ; 38.9 % in the her2-enriched subtype and 31.1 % in the triple negative subtype ( houssami et al . however , although most neoadjuvant chemotherapy trials have shown that pcr is associated with a favorable outcome in terms of drfs and os ( kuerer et al . 1999 ; kaufmann et al . 2006 ; buzdar et al . 2007 ; dawood et al . this discordance is not explained by the definition of pcr we applied , since it is now the most commonly used . different definitions for pcr have been used in different clinical trials , varying according to site ( i.e. , breast only or both breast and axillary ) and residual disease ( i.e. , presence of focal invasive cancer , non - invasive cancer residuals or absence of invasive and non - invasive cancer ) ( sataloff et al . 1995 ; bear et al . . absence of invasive and non - invasive cancer has been reported to be associated with a better prognosis ( von minckwitz et al . the incidence of residual non - invasive cancer in our study ( only six patients had residual in situ ductal carcinoma in the final pathologic examination ) , is too low to justify the poorer outcome observed in the whole population . more importantly , most of the patients in the study ( 211 patients , 51.9 % ) had luminal a or luminal b / her2-negative tumors , a subgroup considered to have slowly proliferating and less chemotherapy responsive tumors . in these patients , pcr has been shown to be not associated with prognosis ( von minckwitz et al . the high number of patients included in the luminal a or luminal b / her2-negative subgroup in our study could have diluted the effect of pcr on outcome . indeed , when these patients were excluded from the analyses , pcr was significantly associated with longer drfs and os . the outcome of patients included in this study was significantly affected by stage at surgery , hr expression and type of surgery . several studies have showed that a higher stage after nst is predictive of poor prognosis ( fisher et al . consistently , we found that patients with stage iii disease had a significantly shorter drfs and os . lack of hr expression is another well - established parameter associated with poor prognosis ( osborne and mcguire 1979 ; mcguire et al . 1986 ) . in our study , after a follow - up of about 12 years , patients with hr - negative tumors had a significantly lower rate of drfs ( 61 vs. 67 % , p < 0.001 ) and os ( 56.6 vs. 65.5 % , p < 0.001 ) compared with patients with hr - positive tumors , independently from her2 status , at least for drfs . this might be explained by the lower responsiveness of hr- and her2-positive tumors to the effect of adjuvant endocrine therapy bcs was carried out in 241 ( 58.9 % ) patients and this is in agreement with the percentage of bcs performed after nst reported in clinical trials ( bear et al . these patients had a significantly better prognosis in terms of drfs and os compared with patients who underwent mastectomy . similar data are presented by other authors who related these findings to patients selection : patients were more likely to have bcs if they presented with earlier stage disease or a clinical complete or greater than 50 % partial response ( schwartz et al . , this advantage was independent of age , histologic type , molecular subtype and surgical stage , but we agree that the achievement of bcs can be considered as an indirect measure of clinical response of the primary tumor , parameter not included in our multivariate analyses since we could not uniformly assess it throughout our patient population . in this study , clinical response was evaluated before , during and after neoadjuvant chemotherapy either by physical examination or by echographic and mammographic measurements , but the lack of standardization did not allow us to include clinical response as a variable for the multivariate analyses . considering achievement of bcs as a surrogate marker of primary tumor response , our results suggest that the clinical response to nst is a strong predictive factor of good outcome . some neoadjuvant trials have provided evidence of the prognostic value of clinical response , even when it was not correlated with pcr ( hortobagyi et al . 1988 ; jacquillat et al . 1991 ; cameron et al . 1997 ; pierga et al . 2003 ) . during follow - up , 25 ( 6.1 % ) patients had lrr , which was related neither to the type of surgery nor to radiotherapy . these data are in agreement with those of other authors reporting a rate of lrr ranging from 6 to 10 % , with a trend toward higher rates in patients with basal - like subtypes ( chen et al . . in conclusion , this retrospective neoadjuvant study , based on a population of patients treated in the practice of clinical medicine , shows that hr negativity , stage iii disease at surgery and failure to achieve bcs after nst are independent factors negatively associated with prognosis . moreover , the results of this study further confirm that pcr is of no prognostic value in patients with luminal a or luminal b / her2-negative tumors . these patients , therefore , should not be included in neoadjuvant clinical trials whose primary end point is pcr , as suggested by other authors ( eiermann et al . 2001 ; berruti et al . none of the authors has any potential financial conflict of interest related to this manuscript . 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 .

purposethe aim of this study is to evaluate the long - term outcome of patients with locally advanced breast cancer treated with neoadjuvant systemic chemotherapy ( nst ) in routine clinical practice.methodsfour hundred and nine patients were identified between january 1999 and december 2011 . all patients received nst followed by surgery , adjuvant treatments and radiotherapy , as appropriate.resultsat kaplan meier analysis , patients with surgical stage iii disease were more likely to develop distant metastasis and die from breast cancer ( p < 0.001 ) . luminal a and luminal b / her2-negative patients had better prognosis ; moreover , patients with hormone receptor ( hr)-positive tumors had a significantly longer drfs ( p < 0.0049 ) and os ( p < 0.0001 ) compared with patients with hr - negative tumors as well as patients who underwent breast - conserving surgery ( drfs and os : p < 0.001 ) . in multivariate analysis , hr negativity ( p < 0.001 for both drfs and os ) , mastectomy ( drfs : p = 0.009 ; os : p = 0.05 ) and stage iii disease ( drfs : p < 0.001 ; os : p = 0.003 ) were associated with shorter drfs and os.conclusionshr negativity , mastectomy and pathological stage iii disease are the variables independently associated with a worse outcome in our cohort of patients . these data are of high interest since they derive from a very heterogeneous group of patients , treated with different neoadjuvant / adjuvant regimens outside of clinical trials and with a long follow - up period .

Background Patients and methods Patient population Pathological assessments Data collection Study endpoints and statistics Results Baseline patient and tumor characteristics Relationship between baseline characteristics and pCR Patients characteristics after NST Survival Discussion Conflict of interest Open Access

however , patients with hormone receptors ( hr)-positive tumors usually have low rates of pcr and maintain a good long - term outcome even in the presence of residual disease ( no pcr ) ( colleoni et al . the aim of this study is to evaluate the long - term outcome in a series of patients with locally advanced breast cancer consecutively treated with nst in our institution . the charts of all patients with locally advanced breast cancer , performance status 02 ( ecog scale ) , consecutively treated with nst at the medical oncology division , university of chieti hospital between january 1999 and december 2011 , were reviewed for this retrospective study . the charts of all patients with locally advanced breast cancer , performance status 02 ( ecog scale ) , consecutively treated with nst at the medical oncology division , university of chieti hospital between january 1999 and december 2011 , were reviewed for this retrospective study . in the multivariate analysis , only hr - negative tumors , independently from her2 status ( her2 enriched : p = 0.043 ; triple negative : p = 0.002 ) and the use of neoadjuvant trastuzumab ( p = 0.035 ) were significantly associated with higher pcr rates ( table 2).table 2association of baseline factors and pcr in multivariate analysisodds ratio95 % cip valueage>35 yearsreference35 years2.0810.42610.175n.s.clinical t3 cmreference3 cm1.0400.4142.611n.s.histologic typeotherreferenceductal2.9650.3276.424n.s.lobular5.9950.21310.785n.s.grade12reference31.2750.4183.885n.s.ki-6714 % reference>14 % 3.6890.89915.132n.s.molecular subtypeluminal a and b / her2 negativereferenceluminal b / her2 positive1.5640.2529.716n.s.her2 enriched6.0901.06234.9210.043triple negative10.6462.30740.1250.002type of nstvarious2.290referencen.s.anthracycline and taxane11.3340.30017.4190.035chemotherapy + trastuzumab1.182108.719no . however , excluding from the analyses patients with luminal a or luminal b / her2-negative tumors ( a group of patients with favorable outcome , representing 51.9 % of the entire study population ) , pcr resulted predictive of better drfs ( p = 0.028 : hr = 0.37 , 95 % ci = 0.190.72 ) with a trend toward significance for os ( p = 0.06 ; hr = 0.34 , 95 % ci = 0.160.77 ) ( fig . patients with higher stage of disease after nst were more likely to develop distant metastasis and die from breast cancer ( fig . patients who achieved pcr ( stage 0 ) had drfs and os rates of 87.1 % ( 95 % ci : 77.396.9 % ) and 92.0 % ( 95 % ci : 84.899.2 % ) , respectively , similar to those of patients with surgical stage i ( drfs : 85.9 % ; 95 % ci : 77.194.7 % and os : 80.4 % ; 95 % ci : 66.993.9 % ) . 2a distant relapse free survival ( drfs ) and b overall survival ( os ) stratified by pathological stage after systemic neoadjuvant chemotherapy a distant relapse free survival ( drfs ) and b overall survival ( os ) stratified by pathological complete response ( pcr ) for the whole population , excluding patients with hr - positive / her2-negative tumors a distant relapse free survival ( drfs ) and b overall survival ( os ) stratified by pathological stage after systemic neoadjuvant chemotherapy significant differences in drfs ( p = 0.006 ) and os ( p = 0.006 ) were observed among patients with different tumor molecular subtypes ( fig . 3a , b ) , the group of patients with luminal a or luminal b / her2-negative tumors showing a better prognosis . moreover , patients with hr - positive tumors had a significant longer drfs ( p < 0.005 : hr = 0.54 , 95 % ci = 0.350.85 ) and os ( p < 0.0001 : hr = 0.34 , 95 % ci = 0.190.63 ) compared with patients with hr - negative tumors ( fig . when survival analysis was stratified according to her2 status , the drfs and os advantage for hr - positive tumors was limited to the her2-negative population ( p = 0.016 : hr = 0.50 , 95 % ci = 0.250.97 and p < 0.0001 : hr = 0.25 , 95 % ci = 0.110.60 , respectively ) ( fig . 3a , c , e distant relapse free survival ( drfs ) and b , d , f overall survival ( os ) stratified by molecular subtypes for the whole population ( a , b ) , by hormone receptor ( hr ) status ( hr+ and hr ) for the whole population ( c , d ) and by hr status for patients with her2-negative tumors ( e , f ) a , c , e distant relapse free survival ( drfs ) and b , d , f overall survival ( os ) stratified by molecular subtypes for the whole population ( a , b ) , by hormone receptor ( hr ) status ( hr+ and hr ) for the whole population ( c , d ) and by hr status for patients with her2-negative tumors ( e , f ) patients who underwent bcs were more likely to have a better drfs ( p < 0.0001 : hr = 0.36 , 95 % ci = 0.230.55 ) and os ( p = 0.0014 : hr = 0.42 , 95 % ci = 0.240.72 ) compared with those who required mastectomy ( fig . bcs breast conservative surgery in multivariate analysis , the variables independently associated with shorter drfs and os were absence of hr expression ( drfs and os : p < 0.001 ) , mastectomy ( drfs : p = 0.009 ; os : p = 0.05 ) and stage iii disease ( drfs : p < 0.001 ; os : p = 0.003 ) , whereas the molecular subtype luminal b / her2-positive tumors reached statistical significance for os ( p = 0.035 ) , but not for drfs ( tables 4 , 5).table 4multivariable proportional hazard regression model predicting drfsparameter estimatehazard ratio95 % cip valueage>35 yearsreference35 years0.1581.1710.4573.003n.s.histologic typelobularreferencen.s.ductal0.0651.0670.5682.006n.s.others0.1871.2060.2475.874molecular subtype prenstluminal a and b / her2 negativereferenceluminal b / her2 positive0.5911.8070.9703.3650.062her2 enriched1.6785.3542.74710.4350.000triple negative1.1413.1301.7035.7520.000type of surgerybcsreferencemastectomy0.6431.9031.1703.0930.009stage0referencei0.1851.2030.4193.454n.s.ii0.7512.1190.8255.443n.s.iii1.8106.1082.37615.7070.000table 5multivariable proportional hazard regression model predicting osparameter estimatehazard ratio95 % cip valueage>35 yearsreferencen.s.35 years0.1781.1950.4043.533histologic typelobularreferenceductal0.4241.5280.5903.955n.s.others0.5091.6640.17016.266n.s.molecular subtype prenstluminal a and b / her2 negativereferenceluminal b / her2 positive0.8462.3301.0625.1110.035her2 enriched1.7515.7612.43813.6100.000triple negative1.6965.4532.62011.3500.000type of surgerybcsreferencemastectomy0.6061.8341.1703.0930.056stage0referencei0.7262.0660.5477.808n.s.ii0.4661.5930.4096.205n.s.iii1.9006.6831.87723.8010.003 multivariable proportional hazard regression model predicting drfs multivariable proportional hazard regression model predicting os the median age was 48.8 years ( range 2580 ) , with 20 ( 4.9 % ) patients being younger than 35 years and 15 ( 3.7 % ) older than 70 years . in the multivariate analysis , only hr - negative tumors , independently from her2 status ( her2 enriched : p = 0.043 ; triple negative : p = 0.002 ) and the use of neoadjuvant trastuzumab ( p = 0.035 ) were significantly associated with higher pcr rates ( table 2).table 2association of baseline factors and pcr in multivariate analysisodds ratio95 % cip valueage>35 yearsreference35 years2.0810.42610.175n.s.clinical t3 cmreference3 cm1.0400.4142.611n.s.histologic typeotherreferenceductal2.9650.3276.424n.s.lobular5.9950.21310.785n.s.grade12reference31.2750.4183.885n.s.ki-6714 % reference>14 % 3.6890.89915.132n.s.molecular subtypeluminal a and b / her2 negativereferenceluminal b / her2 positive1.5640.2529.716n.s.her2 enriched6.0901.06234.9210.043triple negative10.6462.30740.1250.002type of nstvarious2.290referencen.s.anthracycline and taxane11.3340.30017.4190.035chemotherapy + trastuzumab1.182108.719no . the occurrence of local relapse was not correlated with stage of disease at surgery , type of surgery and radiation therapy , while it was more frequent among patients with hr - negative tumors ( p = 0.007 by pearson s ) . however , excluding from the analyses patients with luminal a or luminal b / her2-negative tumors ( a group of patients with favorable outcome , representing 51.9 % of the entire study population ) , pcr resulted predictive of better drfs ( p = 0.028 : hr = 0.37 , 95 % ci = 0.190.72 ) with a trend toward significance for os ( p = 0.06 ; hr = 0.34 , 95 % ci = 0.160.77 ) ( fig . patients with higher stage of disease after nst were more likely to develop distant metastasis and die from breast cancer ( fig . patients who achieved pcr ( stage 0 ) had drfs and os rates of 87.1 % ( 95 % ci : 77.396.9 % ) and 92.0 % ( 95 % ci : 84.899.2 % ) , respectively , similar to those of patients with surgical stage i ( drfs : 85.9 % ; 95 % ci : 77.194.7 % and os : 80.4 % ; 95 % ci : 66.993.9 % ) . 2a distant relapse free survival ( drfs ) and b overall survival ( os ) stratified by pathological stage after systemic neoadjuvant chemotherapy a distant relapse free survival ( drfs ) and b overall survival ( os ) stratified by pathological complete response ( pcr ) for the whole population , excluding patients with hr - positive / her2-negative tumors a distant relapse free survival ( drfs ) and b overall survival ( os ) stratified by pathological stage after systemic neoadjuvant chemotherapy significant differences in drfs ( p = 0.006 ) and os ( p = 0.006 ) were observed among patients with different tumor molecular subtypes ( fig . moreover , patients with hr - positive tumors had a significant longer drfs ( p < 0.005 : hr = 0.54 , 95 % ci = 0.350.85 ) and os ( p < 0.0001 : hr = 0.34 , 95 % ci = 0.190.63 ) compared with patients with hr - negative tumors ( fig . 3a , c , e distant relapse free survival ( drfs ) and b , d , f overall survival ( os ) stratified by molecular subtypes for the whole population ( a , b ) , by hormone receptor ( hr ) status ( hr+ and hr ) for the whole population ( c , d ) and by hr status for patients with her2-negative tumors ( e , f ) a , c , e distant relapse free survival ( drfs ) and b , d , f overall survival ( os ) stratified by molecular subtypes for the whole population ( a , b ) , by hormone receptor ( hr ) status ( hr+ and hr ) for the whole population ( c , d ) and by hr status for patients with her2-negative tumors ( e , f ) patients who underwent bcs were more likely to have a better drfs ( p < 0.0001 : hr = 0.36 , 95 % ci = 0.230.55 ) and os ( p = 0.0014 : hr = 0.42 , 95 % ci = 0.240.72 ) compared with those who required mastectomy ( fig . bcs breast conservative surgery in multivariate analysis , the variables independently associated with shorter drfs and os were absence of hr expression ( drfs and os : p < 0.001 ) , mastectomy ( drfs : p = 0.009 ; os : p = 0.05 ) and stage iii disease ( drfs : p < 0.001 ; os : p = 0.003 ) , whereas the molecular subtype luminal b / her2-positive tumors reached statistical significance for os ( p = 0.035 ) , but not for drfs ( tables 4 , 5).table 4multivariable proportional hazard regression model predicting drfsparameter estimatehazard ratio95 % cip valueage>35 yearsreference35 years0.1581.1710.4573.003n.s.histologic typelobularreferencen.s.ductal0.0651.0670.5682.006n.s.others0.1871.2060.2475.874molecular subtype prenstluminal a and b / her2 negativereferenceluminal b / her2 positive0.5911.8070.9703.3650.062her2 enriched1.6785.3542.74710.4350.000triple negative1.1413.1301.7035.7520.000type of surgerybcsreferencemastectomy0.6431.9031.1703.0930.009stage0referencei0.1851.2030.4193.454n.s.ii0.7512.1190.8255.443n.s.iii1.8106.1082.37615.7070.000table 5multivariable proportional hazard regression model predicting osparameter estimatehazard ratio95 % cip valueage>35 yearsreferencen.s.35 years0.1781.1950.4043.533histologic typelobularreferenceductal0.4241.5280.5903.955n.s.others0.5091.6640.17016.266n.s.molecular subtype prenstluminal a and b / her2 negativereferenceluminal b / her2 positive0.8462.3301.0625.1110.035her2 enriched1.7515.7612.43813.6100.000triple negative1.6965.4532.62011.3500.000type of surgerybcsreferencemastectomy0.6061.8341.1703.0930.056stage0referencei0.7262.0660.5477.808n.s.ii0.4661.5930.4096.205n.s.iii1.9006.6831.87723.8010.003 multivariable proportional hazard regression model predicting drfs multivariable proportional hazard regression model predicting os in this retrospective study , we show that hr negativity , requirement for mastectomy and pathological stage iii disease are independently associated with a worse outcome in breast cancer patients treated with nst in clinical practice . these data are of high interest since they derive from a very heterogeneous group of patients , treated with different neoadjuvant / adjuvant regimens outside of clinical trials and with a long follow - up period . in our study , after a follow - up of about 12 years , patients with hr - negative tumors had a significantly lower rate of drfs ( 61 vs. 67 % , p < 0.001 ) and os ( 56.6 vs. 65.5 % , p < 0.001 ) compared with patients with hr - positive tumors , independently from her2 status , at least for drfs . these patients had a significantly better prognosis in terms of drfs and os compared with patients who underwent mastectomy .

down syndrome ( ds ) is the most common genetic ( chromosomal ) mental retardation syndrome , occurring in 1 in 7001000 live births . in ds , common features include distinctive craniofacial features , congenital heart disease , middle ear disease , and immune and endocrine system abnormalities [ 2 , 3 ] . in recent years , we have witnessed a growing interest in the mass and bone quality of patients with ds ; many studies have evaluated densitometric characteristics via dual - energy x - ray absorptiometry ( dxa ) [ 47 ] , peripheral quantitative computed tomography ( pqct ) , and quantitative ultrasound ( qus ) . however , many studies have focused on adults who live either in the community or in residential institutions . in these patients , several environmental and hormonal factors such as muscle hypotonia , low physical activity , poor calcium and vitamin d intake , hypogonadism , growth retardation , and thyroid dysfunction may contribute to low bone mineral density ( bmd ) [ 4 , 9 ] . these patients may develop reduced bone - mass accrual , predisposing them to fragility , fractures , and osteoporosis . among these factors , vitamin d may play a significant role in the health of patients with ds . vitamin d status varies widely between different countries in europe [ 11 , 12 ] , depending on many factors such as different exposures to sunshine , dietary intake of vitamin d , and the use of supplements . normally , in winter months and with increasing latitude , the amount of ultraviolet radiation reaching the earth 's atmosphere is decreased , because the rays of the sun enter the atmosphere at a more oblique angle . as a consequence moreover , during the winter months , children spend more time indoors and less of their skin is exposed to the sun . this fact may explain the high percentage of ds individuals and controls showing a vitamin d deficiency . vitamin d status is defined according to the serum concentration of 25-hydroxyvitamin d ( 25(oh)d ) . as previously reported , vitamin d deficiency is defined to exist when the serum 25(oh)d level is lower than 25 nmol / l ( 10 ng / ml ) ; vitamin d insufficiency is considered to exist when the serum 25(oh)d is between 25 and 50 nmol / l ( 1020 ng / ml ) [ 14 , 15 ] . however , an evaluation of satisfactory levels of vitamin d in healthy children has not yet been reported by adequate studies . the prevalence of 25(oh)d deficiency varies between 30% and 93% in different studies in adults , even if in norway and sweden the prevalence is rather low . however , in a small italian study , more than 80% of children showed insufficiency or deficiency of 25(oh)d ; the data were confirmed in other , larger studies providing cross - sectional and longitudinal data . an adequate status of 25(oh)d is very important because 25(oh)d deficiency has been shown to be a risk factor for several chronic diseases , in addition to the classic deleterious effects on bone , such as secondary hyperparathyroidism , and reduced bone accrual and mass . in fact , the discovery that most tissues in the body have vitamin d receptors and several have one hydroxylase enzyme to convert 25(oh)d to its active form has provided new insights into the pleiotropic role of this vitamin . in fact , vitamin d receptors are found on several immune cells and vitamin d metabolites seem to modulate t cell proliferation and dendritic cell function [ 21 , 22 ] . however , vitamin d deficiency may be a risk factor for the development of autoimmune diseases and loss of muscle mass and muscle weakness . finally , many data have demonstrated that vitamin d may confer protection against diabetes mellitus ( dm ) type 1 , hypertension , multiple sclerosis , and cancer [ 24 , 25 ] . therefore , vitamin d insufficiency may have important health consequences because of its role in the maintenance of normal bone mass turnover and its role as an immunoregulatory agent . to date , few studies have assessed vitamin d status among children and adults with ds [ 26 , 27 ] . such studies have yielded conflicting results about the beneficial effects of intervention with vitamin d or the lack of prescribing vitamin d when appropriate periods of exposure to sunlight exposure are available . the purpose of the present study is to assess serum 25(oh)d in children and adolescents living in tuscany , italy , ( latitude : 44 north ) , and to identify risk factors for vitamin d deficiency in different age groups of individuals with ds . furthermore , this study also aimed to evaluate whether a normal 25(oh)d value can be restored in 25(oh)d - deficient ds patients in respect to controls with a daily supplement of 400 i.u . we longitudinally evaluated 31 caucasian children and adolescents ( 17 males and 14 females , aged 4.518.9 years ) with ds from tuscany in the central region of italy . all of the subjects were selected among individuals with ds who visited the paediatric endocrinology unit of anna meyer children 's university hospital in florence and the paediatric unit , mugello 's hospital , borgo san lorenzo , italy , between december 2010 and october 2013 . the hospital ethics committees of anna meyer children 's university hospital and mugello 's hospital approved the study , conducted in accordance with the declaration of helsinki guidelines . the present study was a 12-month ( t0-t1 ) controlled study of vitamin d supplementation . for baseline data collection , the ds patients were compared with a 1 : 3 proportion of healthy caucasian controls . of the 38 ds subjects initially recruited to take part in this interventional study , 7 dropped out for various reasons ( noncompliance , lost to follow - up , etc . ) . the final count , 31 individuals ( 81.6% ) , was the subjects who were able to finish the interventional study . at t0 and t1 , clinical and demographic data were collected from both the ds patients and healthy controls , including height , weight , body mass index ( bmi ) , blood pressure , pubertal stage , therapies carried out , family and patient histories of autoimmune diseases and osteoporosis , and time dedicated to outdoor physical activity . furthermore , nutrients diaries were recorded for each patient based on medical charts and standardized interviews . however , during the longitudinal study , the ds subjects and healthy controls were divided into two age groups : children ( 212 years ) and adolescents ( older than 12 years ) . at t0 and t1 , all of the ds patients and healthy controls underwent laboratory tests to measure their plasma 25(oh)d levels , serum calcium and phosphate , bone specific alkaline phosphatase , parathyroid hormone ( pth ) , triglycerides ( tg ) , total cholesterol , and low - density lipoprotein ( ldl ) cholesterol . none of the participants had a recent history of travelling to warmer , sunnier areas prior to and/or during the study . other exclusion criteria included taking calcium , vitamin d supplements , or any drugs affecting calcium or vitamin d metabolism in the past six months , such as a positive history of primary hyperparathyroidism or other skeletal diseases , severe obesity , malabsorptive disorders , and neurological or renal diseases . vitamin d status : serum 25-oh levels were stratified according to the following brackets : 10 , 1120 , and 2130 and > 30 ng / ml , and they were defined as severe deficiency , deficiency , insufficiency , and sufficiency , respectively , according to previously established guidelines for bone health ( in the absence of a consensus regarding appropriate levels for endocrine and extraendocrine health ) [ 28 , 29 ] . however , for evaluating the seasonal variations of 25(oh)d , we divided the year into four seasons : winter ( december march ) , spring ( march june ) , summer ( june september ) , and autumn ( september december ) . evaluation of dietary intake of calcium and vitamin d : dietary intakes of calcium and 25(oh)d were estimated using standardized interviews ( by the parents ) recording race , religion , country of birth , birth weight , type of feeding during the first year ( breast , formula milk , or mixed ) , mother 's use of vitamin supplementation during her pregnancy , child 's use of vitamin d supplementation , and daily intake of cow 's milk ( categorized as more or less than 200 ml per day ) . nutrient analyses were obtained from the food composition database for epidemiological studies in italy . the frequency consumption ( daily , weekly , and monthly ) of each food item was evaluated . outdoor exposure evaluation : outdoor exposure was quantified from both questions regarding each child 's and adolescent 's average number of daily outdoor hours across each season and a prospective daily time - activity diary . for this analysis , we used an activity questionnaire and physical activity was assessed with a modified activity score composed of the scores for outdoor sports / leisure activities ( 0 , < 2 , or > 2 hours per week ) , as previously described . vitamin d intervention : all of the subjects ( ds patients and controls ) were treated with 400 i.u . ( 10 g ) of vitamin d3 ( cholecalciferol ) administered orally once daily from november through may . the administration was based on the recommendation of the american academy of pediatrics that stated a recommended daily intake of vitamin d of 400 i.u./day for all infants , children , and adolescents [ 33 , 34 ] . after 25(oh)d supplementation , 25(oh)d levels , serum calcium and phosphate , alkaline phosphatase , and pth were reevaluated . the mean elapsed duration between the first and the second determinations was 12.3 months ( range : 8.114.7 months ) . compliance was evaluated by written instructions given at the onset of the study and at clinical controls through the delivery of a written questionnaire drawn up by the parents . compliance was further verified by e - mails and/or telephone interviews performed by a study nurse ( to confirm the 25(oh)d intake ) and by the bottle count performed at the end of the study period . control group included 99 ( 84.6% out of 117 recruited subjects initially ) healthy age- and sex - matched subjects ( 51 males and 48 females : age range 4.819.8 years ) seen for noninflammatory musculoskeletal complaints . all of the subjects were evaluated at the time of routine follow - up visits , and parental informed consent was obtained . height was measured using harpenden 's stadiometer in triplicate to the nearest 0.1 cm . however , age - related reference values for height and bmi currently used in italy , obtained in high sample numbers of italian children , were used for comparison between ds subjects and controls . subjects with a bmi over the 95th percentile were considered obese , and subjects with a bmi over the 85th percentile but below the 95th percentile were considered overweight . as described , height and bmi were normalized for chronological age by converting to standard deviation scores ( sdss ) . sdss were calculated according to the following formula : patient value minus mean of age - related reference value / standard deviation of the age - related reference value . pubertal staging was determined at baseline and at each visit and was performed according to the criteria of marshall and tanner [ 39 , 40 ] , using an orchidometer in boys . blood pressure was measured three times by trained personnel by auscultation using a mercury sphygmomanometer on the right arm after the patient has been sitting quietly for 5 minutes , with the back supported , feet on the floor , right arm supported , and cubital fossa at heart level , as previously described . plasma concentrations of calcium , phosphate , and alkaline phosphatase were determined following routine biochemical laboratory protocols . furthermore , the total cholesterol and triglyceride ( tg ) measurements were performed according to routine laboratory methods . low - density lipoprotein ( ldl ) cholesterol was calculated using the friedwald formula : ldl = total cholesterol hdl cholesterol tg/2.2 . sera 25(oh)d and pth were determined by chemiluminescence enzyme - labeled immunometric assays using an immulite 2000 systems analyzer ( siemens , gwynedd , uk ) . the intra- and interassays cvs were < 5% and < 8% and < 8% and < 10% , respectively . statistical analyses were performed using spssx ( spssx inc . , chicago , il , usa ) . clinical variables considered relevant to the study were as follows : sex ( m : f ) , bmi sdss , height sdss , age at onset of puberty , pubertal stage , plasma concentrations of calcium , phosphate , alkaline phosphatase , and sera 25(oh)d and pth at the first and second examinations . the characteristics of the study population were described through frequency distributions for categorical variables and through means and standard deviations ( sds ) , medians , and range for continuous variables . for categorical variables the kolmogorov - smirnov test was used to determine if variables were normally distributed . for continuous variables , groups were compared using student 's t - test and mann - whitney u test , since not all of the continuous variables were normally distributed according to shapiro - wilk 's test . intergroup comparisons for parameters were conducted using analysis of variance ( anova ) or repeated - measures analysis of covariance ( ancova ) , as appropriate . a multiple stepwise regression was performed to investigate factors associated with insufficient vitamin d status , after adjusting for potential confounders ( age , sex , pubertal stage , vitamin d intake , and bmi ) . covariates that were found to be nonsignificant at the 0.05 level were removed from the regression model using a stepwise elimination technique . no statistically significant differences in terms of history of fractures were found between our group of patients with ds and the control group . on the contrary , a statistically significant difference was found regarding height sdss ( 1.5 1.0 versus 0.2 0.8 ; p < 0.005 ) and the bmi sdss ( 1.0 1.4 versus 0.1 0.9 ; p < 0.05 ) , even when considering the children and adolescents separately . furthermore , no statistical differences were found regarding 25(oh)d status in the different seasons between ds patients and controls . evaluating the percentages regarding 25(oh)d sufficiency , insufficiency , and deficiency , 2/31 ( 6.5% ) of ds subjects had sufficient vitamin d levels , 5/31 ( 16.1% ) had insufficient levels , 14/31 ( 45.2% ) showed deficient levels , and 10/31 ( 32.2% ) exhibited a severe deficiency . the percentage of 25(oh)d sufficiency is not significantly different from the controls ( 11/99 : 11.1% ) , but the insufficiency ( 33/99 : 33.3% ; p < 0.005 ) , deficiency ( 35/99 : 35.4% ; p < 0.05 ) , and severe deficiency ( 20/99 : 20.2% ; p < 0.005 ) are significantly different . however , for evaluating the 25(oh)d levels , we show that the ds subjects had significantly reduced 25(oh)d levels compared with the controls ( 14.34 8.31 ng / ml versus 27.04 7.47 ; p < 0.0001 ) ( figure 1(a ) ) . in the ds subjects , 25(oh)d levels were not different between males ( 14.85 8.25 ng / ml ) and females ( 13.75 8.70 ) , children ( 14.26 8.71 ng / ml ) , and adolescents ( 14.45 8.15 ) ; we showed significant statistical differences between ds individuals with normal weights ( 16.93 8.71 ng / ml ) and those who were obese ( 10.20 5.13 ; p < 0.05 ) ( figure 2(a ) ) and ds individuals without ( 19.00 8.06 ng / ml ) and with ( 10.35 6.57 ng / ml ; p < 0.005 ) a history of autoimmune diseases ( figure 3(a ) ) . regarding the effect of the different seasons on 25(oh)d status , the levels of 25(oh)d in ds subjects were significantly reduced in winter , spring , and autumn ( 11.33 5.16 , 7.85 4.67 , and 12.42 4.96 ng / ml , resp . ) with respect to summer value ( 22.53 8.87 ng / ml ; p < 0.005 , p < 0.001 , and p < 0.05 , resp . ) ( figure 4 ) . these results were not significantly different when ds patients were divided into a child group ( winter : 12.46 5.67 ; spring : 8.98 5.02 ; summer : 23.12 8.76 ; autumn : 13.43 5.22 ng / ml ) and an adolescent group ( winter : 10.12 4.89 ; spring : 7.34 4.65 ; summer : 21.00 8.99 ; autumn : 11.76 4.54 ng / ml ) and males ( winter : 11.88 5.34 ; spring : 8.13 4.88 ; summer : 23.65 7.80 ; autumn : 11.37 4.34 ng / ml ) and females ( winter : 10.89 5.00 ; spring : 7.67 4.34 ; summer : 22.02 8.99 ; autumn : 13.13 4.99 ng / ml ) . however , these values are always significantly smaller in ds individuals than in controls ( winter : 18.9 6.3 ng / ml ; p < 0.005 ; spring : 24.82 6.06 ng / ml ; p < 0.0001 ; summer : 30.33 11.03 ng / ml ; p < 0.005 ; autumn : 21.23 6.24 ng / ml ; p < 0.005 ) ( figure 4 ) . ds patients showed normal total calcium levels ( 2.42 0.14 versus 2.51 0.10 mmol / l ; p = ns ) compared with the controls and their phosphate levels were normal . however , ds patients also had significantly higher pth levels compared with the controls ( 54.76 32.15 versus 26.13 10.76 pg / ml ; p < 0.0001 ) ( figure 5(a ) ) . pth levels were higher , but not significantly , in ds adolescents ( 60.50 38.45 pg / ml ) compared with children ( 46.30 27.93 pg / ml ) . these results differ from what is seen in the controls , in which we showed that pth levels in children were 25.23 9.10 pg / ml in adolescents ; a statistical difference between ds individuals and controls was seen in children ( p < 0.0001 ) but not in adolescents . no significant differences were found in calcium intake between ds patients and the controls ( 796 283 versus 821 256 mg / day ) , even if the samples were divided into child ( 810 270 versus 835 285 mg / day ) and adolescent ( 755 290 versus 790 235 mg / day ) groups . nevertheless , despite the dietary calcium intake not being different between ds and control individuals , the amount of cow 's milk drunk per day was related to 25(oh)d levels , with a high percentage ( 18/24 , 75.0% ) of ds patients consuming less milk and showing 25(oh)d levels in the range of a deficiency or severe deficiency . in particular , only 30% of children with a severe deficiency ( n = 3/10 ) consumed more than 200 ml of cow milk per day versus 71.4% of children ( n = 5/7 ) with sufficient or insufficient 25(oh)d levels ( p = 0.002 ) . these data are similar to what is seen in the controls , with a high percentage ( 38/55 , 69.1% ) of individuals consuming less milk showing 25(oh)d levels in the range of a deficiency or severe deficiency . the quantitative assessment of physical activity in patients with ds and the controls showed significant differences between the two groups ; the percentage of current physical activity levels was significantly lower for patients with ds than for the controls ( 0 hours per week group : 56% and 27% , resp . ; however , regarding the effect of hours spent outdoors ( including also physical activity ) on 25(oh)d levels , there was no association between the reported average daily number of hours spent outdoors and baseline 25(oh)d levels ( p = ns ) , even if patients with ds who spent more than 8 hours / week outdoors showed higher 25(oh)d levels than patients with who spent fewer than 4 hours / week outdoors ( 21.75 6.43 versus 9.87 4.35 ng / ml ; p = 0.006 ) . evaluating correlations among 25(oh)d and age , sex , seasons , physical activity , milk intake , pth , bmi , height , total cholesterol , triglycerides , ldl cholesterol , systolic blood pressure , diastolic blood pressure , and autoimmune disease development , we showed that 25(oh)d levels correlated inversely with pth ( r = 0.42 , p < 0.005 ) , bmi ( r = 0.39 , p < 0.005 ) , physical and outdoor activities ( r = 0.31 , p < 0.05 ) , milk intake ( r = 0.30 , p < 0.05 ) , total cholesterol ( r = 0.43 , p < 0.005 ) , ldl cholesterol ( r = 0.28 , p < 0.05 ) , systolic blood pressure ( r = 0.34 , p < 0.005 ) , and autoimmune diseases ( r = 0.56 , p < 0.005 ) . the multivariate linear regression analyses showed that serum 25(oh)d concentration was negatively associated with bmi ( = 0.29 , p < 0.005 ) . after supplementation with 25(oh)d and evaluating the percentages of ds patients and controls with 25(oh)d sufficiencies , insufficiencies , and deficiencies , we show that 7 ( 22.6% versus 6.5% ; p < 0.001 ) ds patients achieved sufficient vitamin d levels , 8 ( 25.8% versus 16.1% ; p < 0.05 ) achieved insufficient levels , 9 ( 29.0% versus 45.2% ; p < 0.001 ) achieved deficient levels , and 7 ( 22.6% versus 32.2% ; p < 0.05 ) still showed a severe deficiency . the results were significantly different for the healthy controls : 26 ( 26.3% versus 11.1% ; p < 0.001 ) reached sufficient vitamin d levels , 43 ( 43.4% versus 33.3% ; p < 0.05 ) reached insufficient levels , 21 ( 21.2% versus 35.4% ; p < 0.005 ) reached deficient levels , and 9 ( 9.1% versus 35.4% ; p < 0.001 ) reached severe deficient levels . however , in terms of 25(oh)d levels in ds subjects at the end of intervention , even if the level was significantly ameliorated ( 20.15 10.88 versus 14.34 8.31 ng / ml ; p < 0.05 ) , these patients still showed extremely reduced levels compared with the controls ( 28.27 7.96 ; p do not continue to be different between males ( 19.35 9.63 ng / ml ) and females ( 21.08 12.55 ; p = ns ) and children ( 19.00 11.49 ng / ml ) and adolescents ( 22.09 9.72 ; p = ns ) , whereas we showed significant statistical differences between ds subjects characterized by a normal weight ( 23.50 11.06 ng / ml ) and individuals who were obese ( 14.80 8.86 ; p < 0.05 ) ( figure 2(b ) ) and ds individuals with ( 12.07 6.81 ng / ml ) and without ( 28.41 8.19 ng / ml ; p < 0.001 ) a history of autoimmune diseases ( figure 3(b ) ) . regarding dietary calcium intake , even if calcium intake was not significantly different between ds patients and controls ( 846 256 versus 889 221 mg / day ) , we showed an increased but nonsignificant change in calcium intake with respect to baseline values . however , the percentage of patients and controls who drank more than 200 ml of cow 's milk per day was significantly increased : in ds patients , 16/31 ( 51.6% ) individuals continued to consume fewer than 200 ml of cow 's milk for day , a better outcome than the 75% of ds patients reported in the first evaluation ( p < 0.0001 ) . this aspect was similar in the controls , with a significant amelioration of the percentage of people consuming more than 200 ml of cow 's milk per day ( 27.2% versus 38.4% ; p < 0.05 ) . nevertheless , milk consumption per day was always related to 25(oh)d levels , with a high percentage ( 14/16 , 87.5% ) of ds patients consuming less milk showing 25(oh)d levels in the range of a deficiency or severe deficiency . the quantitative assessment of physical activity in patients with ds and controls confirmed a significantly lower percentage of physical activity in patients with ds than the controls ( 0 hours per week group : 49% and 28% , resp . ; however , regarding the effect of the number of hours spent outdoors ( including also physical activity ) on 25(oh)d levels , there was no association between the reported average daily hours spent outdoors and baseline 25(oh)d levels ( p = ns ) , even if patients with ds who spent more than 8 hours / week outdoors showed higher 25(oh)d levels than patients with a history spending fewer than 4 hours / week outdoors ( 26.58 9.00 versus 15.64 11.71 ng / ml ; p < 0.005 ) . ds patients still had significantly higher pth levels compared with controls ( 43.57 14.05 versus 26.89 13.56 pg / ml ; p < 0.005 ) ( figure 5(b ) ) . however , ds subjects who were obese still showed significantly higher pth levels ( 54.90 13.45 pg / ml ) than ds individuals with normal weights ( 36.50 9.04 pg / ml ; p < 0.005 ) . however , ds patients with a history of autoimmune diseases showed higher pth levels ( 56.70 11.98 pg / ml ) than patients without a history of autoimmune diseases ( 35.37 7.51 pg / ml ; p < 0.05 ) . in evaluating the correlations among 25(oh)d and age , sex , seasons , physical activity , milk intake , pth , bmi , height , total cholesterol , triglycerides , ldl cholesterol , systolic blood pressure , diastolic blood pressure , and a history of autoimmune disease , we showed that 25(oh)d levels were still inversely correlated with pth ( r = 0.38 , p < 0.05 ) , bmi ( r = 0.43 , p < 0.005 ) , physical and outdoor activity ( r = 0.34 , p < 0.05 ) , total cholesterol ( r = 0.33 , p < 0.05 ) , ldl cholesterol ( r = 0.27 , p < 0.05 ) , systolic blood pressure ( r = 0.30 , p < 0.05 ) , milk intake ( r = 0.36 , p < 0.05 ) , and autoimmune diseases ( r = 0.59 , p < 0.005 ) . our study shows , for the first time , an extensive evaluation of 25(oh)d status in children and adolescents with ds . we demonstrate a very high prevalence of vitamin d deficiency in different age groups , revealing an important health problem in these patients . in the control subjects , different seasons influenced vitamin d status [ 42 , 43 ] . as with general population , for ds individuals , the 25(oh)d values differed according to the seasons , even if these values remain always less than in the control population , demonstrating the role of many different determinants and/or more determinants that more severely affected vitamin d status in these patients . possible reasons for this very high and important prevalence of hypovitaminosis d in these subjects , such as seen in the general population , may include increased urbanization , an increased time spent indoors , and extensive use of sunscreens but also a lower intake of calcium and vitamin d. our data confirm that ds subjects commonly spent less time outdoors and less time being physically active , important contributors to being overweight and/or obese , all factors contributing to reduced 25(oh)d values . our data also show that ds subjects who are obese with a history of autoimmune diseases showed very reduced 25(oh)d levels , conditions very frequently seen in these patients . consistent with other authors [ 46 , 47 ] , we also demonstrate an inverse association between milk intake and a 25(oh)d deficit , although our result is of limited statistical significance due to the small - number statistics of our study . as shown in recent studies in obese children without ds , we also show that obese ds children and adolescents were at a higher risk of a more severe vitamin d deficiency . the explanation for this deficiency , shared in common with the general population , stems from the decreased vitamin d bioavailability from cutaneous and dietary sources because of its deposition in body fat and because obese children may lead a more sedentary , indoor lifestyle [ 48 , 49 ] . in a longitudinal study conducted with 12 ds subjects , zubillaga et al of 25(oh)d plus 1 g of calcium once daily may yield an improvement in the biochemical markers related to the phosphocalcium metabolism and bone remodelling . however , del arco et al . , studying 21 patients with ds , found no child with ds exhibiting values below the normal range , either in vitamin d metabolites or in the other parameters of calcium metabolism . interestingly , the authors also found that the normal increment of 25(oh)d values from march to october was not observed in five children . we do not know if these subjects were obese or had a history of autoimmune diseases . in ds individuals , our data show that vitamin d supplementation did not appear to be sufficient , even if 25(oh)d levels increased significantly after supplementation . however , patients with ds who were also obese and/or had a history of autoimmune diseases seem to need more 25(oh)d supplementation . these data confirm the need to extend vitamin d prophylaxis in all ds children , particularly for the high - risk population of obese individuals and subjects with autoimmune diseases . in this group of patients , we suggest using a higher dose of 25(oh)d than 400 i.u . finally , our data showed that a 25(oh)d deficiency was associated with elevated pth hormone levels , thus confirming the importance of a sufficient vitamin d status to maintain a normal bone metabolism . furthermore , we found that our data showed a correlation between 25(oh)d deficiency and other cardiovascular risk factors ( systolic blood pressure and ldl cholesterol level ) . in fact , vitamin d deficiency has been added as a novel risk factor for cardiovascular disease [ 5052 ] , possibly by the downregulation of many genes , including those involved in renin production , proliferation of cardiac and vascular muscle cells , downregulation of c reactive protein and other proinflammatory markers . vitamin d deficiency has also been reported to be associated with a higher risk of metabolic syndrome and hypertension [ 50 , 53 ] . however , epidemiological and observational studies have kindled a growing interest in the potential role of vitamin d and inflammatory process in the pathogenesis , prevention , and control of many autoimmune diseases , such as type i dm , multiple sclerosis , crohn 's disease , or rheumatoid arthritis . epidemiological evidence suggests that adults with high blood levels have the lowest risk of developing multiple sclerosis or rheumatoid arthritis . however , animal and human studies seem to suggest that vitamin d is a potential modifier of diabetes [ 5557 ] , showing the possible immunomodulatory and anti - inflammatory effects of vitamin d in the reduction of autoimmune insulitis of type i dm [ 34 , 35 ] . moreover , children who show signs of vitamin d deficiency have a 2.4-fold increased risk of developing type i dm . therefore , in ds patients , reduced levels of 25(oh)d may predispose individuals to developing autoimmune diseases . however , it is also interesting to note that the presence of autoimmune disorders may increase this defect , causing other health problems in these subjects . finally , 25(oh)d and pth are important to determine normal bone modeling and remodeling and insure normal bone accrual and muscle - skeletal function . in ds individuals , muscle hypotonia , low levels of physical activity , poor calcium and vitamin d intake , hypogonadism , growth retardation , and thyroid dysfunction may all contribute to substantial impairments in skeletal maturation and bone - mass accrual , potentially predisposing these patients to fragility and fractures . however , it is interesting to note that more and more data in recent years have showed that ds is surely a genetic form associated with an impaired bone status , such as demonstrated by densitometric data evaluated by dxa [ 47 ] , pqct , and qus , although many studies have focused on adults who live either in the community or in residential institutions . in fact , in the murine ds model ts65dn , the low bmd was correlated with significantly decreased osteoblast and osteoclast development , decreased bone biochemical markers , and a diminished bone formation rate . in these mice , recently , low bmd in adults with ds has been discovered to be correlated with a significant decrease in bone formation markers , compared to controls without ds , suggesting a diminished osteoblastic bone formation and inadequate accrual of bone mass . in conclusion , our results indicate that hypovitaminosis d is very frequent in ds individuals and that it is critical to assess the importance of vitamin d prophylaxis in these subjects , in particular individuals who are obese and have a history of autoimmune diseases . the reduced 25(oh)d levels seem to be also related to reduced dietary intake and outdoor activity levels . ds patients who are obese and who have a history of autoimmune diseases may need more 25(oh)d supplementation .

background . poor studies have evaluated 25-hydroxycholecalciferol ( 25(oh)d ) levels in down syndrome ( ds ) . objective . to assess in ds subjects serum 25(oh)d value , to identify risk factors for vitamin d deficiency , and to evaluate whether a normal 25(oh)d value can be restored with a 400 i.u . daily supplement of cholecalciferol in respect to controls . methods . we have longitudinally evaluated 31 ds patients ( aged 4.518.9 years old ) and 99 age- and sex - matched healthy controls . in these subjects , we analysed calcium , phosphate , parathyroid hormone ( pth ) , 25(oh)d concentrations , and calcium and 25(oh)d dietary intakes , and we quantified outdoor exposure . after 12.3 months ( range 8.114.7 months ) of 25(oh)d supplementation , we reevaluated these subjects . results . ds subjects showed reduced 25(oh)d levels compared to controls ( p < 0.0001 ) , in particular ds subjects with obesity ( p < 0.05 ) and autoimmune diseases history ( p < 0.005 ) . pth levels were significantly higher in ds subjects than controls ( p < 0.0001 ) . after cholecalciferol supplementation , 25(oh)d levels were significantly ameliorated ( p < 0.05 ) , even if reduced compared to controls ( p < 0.0001 ) , in particular in ds subjects with obesity ( p < 0.05 ) and autoimmune diseases ( p < 0.001 ) . conclusions . hypovitaminosis d is very frequent in ds subjects , in particular in presence of obesity and autoimmune diseases . in these subjects , there could be a need for higher cholecalciferol supplementation .

1. Introduction 2. Methods 3. Results 4. Conclusions

in recent years , we have witnessed a growing interest in the mass and bone quality of patients with ds ; many studies have evaluated densitometric characteristics via dual - energy x - ray absorptiometry ( dxa ) [ 47 ] , peripheral quantitative computed tomography ( pqct ) , and quantitative ultrasound ( qus ) . an adequate status of 25(oh)d is very important because 25(oh)d deficiency has been shown to be a risk factor for several chronic diseases , in addition to the classic deleterious effects on bone , such as secondary hyperparathyroidism , and reduced bone accrual and mass . the purpose of the present study is to assess serum 25(oh)d in children and adolescents living in tuscany , italy , ( latitude : 44 north ) , and to identify risk factors for vitamin d deficiency in different age groups of individuals with ds . furthermore , this study also aimed to evaluate whether a normal 25(oh)d value can be restored in 25(oh)d - deficient ds patients in respect to controls with a daily supplement of 400 i.u . we longitudinally evaluated 31 caucasian children and adolescents ( 17 males and 14 females , aged 4.518.9 years ) with ds from tuscany in the central region of italy . at t0 and t1 , clinical and demographic data were collected from both the ds patients and healthy controls , including height , weight , body mass index ( bmi ) , blood pressure , pubertal stage , therapies carried out , family and patient histories of autoimmune diseases and osteoporosis , and time dedicated to outdoor physical activity . at t0 and t1 , all of the ds patients and healthy controls underwent laboratory tests to measure their plasma 25(oh)d levels , serum calcium and phosphate , bone specific alkaline phosphatase , parathyroid hormone ( pth ) , triglycerides ( tg ) , total cholesterol , and low - density lipoprotein ( ldl ) cholesterol . vitamin d status : serum 25-oh levels were stratified according to the following brackets : 10 , 1120 , and 2130 and > 30 ng / ml , and they were defined as severe deficiency , deficiency , insufficiency , and sufficiency , respectively , according to previously established guidelines for bone health ( in the absence of a consensus regarding appropriate levels for endocrine and extraendocrine health ) [ 28 , 29 ] . however , for evaluating the seasonal variations of 25(oh)d , we divided the year into four seasons : winter ( december march ) , spring ( march june ) , summer ( june september ) , and autumn ( september december ) . evaluation of dietary intake of calcium and vitamin d : dietary intakes of calcium and 25(oh)d were estimated using standardized interviews ( by the parents ) recording race , religion , country of birth , birth weight , type of feeding during the first year ( breast , formula milk , or mixed ) , mother 's use of vitamin supplementation during her pregnancy , child 's use of vitamin d supplementation , and daily intake of cow 's milk ( categorized as more or less than 200 ml per day ) . vitamin d intervention : all of the subjects ( ds patients and controls ) were treated with 400 i.u . the administration was based on the recommendation of the american academy of pediatrics that stated a recommended daily intake of vitamin d of 400 i.u./day for all infants , children , and adolescents [ 33 , 34 ] . after 25(oh)d supplementation , 25(oh)d levels , serum calcium and phosphate , alkaline phosphatase , and pth were reevaluated . the mean elapsed duration between the first and the second determinations was 12.3 months ( range : 8.114.7 months ) . plasma concentrations of calcium , phosphate , and alkaline phosphatase were determined following routine biochemical laboratory protocols . clinical variables considered relevant to the study were as follows : sex ( m : f ) , bmi sdss , height sdss , age at onset of puberty , pubertal stage , plasma concentrations of calcium , phosphate , alkaline phosphatase , and sera 25(oh)d and pth at the first and second examinations . on the contrary , a statistically significant difference was found regarding height sdss ( 1.5 1.0 versus 0.2 0.8 ; p < 0.005 ) and the bmi sdss ( 1.0 1.4 versus 0.1 0.9 ; p < 0.05 ) , even when considering the children and adolescents separately . evaluating the percentages regarding 25(oh)d sufficiency , insufficiency , and deficiency , 2/31 ( 6.5% ) of ds subjects had sufficient vitamin d levels , 5/31 ( 16.1% ) had insufficient levels , 14/31 ( 45.2% ) showed deficient levels , and 10/31 ( 32.2% ) exhibited a severe deficiency . the percentage of 25(oh)d sufficiency is not significantly different from the controls ( 11/99 : 11.1% ) , but the insufficiency ( 33/99 : 33.3% ; p < 0.005 ) , deficiency ( 35/99 : 35.4% ; p < 0.05 ) , and severe deficiency ( 20/99 : 20.2% ; p < 0.005 ) are significantly different . however , for evaluating the 25(oh)d levels , we show that the ds subjects had significantly reduced 25(oh)d levels compared with the controls ( 14.34 8.31 ng / ml versus 27.04 7.47 ; p < 0.0001 ) ( figure 1(a ) ) . in the ds subjects , 25(oh)d levels were not different between males ( 14.85 8.25 ng / ml ) and females ( 13.75 8.70 ) , children ( 14.26 8.71 ng / ml ) , and adolescents ( 14.45 8.15 ) ; we showed significant statistical differences between ds individuals with normal weights ( 16.93 8.71 ng / ml ) and those who were obese ( 10.20 5.13 ; p < 0.05 ) ( figure 2(a ) ) and ds individuals without ( 19.00 8.06 ng / ml ) and with ( 10.35 6.57 ng / ml ; p < 0.005 ) a history of autoimmune diseases ( figure 3(a ) ) . regarding the effect of the different seasons on 25(oh)d status , the levels of 25(oh)d in ds subjects were significantly reduced in winter , spring , and autumn ( 11.33 5.16 , 7.85 4.67 , and 12.42 4.96 ng / ml , resp . ) with respect to summer value ( 22.53 8.87 ng / ml ; p < 0.005 , p < 0.001 , and p < 0.05 , resp . ) however , these values are always significantly smaller in ds individuals than in controls ( winter : 18.9 6.3 ng / ml ; p < 0.005 ; spring : 24.82 6.06 ng / ml ; p < 0.0001 ; summer : 30.33 11.03 ng / ml ; p < 0.005 ; autumn : 21.23 6.24 ng / ml ; p < 0.005 ) ( figure 4 ) . however , ds patients also had significantly higher pth levels compared with the controls ( 54.76 32.15 versus 26.13 10.76 pg / ml ; p < 0.0001 ) ( figure 5(a ) ) . pth levels were higher , but not significantly , in ds adolescents ( 60.50 38.45 pg / ml ) compared with children ( 46.30 27.93 pg / ml ) . these results differ from what is seen in the controls , in which we showed that pth levels in children were 25.23 9.10 pg / ml in adolescents ; a statistical difference between ds individuals and controls was seen in children ( p < 0.0001 ) but not in adolescents . no significant differences were found in calcium intake between ds patients and the controls ( 796 283 versus 821 256 mg / day ) , even if the samples were divided into child ( 810 270 versus 835 285 mg / day ) and adolescent ( 755 290 versus 790 235 mg / day ) groups . nevertheless , despite the dietary calcium intake not being different between ds and control individuals , the amount of cow 's milk drunk per day was related to 25(oh)d levels , with a high percentage ( 18/24 , 75.0% ) of ds patients consuming less milk and showing 25(oh)d levels in the range of a deficiency or severe deficiency . in particular , only 30% of children with a severe deficiency ( n = 3/10 ) consumed more than 200 ml of cow milk per day versus 71.4% of children ( n = 5/7 ) with sufficient or insufficient 25(oh)d levels ( p = 0.002 ) . these data are similar to what is seen in the controls , with a high percentage ( 38/55 , 69.1% ) of individuals consuming less milk showing 25(oh)d levels in the range of a deficiency or severe deficiency . ; however , regarding the effect of hours spent outdoors ( including also physical activity ) on 25(oh)d levels , there was no association between the reported average daily number of hours spent outdoors and baseline 25(oh)d levels ( p = ns ) , even if patients with ds who spent more than 8 hours / week outdoors showed higher 25(oh)d levels than patients with who spent fewer than 4 hours / week outdoors ( 21.75 6.43 versus 9.87 4.35 ng / ml ; p = 0.006 ) . evaluating correlations among 25(oh)d and age , sex , seasons , physical activity , milk intake , pth , bmi , height , total cholesterol , triglycerides , ldl cholesterol , systolic blood pressure , diastolic blood pressure , and autoimmune disease development , we showed that 25(oh)d levels correlated inversely with pth ( r = 0.42 , p < 0.005 ) , bmi ( r = 0.39 , p < 0.005 ) , physical and outdoor activities ( r = 0.31 , p < 0.05 ) , milk intake ( r = 0.30 , p < 0.05 ) , total cholesterol ( r = 0.43 , p < 0.005 ) , ldl cholesterol ( r = 0.28 , p < 0.05 ) , systolic blood pressure ( r = 0.34 , p < 0.005 ) , and autoimmune diseases ( r = 0.56 , p < 0.005 ) . the multivariate linear regression analyses showed that serum 25(oh)d concentration was negatively associated with bmi ( = 0.29 , p < 0.005 ) . after supplementation with 25(oh)d and evaluating the percentages of ds patients and controls with 25(oh)d sufficiencies , insufficiencies , and deficiencies , we show that 7 ( 22.6% versus 6.5% ; p < 0.001 ) ds patients achieved sufficient vitamin d levels , 8 ( 25.8% versus 16.1% ; p < 0.05 ) achieved insufficient levels , 9 ( 29.0% versus 45.2% ; p < 0.001 ) achieved deficient levels , and 7 ( 22.6% versus 32.2% ; p < 0.05 ) still showed a severe deficiency . the results were significantly different for the healthy controls : 26 ( 26.3% versus 11.1% ; p < 0.001 ) reached sufficient vitamin d levels , 43 ( 43.4% versus 33.3% ; p < 0.05 ) reached insufficient levels , 21 ( 21.2% versus 35.4% ; p < 0.005 ) reached deficient levels , and 9 ( 9.1% versus 35.4% ; p < 0.001 ) reached severe deficient levels . however , in terms of 25(oh)d levels in ds subjects at the end of intervention , even if the level was significantly ameliorated ( 20.15 10.88 versus 14.34 8.31 ng / ml ; p < 0.05 ) , these patients still showed extremely reduced levels compared with the controls ( 28.27 7.96 ; p do not continue to be different between males ( 19.35 9.63 ng / ml ) and females ( 21.08 12.55 ; p = ns ) and children ( 19.00 11.49 ng / ml ) and adolescents ( 22.09 9.72 ; p = ns ) , whereas we showed significant statistical differences between ds subjects characterized by a normal weight ( 23.50 11.06 ng / ml ) and individuals who were obese ( 14.80 8.86 ; p < 0.05 ) ( figure 2(b ) ) and ds individuals with ( 12.07 6.81 ng / ml ) and without ( 28.41 8.19 ng / ml ; p < 0.001 ) a history of autoimmune diseases ( figure 3(b ) ) . regarding dietary calcium intake , even if calcium intake was not significantly different between ds patients and controls ( 846 256 versus 889 221 mg / day ) , we showed an increased but nonsignificant change in calcium intake with respect to baseline values . however , the percentage of patients and controls who drank more than 200 ml of cow 's milk per day was significantly increased : in ds patients , 16/31 ( 51.6% ) individuals continued to consume fewer than 200 ml of cow 's milk for day , a better outcome than the 75% of ds patients reported in the first evaluation ( p < 0.0001 ) . this aspect was similar in the controls , with a significant amelioration of the percentage of people consuming more than 200 ml of cow 's milk per day ( 27.2% versus 38.4% ; p < 0.05 ) . nevertheless , milk consumption per day was always related to 25(oh)d levels , with a high percentage ( 14/16 , 87.5% ) of ds patients consuming less milk showing 25(oh)d levels in the range of a deficiency or severe deficiency . ; however , regarding the effect of the number of hours spent outdoors ( including also physical activity ) on 25(oh)d levels , there was no association between the reported average daily hours spent outdoors and baseline 25(oh)d levels ( p = ns ) , even if patients with ds who spent more than 8 hours / week outdoors showed higher 25(oh)d levels than patients with a history spending fewer than 4 hours / week outdoors ( 26.58 9.00 versus 15.64 11.71 ng / ml ; p < 0.005 ) . ds patients still had significantly higher pth levels compared with controls ( 43.57 14.05 versus 26.89 13.56 pg / ml ; p < 0.005 ) ( figure 5(b ) ) . however , ds subjects who were obese still showed significantly higher pth levels ( 54.90 13.45 pg / ml ) than ds individuals with normal weights ( 36.50 9.04 pg / ml ; p < 0.005 ) . however , ds patients with a history of autoimmune diseases showed higher pth levels ( 56.70 11.98 pg / ml ) than patients without a history of autoimmune diseases ( 35.37 7.51 pg / ml ; p < 0.05 ) . in evaluating the correlations among 25(oh)d and age , sex , seasons , physical activity , milk intake , pth , bmi , height , total cholesterol , triglycerides , ldl cholesterol , systolic blood pressure , diastolic blood pressure , and a history of autoimmune disease , we showed that 25(oh)d levels were still inversely correlated with pth ( r = 0.38 , p < 0.05 ) , bmi ( r = 0.43 , p < 0.005 ) , physical and outdoor activity ( r = 0.34 , p < 0.05 ) , total cholesterol ( r = 0.33 , p < 0.05 ) , ldl cholesterol ( r = 0.27 , p < 0.05 ) , systolic blood pressure ( r = 0.30 , p < 0.05 ) , milk intake ( r = 0.36 , p < 0.05 ) , and autoimmune diseases ( r = 0.59 , p < 0.005 ) . as with general population , for ds individuals , the 25(oh)d values differed according to the seasons , even if these values remain always less than in the control population , demonstrating the role of many different determinants and/or more determinants that more severely affected vitamin d status in these patients . possible reasons for this very high and important prevalence of hypovitaminosis d in these subjects , such as seen in the general population , may include increased urbanization , an increased time spent indoors , and extensive use of sunscreens but also a lower intake of calcium and vitamin d. our data confirm that ds subjects commonly spent less time outdoors and less time being physically active , important contributors to being overweight and/or obese , all factors contributing to reduced 25(oh)d values . our data also show that ds subjects who are obese with a history of autoimmune diseases showed very reduced 25(oh)d levels , conditions very frequently seen in these patients . in ds individuals , our data show that vitamin d supplementation did not appear to be sufficient , even if 25(oh)d levels increased significantly after supplementation . in this group of patients , we suggest using a higher dose of 25(oh)d than 400 i.u . however , epidemiological and observational studies have kindled a growing interest in the potential role of vitamin d and inflammatory process in the pathogenesis , prevention , and control of many autoimmune diseases , such as type i dm , multiple sclerosis , crohn 's disease , or rheumatoid arthritis . therefore , in ds patients , reduced levels of 25(oh)d may predispose individuals to developing autoimmune diseases . in ds individuals , muscle hypotonia , low levels of physical activity , poor calcium and vitamin d intake , hypogonadism , growth retardation , and thyroid dysfunction may all contribute to substantial impairments in skeletal maturation and bone - mass accrual , potentially predisposing these patients to fragility and fractures . in these mice , recently , low bmd in adults with ds has been discovered to be correlated with a significant decrease in bone formation markers , compared to controls without ds , suggesting a diminished osteoblastic bone formation and inadequate accrual of bone mass . in conclusion , our results indicate that hypovitaminosis d is very frequent in ds individuals and that it is critical to assess the importance of vitamin d prophylaxis in these subjects , in particular individuals who are obese and have a history of autoimmune diseases .

integrating sensory information and producing the appropriate motor output are the basic functions of the nervous system , and the neural networks underlying these two functions are tightly linked . the basal ganglia are involved in various functions , including motor learning , planning , and execution , as well as in decision making and reward ( haber , 2008 ; middleton and strick , 2000 ; schultz et al . , the input layer of the basal ganglia , striatum , receives glutamatergic inputs from multiple cortical areas , including sensory , motor , and prefrontal cortices ( alloway et al . , 2009 ; these projections are characterized by a high degree of divergence and convergence ( flaherty and graybiel , 1991 ) , enabling striatal neurons to integrate inputs from different cortical areas and modalities ( chudler et al . , 1995 ; nagy et al . , 2005 , 2006 ; wilson et al . , 1983 ) . corticostriatal projections originate from both hemispheres and are mediated by different subtypes of pyramidal neurons ( carman et al . , 1965 ; kress et al . , 2013 ; knzle , 1975 ; lei et al . , 2004 ; wall et al . , 2013 ) , suggesting that striatal neurons may receive bilateral sensory input with different synaptic properties . striatal neurons respond to sensory input from different modalities such as tactile , auditory , and visual input ( brown et al . , 1996 ; nagy et al . , 2005 ; schulz et al . , 2009 ; wilson et al . , 1983 ) . due to the high convergence in the corticostriatal pathway and the loose topographical correspondence ( kincaid et al . , 1998 ) , individual striatal neurons may be involved in tactile - visual sensory integration ; however , such synaptic integration at the single neuron level has not yet been shown in striatum . striatal projection neurons ( msns ) are divided into two main subpopulations according to their projection via the direct or indirect pathway ( alexander and crutcher , 1990 ; smith et al . , 1998 ) , with direct pathway neurons facilitating motor activity and indirect pathway neurons inhibiting it ( albin et al . , 1989 ; kravitz et al . , both subpopulations receive cortical as well as thalamic inputs ( doig et al . , 2010 ) ; however , it has been debated whether contralateral and ipsilateral corticostriatal projections are selective or biased in targeting direct and indirect pathway msns , respectively ( kress et al . , 2013 ; lei et al . , 2004 corticostriatal synapses formed onto direct and indirect pathway neurons and interneurons have different properties ( calabresi et al . , 1996 ; fino and venance , 2011 ; kreitzer and malenka , 2008 ; surmeier et al . , 2007 ) , suggesting that striatal neurons of different types may also respond differently to sensory input in vivo . msns recorded in vivo are characterized by low discharge frequencies ( adler et al . , 2012 ; berke et al . , 2004 ; wilson , 1993 ) , suggesting that a large fraction of their synaptic inputs are subthreshold and do not often contribute to action potential discharge . we therefore used whole - cell striatal recordings in order to study synaptic responses to tactile and visual stimuli . we show that neurons throughout dorsal striatum respond to bilateral whisker stimulation in a type - dependent manner and that neurons in dorsomedial striatum perform multisensory integration . whole - cell patch - clamp recordings were obtained from neurons in dorsolateral striatum ( n = 59 neurons ) , dorsomedial striatum ( n = 50 neurons ) , and layer v of s1 barrel field ( n = 20 neurons ) . of all recorded neurons ( n = 129 ) , 45 were stained and morphologically reconstructed ( see experimental procedures ) , three of which were cortical pyramidal neurons and 42 were striatal neurons . striatal neurons were located between 1,854 and 2,613 m below the pia , and the average recording time for all neurons was 48 20 min ( minimum = 9 min , maximum = 100 min ; n = 129 ) . the striatal regions targeted for recordings were selected according to the existence of corticostriatal projections from primary somatosensory and visual cortices ( s1 and v1 , respectively ) following anterograde tracing ( see experimental procedures ; figures 1a , 1b , 3a , and 3b ) . as described in previous studies ( alloway et al . , 1999 , 2006 ; 2001 ) , projections from s1 ( barrel field ) were found throughout the dorsal striatum , with higher concentration in dorsolateral striatum ( figures 1b ; figure s1 available online ) . projections from visual cortex , on the other hand , were located dorsomedially in proximity to the lateral ventricle ( figure 3b ) , in agreement with previous reports in other species ( donoghue and herkenham , 1986 ; faull et al . , 1986 ; norita et al . , 1991 ; serizawa et al . , 1994 ) . all recorded cortical and striatal neurons exhibited slow wave oscillations ( figures 2a , 3c , s4 , s6 , and s7 ) with bimodal distribution of the membrane potential ( figures s4 and s7 ) , as previously described ( wilson and kawaguchi , 1996 ) . striatal neurons , however , had longer up state durations than cortical neurons and rarely discharged action potentials , unlike cortical neurons , which discharged several aps during up states ( figure s4 ) . the input resistance of recorded neurons was extracted using step current injections and was calculated separately for up and down states ( figures s4f , s4h , and s4i ) . unlike cortical neurons , striatal neurons had higher input resistance at up states and at more depolarized membrane potentials ( figure s4f ) , suggesting that inward rectification in msns ( kita et al . , 1984 ; nisenbaum and wilson , 1995 ) is the dominant factor determining their input resistance as recorded at the soma , even in the presence of the synaptic barrages occurring during the up states . in order to study bilateral sensory integration , we delivered brief air puffs to the whisker pads on both sides ( see experimental procedures ) and recorded subthreshold responses to ipsilateral , contralateral , and bilateral stimulation in dorsolateral striatal neurons . using the same stimulation protocol , we compared the whisker - evoked responses in striatal msns to cortical regular spiking neurons ( putative pyramidal cells ) in layer 5 of the barrel cortex ( figures 1 and s2 ; table 1 ) . all recorded neurons responded to both ipsilateral and contralateral whisker stimulation ( cortical n = 17 ; striatal msns n = 20 ) . sensory responses were classified according to those occurring during up or down states , including cases in which sensory stimulation triggered state transitions ( reig and sanchez - vives , 2007 ) ( figure 1 and s2 , respectively ) . response onset delays were significantly longer for ipsilateral than contralateral whisker stimulation in both cortical and striatal neurons ( bf ipsilateral : 25.11 6.49 ms , contralateral : 13.32 3.61 ms , p < 0.001 , n = 17 ; dorsolateral striatum ipsilateral : 28.45 6.94 ms , contralateral : 19.78 3.42 ms , p < 0.001 , n = 20 ) ( figure 1f ) . corresponding differences were observed also for response peak latencies ( bf ipsilateral : 71.13 21.98 ms , contralateral : 37.75 8.52 ms , p < 0.001 , n = 17 ; striatum ipsilateral : 66.88 26.07 ms , contralateral : 52.62 15.82 ms , p < 0.01 , n = 20 ) ( figure 1 g ) . onset and peak latencies in response to contralateral and bilateral stimulation were significantly shorter in cortical neurons ( figures 1f and 1 g ) . response amplitudes for both cortical and striatal neurons were always larger when occurring during down states than during up states ( figures 1h and s2f , respectively ) . contralateral whisker stimulation evoked larger amplitudes than ipsilateral stimulation in both cortical and striatal neurons ( cortex during down states , ipsilateral : 9.11 3.41 mv , contralateral : 14.07 5.67 mv , p < 0.001 ; striatum during down states , ipsilateral : 10.27 3.27 mv , contralateral : 15.65 6.90 mv , p < 0.01 ) ( figure 1h ) . however , when stimuli were delivered simultaneously to both sides , a significant increase in amplitude was observed in striatal responses but not in cortical ones ( cortex bilateral : 15.72 7.09 mv ; striatum bilateral : 20.37 6.52 mv , p < 0.05 ) ( figure 1h ) . the increase in amplitude in the striatal msns is also reflected in the slopes of responses to bilateral stimulation ( figure 1i ) , both suggesting a higher sensitivity to bilateral input in striatal msns than that observed in s1 layer 5 pyramidal cells . this difference could be explained by the higher temporal separation between ipsilateral and contralateral responses in cortical neurons compared to msns ( response peak latency : cortex = 33.38 21.46 ms ; striatum = 14.26 24.96 ms , p < 0.05 , data not shown ) . sensory and electrically evoked excitation of striatal neurons is accompanied by inhibition ( pidoux et al . , 2011 ) , originating from striatal interneurons and msn collaterals ( kos and tepper , 1999 ; tunstall et al . , 2002 ) . in order to decompose the excitatory and inhibitory response components , we used a low - chloride intracellular solution ( see experimental procedures ) enabling us to hold recorded neurons at the reversal potential for excitation ( 5 mv ) or gabaa inhibition ( 70 mv , figures 2a2d ) . in all recorded msns , excitation preceded inhibition for ipsilateral , contralateral , and bilateral whisker stimulation ( onset delays : ipsilateral excitation 24.63 3.76 ms , inhibition 40.6 9.7ms , p < 0.01 ; contralateral excitation 19.16 2.8 ms , inhibition 28.98 1.93 ms , p < 0.001 ; bilateral excitation 19.71 3.27 ms , inhibition 25.61 3 ms , p < 0.01 , n = 8) ( figure 2c ) . regardless of the stimulus condition ( ipsilateral , contralateral , or bilateral whisker stimulation ) , larger excitatory responses were matched by larger inhibitory ones ( figure 2d ) . spontaneous activity was also mediated by mixed excitatory and inhibitory synaptic barrages during up states , as recorded at the respective reversal potentials ( figures 2a and s4 ) . we next wanted to assess the stimulus intensity dependence of the different response conditions . to that end , we altered the pressure generating the air puff , resulting in different deflection intensities . the air puff duration remained constant ( 15 ms ) , and the air pressure was changed in a range between 0 and 30 psi ( 0 , 2 , 5 , 10 , 20 , and 30 psi ) ( figure 2e ) . for all three stimulus conditions , onset and peak delays were shortened , and amplitudes and slopes increased when air puff pressure was increased from 2 to 20 psi ( figures 2e2i ) . interestingly , response durations , measured as the width at 75% amplitude , were inversely related to the amplitudes and stimulus strength ( figures 2j2l ) , possibly due to curtailing of responses by the increased inhibitory component ( figure 2d ) . the relationship between ipsilateral and contralateral responses was maintained for different stimulus intensities , with contralateral ( and bilateral ) stimulation evoking earlier onset and peak response latencies ( figures 2f and 2 g ) , with larger amplitudes and rise slopes ( figures 2h and 2i ) . in order to study responses of striatal neurons to visual stimulus , we obtained whole - cell recordings from neurons in dorsomedial striatum ( figure 3 ) . the recording area was selected according to the presence of axonal projections from cortical v1 ( figures 3a and 3b , n = 5 ) , which were clustered in dorsomedial portions of the striatum , near the lateral ventricle ( figure 3b ) . visual stimuli were presented to the contralateral eye as brief light flashes from a white led ( see experimental procedures ) during whole - cell recordings in striatum and lfp recordings from v1 . as described above , responses were sorted offline to those occurring during up or down states . the onset delay for contralateral visual responses during down states was 98.43 19.05 ms ( ranging between 56.7 and 141.0 ms , n = 25 ) , almost five times longer than the onset response to whisker stimulation ( 19.78 3.42 ms , figure 1f ) and similar to previously reported visual responses ( schulz et al . , 2009 , 2011 ) . such delays are expected when comparing to the long and variable delays ( 50130 ms ) described for visual responses in mouse visual cortex ( niell and stryker , 2008 ; takagaki et al . , 2008 ) . in order to verify the occurrence of cortical visual responses , we obtained extracellular recordings ( lfp ) in v1 simultaneously with the striatal whole - cell recordings ( n = 16 , figures 3c and 3d ) . onset and peak latencies were slightly earlier , although not significantly different between cortical and striatal visual responses ( figures 3e and 3f ) , which may be due to the large response variability . the amplitude of visual responses was 13.28 5.38 mv and slopes 0.14 0.15 mv / ms ( data not shown , n = 25 ) . visual responses were evoked in 25 of dorsomedial neurons ( 66% ) and in none of the dorsolateral ones ; however , all neurons in both regions responded to bilateral whisker stimulation ( figure 4 ) . figure 4c depicts the position of anatomically reconstructed neurons , in which both tactile and visual stimulation were presented ( n = 30 ) . all neurons that responded to visual stimuli were located medially ( figure 4c , orange points ) , close to the area receiving axonal projections from v1 ( figure 3b ) . the differences in onset and peak delays between whisker and visual sensory responses suggested that the strongest responses in striatal neurons would not occur when stimuli are presented simultaneously , but rather when visual stimulation precedes tactile stimulation . to test this prediction , we presented the whisker stimuli at different time intervals with relation to the visual stimuli ( figure 4d ) . indeed , in all cases ( n = 7 ) maximal response amplitudes were evoked when whisker deflections followed the visual stimuli synchronized to the respective response onsets ( relative interval 102.11 20.98 ms , figure 4e ) . multisensory responses did not summate linearly , with maximal response amplitudes smaller and earlier than those predicted by the linear sum of unimodal responses ( independent whisker 13.73 3.15 mv , independent visual 13.23 4.66 mv , synchronized onset 18.19 4.54 mv , n = 7 ) ( figures 4d and 4e ) . this sublinear summation is expected due to the proportional inhibitory component of sensory responses ( figures 2a2d ) but may also be mediated by activation of common cortical association areas ( olcese et al . , 2013 ) . as shown above neurons in both dorsolateral and dorsomedial striatum responded to whisker stimulation ; however , there were notable differences in responses recorded in both striatal regions . dorsolateral msns had stronger and faster responses ( figure 4f ) , as reflected in response amplitudes ( contralateral whisker , dorsolateral striatum : 15.6 6.9 mv , dorsomedial 8.4 3.67 mv ; bilateral dorsolateral 20.37 6.52 mv , dorsomedial 10.29 4.42 mv ) ( figure 4h ) and slopes ( ipsi - dorsolateral 0.36 0.24 mv / ms , dorsomedial 0.16 0.08 mv / ms ; contralateral dorsolateral 0.63 0.46 mv / ms , dorsomedial 0.17 0.09 mv / ms ; bilateral dorsolateral 1.08 0.63 mv / ms , dorsomedial 0.28 0.18 mv / ms , dorsolateral n = 20 , dorsomedial n = 24 ) ( figure 4i ) . there were no differences in onset delays ( ipsi - dorsolateral 28.45 6.94 ms , dorsomedial 31.8 8.35 ms , p = 0.16 ; contra - dorsolateral 19.78 5.09 ms , dorsomedial 18.24 6.68 ms , p = 0.41 ; bilateral dorsolateral 20.49 5.19 ms , dorsomedial 18.39 5.71 ms , p = 0.23 , data not shown ) ; however , there were significant differences for peak delays in all conditions , reflecting the slower response rising slopes ( figure 4 g ) . these differences in sensory responses correlate to the differences between dorsolateral and dorsomedial striatum in the density of axonal projections from primary somatosensory cortex ( figures 1b and s1 ) . in summary , neurons located in the dorsomedial striatum integrate tactile and visual sensory inputs , with maximal responses when the respective response onsets are aligned . dorsolateral msns do not respond to visual stimulation but have larger and faster responses to whisker stimulation than dorsomedial ones . the large majority of striatal neurons are msns , projecting via the striatonigral ( direct ) and striatopallidal ( indirect ) pathways . since these projection pathways are believed to have different roles in basal ganglia function , it is important to understand whether and how they differ in their integration of sensory input . to that end , whole - cell recorded and electrophysiological identified msns were subsequently immunostained with d1 antibody ( see experimental procedures ; figures 5a , s5 , and s6 ) in order to classify them as direct or indirect pathway msns . the staining allowed us to classify msns into d1-expressing and putative d2-expressing msns , which we refer to below as d2 msns . using a drd2 bac transgenic mouse we verified that the d1 antibody did not stain d2-egfp msns ( figure s5 ) , suggesting that recorded msns that were d1 negative are indeed d2 msns . msn subtype identification was obtained for 28 msns : 15 d1 positive and 13 d2 neurons ( figures 5a and s6 ) . while similar in most of their electrophysiological properties , d2 msns had higher input resistance than d1 msns , as measured by injection of a depolarizing and hyperpolarizing current step during down states ( in mohm , depolarized d1 112.85 25.76 and d2 158.81 28.93 , p < 0.001 ; hyperpolarized d1 88.13 27.7 and d2 107.81 39.01 , p < 0.05 ) ( figure 5c ) . similar results were reported for d1 and d2 msns in recent slice studies ( gertler et al . the differences in onset delays between contralateral and ipsilateral responses were significantly larger in d1 msns ( d1 ipsi : 31.18 6.49 ms , contra : 17.07 7.97 ms , p < 0.001 ; d2 ipsi : 25.16 3.68 ms , contra : 20.38 5.35 ms , p < this difference was caused by the response onset for ipsilateral stimulation that was longer in d1 than d2 msns ( d1 : 31.18 6.49 ms , d2 25.16 3.68 ms , p < peak responses to contralateral and ipsilateral stimulation also had different latencies in d1 msns ( ipsi 89.85 44.44 ms , contra 63.08 24.87 ms , p < 0.01 ) but were similar in d2 msns ( ipsi 70.33 23.47 ms , contra 63.71 23.52 ms , p = 0.192 ) ( figures 5d , 5f and 5 g ) . response amplitudes were overall larger in d1 msns than d2 msns , significantly so for contralateral whisker stimulation ( d1 msns 15.14 5.47 mv , d2 msns 10 4.93 mv , p < 0.05 ) ( figure 5h ) . the two msn subpopulations also had differences in the response slopes , where d1 msns had faster slopes for contralateral versus ipsilateral stimuli ( p < 0.05 ) but not d2 msns ( p = 0.13 , see table 2 ; figure 5i ) . both d1 and d2 msns had the fastest slopes for bilateral stimulation ( see table 2 ; figure 5i ) . differences between d1 and d2 msns in their responses to whisker stimulation persisted also when occurring during up states ( figure s6 ) ; however , no differences were observed in spontaneous up states amplitudes or durations ( vm down states d1 72.28 5.44 mv , d2 72.66 3.02 mv ; ap threshold d1 46.24 3.31 mv , d2 45.71 3.73 mv ; up state amplitude d1 11.41 3.16 mv , d2 12.24 4.6 mv ; up state duration d1 0.62 0.18 s , d2 0.61 0.26 s ; ap frequency d1 0.16 0.31 hz , d2 0.10 0.13 hz ) ( figure s6 ) . we also recorded from identified msns in dorsomedial striatum that responded to whisker and visual stimulation ( five d1-msns and three d2-msns ) , thus showing that both msn types integrate multimodal sensory inputs ( figures 5 , 5j , and 5k ) . the small sample size of type - identified visually responding msns prevents us from drawing conclusions regarding their respective integration properties . in summary , msns belonging to the direct and indirect pathways exhibited significant differences in their bilateral integration of tactile sensory inputs . direct pathway msns ( d1 ) responded earlier ( onset delay ) , stronger ( amplitude ) , and faster ( slope ) to contralateral compared to ipsilateral whisker stimulation . in contrast , in d2 msns contralateral and ipsilateral stimulation resulted in more similar response properties , with ipsilateral responses being earlier ( onset ) and faster ( slope ) . these results suggest that d1 and d2 msns have different roles in their sensory integration , d1 msns tuned to detect differences between ipsilateral and contralateral whiskers than d2 msns , which act as integrators of bilateral inputs . striatal interneurons form a small albeit diverse minority in the striatal microcircuitry ; therefore , our method of blind whole - cell patch - clamping resulted in a rather small number of interneurons . out of 109 recorded striatal neurons , four were classified as interneurons , of which two were fast spiking ( figures s7a s7d ) and two others were cholinergic interneurons ( figures s7e s7h ) . neurons were initially classified according to their recorded electrical properties and following morphological staining , according to the aspiny dendrites and the large soma size ( in the case of the cholinergic interneurons ) . fs interneurons displayed narrow action potentials , relatively depolarized rest potential , high discharge rate of action potentials during up states , and no apparent inward rectification . cholinergic interneurons were characterized by their voltage sag response to current step injections , depolarized membrane potential , and spontaneous discharge activity . they also displayed spontaneous slow wave activity , although the amplitude range was not as wide as in fs interneurons or msns yet was sufficient to phase - lock spontaneous discharge to the cortical oscillations as recorded simultaneously in s1 and v1 ( figure s7 ) . as in all other recorded neurons , interneurons responded to either whisker stimulation with stronger and earlier responses to the contralateral whisker deflection ( figures s7c , s7 g , and s7i ) , and in one interneuron where visual responses were tested , the recorded fs responded to visual input ( figure s7c ) . in this study we used whole - cell patch - clamp recordings to study the integration of bilateral and multimodal sensory information in striatal neurons . we show that individual striatal neurons integrate bilateral as well as multisensory inputs , that both spontaneous and sensory evoked inputs are comprised of overlapping excitatory and inhibitory synaptic input , and that msns of the direct and indirect pathways differ in the way they integrate bilateral sensory input . all neurons recorded in dorsal striatum , including projection neurons and interneurons , responded to bilateral whisker stimulation in a type - dependent manner , and a population of dorsomedial neurons also responded to visual input . recordings were obtained under anesthesia , enabling activation of sensory pathways while avoiding interference with motor related inputs . in future studies it would be of interest to study these sensorimotor interactions in the unanesthetized , behaving animal . all neurons recorded in the dorsal striatum responded to stimulation of whiskers of both sides . the responses differed in latency , slope , and amplitude , with contralateral whisker stimulation inducing larger and earlier responses as seen also in the recordings from cortical neurons . there are , however , notable differences between the striatal and cortical responses , suggesting different integration properties . onset delays for contralateral and bilateral responses in dorsolateral msns were 6 to 7 ms longer than the cortical ones ( figure 1f ) . this result , together with the anatomical tracing data , supports the idea that the responses in striatal neurons under our experimental conditions are generated primarily by cortical inputs without engaging a thalamic shortcut ( mowery et al . , 2011 ) . bilateral responses were relatively larger in striatal neurons than in cortical ones ( figure 1h ) , showing that striatal msns act as integrators of bilateral sensory input to a higher degree than cortical neurons . this result can be explained by the time differences between ipsilateral and contralateral onset latencies , which were shorter in striatal neurons , in particular d2 msns ( figure 5 g , inset ) . the sensory responses we observed were almost entirely subthreshold , compared to a larger fraction of suprathreshold responses to contralateral whisker stimulation recently reported in rats ( pidoux et al . , 2011 ) . the discrepancy in the measured contralateral responses may be explained by differences in the air puff duration and pressure settings ( see experimental procedures ) , striatal coordinates ( medial - lateral or rostro - caudal axes ) , and species ( rat and mouse ) , but it may also be attributed to differences between intracellular sharp and whole - cell recordings ( staley et al . , 1992 ) . neurons in both dorsolateral and dorsomedial striatum responded to bilateral whisker stimulation ; however , these responses differed in several aspects . response amplitudes and slopes were larger in dorsolateral striatum ( figures 4f4i ) ; however , onset latencies were similar between the two regions , suggesting that both receive monosynaptic inputs from s1 . these results together with the anatomical results ( figure 1b ) suggest that the primary target receiving whisker information is indeed the dorsolateral striatum . a larger striatal area , however , receives the sensory input generating an attenuated and slower response , which , in the case of the dorsomedial striatum , also receives sensory information from a different modality ( figures 4a4e ) . these differences in sensory input to dorsolateral and dorsomedial striatal regions may underlie the differences in their discharge pattern as recorded during task performance ( thorn et al . , 2010 ) . sensory - evoked responses in striatal neurons in all tested cases were composed of excitatory and inhibitory components ( figure 2 ) . inhibition followed excitation by a few milliseconds , suggesting that it was mediated by intrastriatal gabaergic neurons driven by the same excitatory input . although the inhibitory component was smaller in amplitude than the excitatory input , it could be strong enough to shape striatal output by preventing or delaying msn discharge ( kos and tepper , 1999 ) . in vivo studies in neocortex have shown that visual and tactile sensory input induces temporally complex inhibitory inputs mediated by gabaergic interneurons ( haider et al . , 2013 ; monier et al . , 2003 is mediated by gabaergic interneurons , in the striatum at least part of the inhibitory component may arise from msn collaterals ( tunstall et al . , 2002 ) , in addition to that from gabaergic interneurons . it is not known which interneurons provide the observed inhibition onto msns ; however , likely candidates are parvalbumin - expressing fs interneurons ( gerfen et al . , 1985 ) . , 2010 ; kos and tepper , 1999 ; planert et al . , 2010 ) and are the first neurons to be activated by cortical input , even before neighboring msns ( mallet et al . , 2005 ) . a similar form of feedforward inhibition from fs interneurons onto projection neurons exists in the thalamocortical pathway , where an early activation of fs interneurons by thalamic synaptic input provides rapid disynaptic inhibition of excitatory neurons ( cruikshank et al . , 2007 ) . inhibition was also present during ongoing activity , in particular during up states ( figures 2a and s4 ) , as reported also in cortical up states ( haider et al . this form of inhibition is likely to originate from striatal neurons that are active during up states ; however , the identity of these neurons is not clear . in our recordings , only a small fraction of msns discharged spontaneous action potentials , whereas the small sample of recorded fs interneurons were more spontaneously active ( figure s7 ) . striatal inhibition may also arise from external sources such as neurons in globus pallidus , which increase their discharge rate during striatal up states ( goldberg et al . , 2003 ) . a particularly robust pallidostriatal inhibitory pathway is mediated by the recently described arkypallidal neurons ( mallet et al . , 2012 ) . the source of inhibition during both spontaneous activity and sensory - evoked responses should be addressed in future studies using cell - type - specific manipulations . visual responses were seen in neurons recorded in the dorsomedial striatum in a relatively large yet restricted striatal region that also received axonal projections from visual cortex , thus suggesting that at least part of the response was mediated by excitatory projections from visual cortex ( figure 3 ) . visually evoked responses were recently described in neurons from rat dorsal striatum , mediated mainly by subcortical inputs following disinhibition of superior colliculus ( schulz et al . recorded neurons were located more laterally than in the present study , which together with the different species and anesthesia may explain the different visual responsiveness . as in the current study , responses were almost entirely subthreshold and had similar latencies ( schulz et al . , 2009 ) , suggesting that the visual responses in our recordings may originate from multiple afferent pathways , both cortical and subcortical . multisensory responses have been described in different basal ganglia nuclei including the striatum using intracellular , extracellular , and optical recordings ( chudler et al . , 1995 ; cui et al . , 2013 ; hikosaka et al . , 1989 ; nagy et al . , 2005 , 2006 ; wilson et al . , 1983 ) . those studies show that a fraction of striatal neurons changed their discharge rate when presented with sensory input of different modalities , mainly somatosensory and auditory . our findings support these studies and describe the synaptic input underlying such cross modal interactions . the striatal neurons we recorded in dorsal striatum showed clear preference for whisker stimulation , while only a fraction of them responded to both tactile and visual input , depending on their topographic location ( figure 4c ) . responses to whisker stimulation were significantly earlier than those to visual stimulation ( figure 4 ) . in the neocortex , whisker responses also show shorter latencies ( manns et al . , 2004 ) the longer latencies in visual responses are in part attributed to retinal processing and may functionally be compensated via retinal motion prediction ( berry et al . , 1999 ) . another possible explanation may lie in the nature of these stimuli , with tactile stimulation originating from nearby objects , whereas visual input would originate from more distal objects , before touching the whiskers . these differences in processing time between visual and tactile stimuli suggest that introducing a time lag between tactile and visual stimuli would result in increased responsiveness in the striatum , as observed in cortical multisensory areas ( olcese et al . , 2013 ) . in agreement with the cortical studies , we showed that maximal response amplitude occurred when visual and whisker inputs simultaneously impinged onto postsynaptic msns . in the current study we only studied visual and tactile sensory integration ; however , the striatum also integrates other sensory modalities such as auditory ( bordi and ledoux , 1992 ) and olfactory ( mcdonald , 1991 ; novejarque et al . , we obtained recordings under anesthesia , enabling the selective activation of sensory pathways , without contamination by motor related interactions . further research addressing multisensory information should also consider other sensory modalities as well as the effects of anesthesia , brain - state , and motor activity on sensory integration ( haider et al . we recorded from several neuronal subtypes and observed differences in their spontaneous activity and response to sensory stimuli . differences in input resistance between direct and indirect pathway msns have been observed in slices ( gertler et al . , 2008 ; we found similar results in vivo , showing higher input resistance in d2 msns , which supports the higher excitability and activity rate of this subpopulation ( cui et al . bilateral cortical input has been reported for both direct and indirect pathway msns ; however , there is a debate regarding the bias in target preference for the different cell types ( kress et al . , 2013 ; lei et al . , 2004 ; reiner et al . , 2010 ; wall et al . , the difference we observed in the bilateral integration between d1 and d2 msns may reflect differences between the afferent corticostriatal pathways to the two populations . in this case , our results would support the findings describing ipsilateral as well as contralateral corticostriatal projection onto both d1 and d2 msns and a stronger ipsilateral corticostriatal projection to d1 than d2 msns ( kress et al . , another explanation may lay in the intrinsic properties of msns subpopulations , in particular the increased excitability of d2 msns and their calcium - mediated dendritic depolarization , which were shown to be different from d1 msns ( day et al . , 2008 ) . although our data set is too small to enable a quantitative characterization , a few observations are important to note . all interneurons displayed the slow wave oscillations as was the case for msns and cortical pyramidal cells . as seen in figures s4 and s7 , interneurons differed in the oscillation amplitudes and shape , suggesting different connectivity patterns conveying the afferent inputs to these neurons . all interneurons responded to bilateral whisker stimulation , and in a single fs interneuron tested for visual stimulation , such responses were indeed observed , showing that fs interneurons as well as msns perform multisensory integration . further studies should elucidate the functional role of the different interneuron types in sensory integration using the large and growing arsenal of molecular tools ( fenno et al . , 2011 ) . all experiments were performed according to the guidelines of the stockholm municipal committee for animal experiments . adult c57bl6 mice of both sexes between 2 and 6 months of age were used to perform the experiments ( n = 92 ) . anesthesia was induced by intraperitoneal injection of ketamine ( 75 mg / kg ) and medetomidine ( 1 mg / kg ) diluted in 0.9% nacl . temperature was maintained between 36c37.5c using a feedback - controlled heating pad ( fhc inc . ) . craniotomies were made at five sites for patch - clamp and extracellular recordings : ap 0 mm from bregma , l 2.5 mm ( dorsomedial striatum ) ; ap 0 mm from bregma , l 3.75 mm ( dorsolateral striatum ) ; ap 1.5 mm , l 3.25 mm ( s1 ) ; ap 1.5 mm , l 2.0 mm ( m1 ) ; ap 3.5 mm , l 2.5 mm ( v1 ) ( following paxinos and franklin , 2001 ) . whole - cell recordings were obtained from dorsolateral striatum between 1,8542,613 m deep and in layer v of cortical barrel field between 617863 m from the pia , in a perpendicular penetration angle . signals were amplified using multiclamp 700b amplifier ( molecular devices ) and digitized at 20 khz with a ced acquisition board and spike 2 software ( cambridge electronic design ) . patch pipettes were pulled with a flaming / brown micropipette puller p-87 ( sutter instruments ) and had an initial resistance of 512 m. extracellular recordings were obtained using tungsten electrodes with impedances of 1 to 2 m. the electrodes were placed in infragranular layers in somatosensory ( bf ) , motor ( m1 ) , and visual ( v1 ) cortex with an angle between 15 and 25. recordings were amplified using a differential ac amplifier model 1700 ( a - m systems ) and digitized at 20 khz with ced and spike-2 simultaneously with the whole - cell recording . whisker stimulation was obtained by brief air puffs delivered by a picospritzer unit ( picospritzer iii , parker hannifin ) via 1 mm diameter plastic tubes placed at 20 mm in front of the whiskers of both sides . air puffs ( 15 ms duration ) were given at least 40 times for each stimulus condition ( ipsilateral , contralateral , or bilateral stimulation ) in a random order , with 5 s of interstimulus interval . visual stimulation was delivered by a white led positioned 50 mm from the contralateral eye . stimulus duration was 10 ms and was delivered with interstimulus intervals of at least 5 s. the eye was covered with vaseline in order to prevent drying , as previously described ( holtmaat et al . , 2009 ) . visual responses were confirmed by monitoring the activation of the contralateral visual cortex using extracellular recordings ( figure 3 ) . tracer injections were made using glass pipettes ( borosilicate , od = 1.5 mm , i d = 1.18 mm ) with a tip diameter of 510 m . a total of 150250 nl of bda 10% ( 10,000 mw lysine - fixable biotin dextran amine , molecular probes ) was dissolved in 0.9% nacl and fast green ( to aid visualization of the injected tracer ) . a single injection was done for each cortical area and animal using the coordinates described above , as taken from paxinos and franklin ( 2001 ) . three to six days following injections , animals were transcardially perfused with a solution containing 4% formalin and 14% saturated picric acid dissolved in 0.1 m phosphate buffer ( pb ) ( ph 7.4 ) . coronal slices ( 20 m thick ) of both hemispheres containing the entire striatum ( from ap 1.7 mm to ap 2.3 mm , following paxinos and franklin , 2001 ) were obtained using a cryostat and collected on gelatin - coated slides . sections were incubated over night with cy3-conjugated streptavidin ( jackson immunoresearch laboratories ) and neurotrace 500/525 green fluorescent nissl stain ( invitrogen ) diluted ( 1:1,000 ) in 1% bsa , 0.3% triton x-100 in 0.1 m pb . at the end of each electrophysiological experiment the mouse was perfused and the brain was placed in fixative solution for 1 to 2 hr in order to stain the neurobiotin - filled neurons ( same procedures and solution described above ) . sections ( 1012 m thick ) mounted on gelatin - coated slides were incubated overnight with cy2-conjugated streptavidin ( jackson immunoresearch laboratories ) diluted ( 1:500 ) in 1% bsa , 0.3% triton x-100 in 0.1 m pb . in between all experimental procedures , slices were washed with 0.01 m pbs . the shortest recording duration for a stained neuron was 24 min , and the average was 55.44 17.87 min ( n = 45 ) . reconstructed striatal neurons were immunolabeled for the detection of d1 dopamine receptors , where we found msns that clearly expressed d1 or not ( d1 n = 15 ; d2 n = 13 ) . primary and secondary antibodies were diluted in 1% bsa , 0.3% triton x-100 in 0.01 m pbs . we used fluorescent and confocal microscopy to recognize the msn receptor expression ( d1 ) . in order to control for the efficacy of the d1 receptor expression described above , we stained slices from d2 egfp mice , showing that d2 expressing neurons were not stained by the antibody ( figure s5 ) .

summarythe basal ganglia are involved in sensorimotor functions and action selection , both of which require the integration of sensory information . in order to determine how such sensory inputs are integrated , we obtained whole - cell recordings in mouse dorsal striatum during presentation of tactile and visual stimuli . all recorded neurons responded to bilateral whisker stimulation , and a subpopulation also responded to visual stimulation . neurons responding to both visual and tactile stimuli were located in dorsomedial striatum , whereas those responding only to whisker deflections were located dorsolaterally . responses were mediated by overlapping excitation and inhibition , with excitation onset preceding that of inhibition by several milliseconds . responses differed according to the type of neuron , with direct pathway msns having larger responses and longer latencies between ipsilateral and contralateral responses than indirect pathway msns . our results suggest that striatum acts as a sensory hub with specialized functional roles for the different neuron types .

Introduction Results Discussion Experimental Procedures

integrating sensory information and producing the appropriate motor output are the basic functions of the nervous system , and the neural networks underlying these two functions are tightly linked . the basal ganglia are involved in various functions , including motor learning , planning , and execution , as well as in decision making and reward ( haber , 2008 ; middleton and strick , 2000 ; schultz et al . , the input layer of the basal ganglia , striatum , receives glutamatergic inputs from multiple cortical areas , including sensory , motor , and prefrontal cortices ( alloway et al . striatal neurons respond to sensory input from different modalities such as tactile , auditory , and visual input ( brown et al . striatal projection neurons ( msns ) are divided into two main subpopulations according to their projection via the direct or indirect pathway ( alexander and crutcher , 1990 ; smith et al . , 1998 ) , with direct pathway neurons facilitating motor activity and indirect pathway neurons inhibiting it ( albin et al . , 2010 ) ; however , it has been debated whether contralateral and ipsilateral corticostriatal projections are selective or biased in targeting direct and indirect pathway msns , respectively ( kress et al . we therefore used whole - cell striatal recordings in order to study synaptic responses to tactile and visual stimuli . we show that neurons throughout dorsal striatum respond to bilateral whisker stimulation in a type - dependent manner and that neurons in dorsomedial striatum perform multisensory integration . whole - cell patch - clamp recordings were obtained from neurons in dorsolateral striatum ( n = 59 neurons ) , dorsomedial striatum ( n = 50 neurons ) , and layer v of s1 barrel field ( n = 20 neurons ) . of all recorded neurons ( n = 129 ) , 45 were stained and morphologically reconstructed ( see experimental procedures ) , three of which were cortical pyramidal neurons and 42 were striatal neurons . striatal neurons were located between 1,854 and 2,613 m below the pia , and the average recording time for all neurons was 48 20 min ( minimum = 9 min , maximum = 100 min ; n = 129 ) . the striatal regions targeted for recordings were selected according to the existence of corticostriatal projections from primary somatosensory and visual cortices ( s1 and v1 , respectively ) following anterograde tracing ( see experimental procedures ; figures 1a , 1b , 3a , and 3b ) . , 1999 , 2006 ; 2001 ) , projections from s1 ( barrel field ) were found throughout the dorsal striatum , with higher concentration in dorsolateral striatum ( figures 1b ; figure s1 available online ) . projections from visual cortex , on the other hand , were located dorsomedially in proximity to the lateral ventricle ( figure 3b ) , in agreement with previous reports in other species ( donoghue and herkenham , 1986 ; faull et al . all recorded cortical and striatal neurons exhibited slow wave oscillations ( figures 2a , 3c , s4 , s6 , and s7 ) with bimodal distribution of the membrane potential ( figures s4 and s7 ) , as previously described ( wilson and kawaguchi , 1996 ) . the input resistance of recorded neurons was extracted using step current injections and was calculated separately for up and down states ( figures s4f , s4h , and s4i ) . in order to study bilateral sensory integration , we delivered brief air puffs to the whisker pads on both sides ( see experimental procedures ) and recorded subthreshold responses to ipsilateral , contralateral , and bilateral stimulation in dorsolateral striatal neurons . all recorded neurons responded to both ipsilateral and contralateral whisker stimulation ( cortical n = 17 ; striatal msns n = 20 ) . sensory responses were classified according to those occurring during up or down states , including cases in which sensory stimulation triggered state transitions ( reig and sanchez - vives , 2007 ) ( figure 1 and s2 , respectively ) . contralateral whisker stimulation evoked larger amplitudes than ipsilateral stimulation in both cortical and striatal neurons ( cortex during down states , ipsilateral : 9.11 3.41 mv , contralateral : 14.07 5.67 mv , p < 0.001 ; striatum during down states , ipsilateral : 10.27 3.27 mv , contralateral : 15.65 6.90 mv , p < 0.01 ) ( figure 1h ) . however , when stimuli were delivered simultaneously to both sides , a significant increase in amplitude was observed in striatal responses but not in cortical ones ( cortex bilateral : 15.72 7.09 mv ; striatum bilateral : 20.37 6.52 mv , p < 0.05 ) ( figure 1h ) . the increase in amplitude in the striatal msns is also reflected in the slopes of responses to bilateral stimulation ( figure 1i ) , both suggesting a higher sensitivity to bilateral input in striatal msns than that observed in s1 layer 5 pyramidal cells . this difference could be explained by the higher temporal separation between ipsilateral and contralateral responses in cortical neurons compared to msns ( response peak latency : cortex = 33.38 21.46 ms ; striatum = 14.26 24.96 ms , p < 0.05 , data not shown ) . in order to decompose the excitatory and inhibitory response components , we used a low - chloride intracellular solution ( see experimental procedures ) enabling us to hold recorded neurons at the reversal potential for excitation ( 5 mv ) or gabaa inhibition ( 70 mv , figures 2a2d ) . in all recorded msns , excitation preceded inhibition for ipsilateral , contralateral , and bilateral whisker stimulation ( onset delays : ipsilateral excitation 24.63 3.76 ms , inhibition 40.6 9.7ms , p < 0.01 ; contralateral excitation 19.16 2.8 ms , inhibition 28.98 1.93 ms , p < 0.001 ; bilateral excitation 19.71 3.27 ms , inhibition 25.61 3 ms , p < 0.01 , n = 8) ( figure 2c ) . regardless of the stimulus condition ( ipsilateral , contralateral , or bilateral whisker stimulation ) , larger excitatory responses were matched by larger inhibitory ones ( figure 2d ) . the relationship between ipsilateral and contralateral responses was maintained for different stimulus intensities , with contralateral ( and bilateral ) stimulation evoking earlier onset and peak response latencies ( figures 2f and 2 g ) , with larger amplitudes and rise slopes ( figures 2h and 2i ) . in order to study responses of striatal neurons to visual stimulus , we obtained whole - cell recordings from neurons in dorsomedial striatum ( figure 3 ) . the recording area was selected according to the presence of axonal projections from cortical v1 ( figures 3a and 3b , n = 5 ) , which were clustered in dorsomedial portions of the striatum , near the lateral ventricle ( figure 3b ) . visual stimuli were presented to the contralateral eye as brief light flashes from a white led ( see experimental procedures ) during whole - cell recordings in striatum and lfp recordings from v1 . the onset delay for contralateral visual responses during down states was 98.43 19.05 ms ( ranging between 56.7 and 141.0 ms , n = 25 ) , almost five times longer than the onset response to whisker stimulation ( 19.78 3.42 ms , figure 1f ) and similar to previously reported visual responses ( schulz et al . such delays are expected when comparing to the long and variable delays ( 50130 ms ) described for visual responses in mouse visual cortex ( niell and stryker , 2008 ; takagaki et al . in order to verify the occurrence of cortical visual responses , we obtained extracellular recordings ( lfp ) in v1 simultaneously with the striatal whole - cell recordings ( n = 16 , figures 3c and 3d ) . visual responses were evoked in 25 of dorsomedial neurons ( 66% ) and in none of the dorsolateral ones ; however , all neurons in both regions responded to bilateral whisker stimulation ( figure 4 ) . figure 4c depicts the position of anatomically reconstructed neurons , in which both tactile and visual stimulation were presented ( n = 30 ) . all neurons that responded to visual stimuli were located medially ( figure 4c , orange points ) , close to the area receiving axonal projections from v1 ( figure 3b ) . to test this prediction , we presented the whisker stimuli at different time intervals with relation to the visual stimuli ( figure 4d ) . indeed , in all cases ( n = 7 ) maximal response amplitudes were evoked when whisker deflections followed the visual stimuli synchronized to the respective response onsets ( relative interval 102.11 20.98 ms , figure 4e ) . this sublinear summation is expected due to the proportional inhibitory component of sensory responses ( figures 2a2d ) but may also be mediated by activation of common cortical association areas ( olcese et al . as shown above neurons in both dorsolateral and dorsomedial striatum responded to whisker stimulation ; however , there were notable differences in responses recorded in both striatal regions . in summary , neurons located in the dorsomedial striatum integrate tactile and visual sensory inputs , with maximal responses when the respective response onsets are aligned . dorsolateral msns do not respond to visual stimulation but have larger and faster responses to whisker stimulation than dorsomedial ones . since these projection pathways are believed to have different roles in basal ganglia function , it is important to understand whether and how they differ in their integration of sensory input . to that end , whole - cell recorded and electrophysiological identified msns were subsequently immunostained with d1 antibody ( see experimental procedures ; figures 5a , s5 , and s6 ) in order to classify them as direct or indirect pathway msns . differences between d1 and d2 msns in their responses to whisker stimulation persisted also when occurring during up states ( figure s6 ) ; however , no differences were observed in spontaneous up states amplitudes or durations ( vm down states d1 72.28 5.44 mv , d2 72.66 3.02 mv ; ap threshold d1 46.24 3.31 mv , d2 45.71 3.73 mv ; up state amplitude d1 11.41 3.16 mv , d2 12.24 4.6 mv ; up state duration d1 0.62 0.18 s , d2 0.61 0.26 s ; ap frequency d1 0.16 0.31 hz , d2 0.10 0.13 hz ) ( figure s6 ) . we also recorded from identified msns in dorsomedial striatum that responded to whisker and visual stimulation ( five d1-msns and three d2-msns ) , thus showing that both msn types integrate multimodal sensory inputs ( figures 5 , 5j , and 5k ) . in summary , msns belonging to the direct and indirect pathways exhibited significant differences in their bilateral integration of tactile sensory inputs . direct pathway msns ( d1 ) responded earlier ( onset delay ) , stronger ( amplitude ) , and faster ( slope ) to contralateral compared to ipsilateral whisker stimulation . these results suggest that d1 and d2 msns have different roles in their sensory integration , d1 msns tuned to detect differences between ipsilateral and contralateral whiskers than d2 msns , which act as integrators of bilateral inputs . striatal interneurons form a small albeit diverse minority in the striatal microcircuitry ; therefore , our method of blind whole - cell patch - clamping resulted in a rather small number of interneurons . neurons were initially classified according to their recorded electrical properties and following morphological staining , according to the aspiny dendrites and the large soma size ( in the case of the cholinergic interneurons ) . as in all other recorded neurons , interneurons responded to either whisker stimulation with stronger and earlier responses to the contralateral whisker deflection ( figures s7c , s7 g , and s7i ) , and in one interneuron where visual responses were tested , the recorded fs responded to visual input ( figure s7c ) . in this study we used whole - cell patch - clamp recordings to study the integration of bilateral and multimodal sensory information in striatal neurons . we show that individual striatal neurons integrate bilateral as well as multisensory inputs , that both spontaneous and sensory evoked inputs are comprised of overlapping excitatory and inhibitory synaptic input , and that msns of the direct and indirect pathways differ in the way they integrate bilateral sensory input . all neurons recorded in dorsal striatum , including projection neurons and interneurons , responded to bilateral whisker stimulation in a type - dependent manner , and a population of dorsomedial neurons also responded to visual input . all neurons recorded in the dorsal striatum responded to stimulation of whiskers of both sides . the responses differed in latency , slope , and amplitude , with contralateral whisker stimulation inducing larger and earlier responses as seen also in the recordings from cortical neurons . this result can be explained by the time differences between ipsilateral and contralateral onset latencies , which were shorter in striatal neurons , in particular d2 msns ( figure 5 g , inset ) . the discrepancy in the measured contralateral responses may be explained by differences in the air puff duration and pressure settings ( see experimental procedures ) , striatal coordinates ( medial - lateral or rostro - caudal axes ) , and species ( rat and mouse ) , but it may also be attributed to differences between intracellular sharp and whole - cell recordings ( staley et al . neurons in both dorsolateral and dorsomedial striatum responded to bilateral whisker stimulation ; however , these responses differed in several aspects . a larger striatal area , however , receives the sensory input generating an attenuated and slower response , which , in the case of the dorsomedial striatum , also receives sensory information from a different modality ( figures 4a4e ) . in vivo studies in neocortex have shown that visual and tactile sensory input induces temporally complex inhibitory inputs mediated by gabaergic interneurons ( haider et al . visual responses were seen in neurons recorded in the dorsomedial striatum in a relatively large yet restricted striatal region that also received axonal projections from visual cortex , thus suggesting that at least part of the response was mediated by excitatory projections from visual cortex ( figure 3 ) . visually evoked responses were recently described in neurons from rat dorsal striatum , mediated mainly by subcortical inputs following disinhibition of superior colliculus ( schulz et al . recorded neurons were located more laterally than in the present study , which together with the different species and anesthesia may explain the different visual responsiveness . multisensory responses have been described in different basal ganglia nuclei including the striatum using intracellular , extracellular , and optical recordings ( chudler et al . the striatal neurons we recorded in dorsal striatum showed clear preference for whisker stimulation , while only a fraction of them responded to both tactile and visual input , depending on their topographic location ( figure 4c ) . responses to whisker stimulation were significantly earlier than those to visual stimulation ( figure 4 ) . another possible explanation may lie in the nature of these stimuli , with tactile stimulation originating from nearby objects , whereas visual input would originate from more distal objects , before touching the whiskers . these differences in processing time between visual and tactile stimuli suggest that introducing a time lag between tactile and visual stimuli would result in increased responsiveness in the striatum , as observed in cortical multisensory areas ( olcese et al . in agreement with the cortical studies , we showed that maximal response amplitude occurred when visual and whisker inputs simultaneously impinged onto postsynaptic msns . , we obtained recordings under anesthesia , enabling the selective activation of sensory pathways , without contamination by motor related interactions . differences in input resistance between direct and indirect pathway msns have been observed in slices ( gertler et al . bilateral cortical input has been reported for both direct and indirect pathway msns ; however , there is a debate regarding the bias in target preference for the different cell types ( kress et al . in this case , our results would support the findings describing ipsilateral as well as contralateral corticostriatal projection onto both d1 and d2 msns and a stronger ipsilateral corticostriatal projection to d1 than d2 msns ( kress et al . all interneurons responded to bilateral whisker stimulation , and in a single fs interneuron tested for visual stimulation , such responses were indeed observed , showing that fs interneurons as well as msns perform multisensory integration . all experiments were performed according to the guidelines of the stockholm municipal committee for animal experiments . whole - cell recordings were obtained from dorsolateral striatum between 1,8542,613 m deep and in layer v of cortical barrel field between 617863 m from the pia , in a perpendicular penetration angle . patch pipettes were pulled with a flaming / brown micropipette puller p-87 ( sutter instruments ) and had an initial resistance of 512 m. extracellular recordings were obtained using tungsten electrodes with impedances of 1 to 2 m. the electrodes were placed in infragranular layers in somatosensory ( bf ) , motor ( m1 ) , and visual ( v1 ) cortex with an angle between 15 and 25. recordings were amplified using a differential ac amplifier model 1700 ( a - m systems ) and digitized at 20 khz with ced and spike-2 simultaneously with the whole - cell recording . stimulus duration was 10 ms and was delivered with interstimulus intervals of at least 5 s. the eye was covered with vaseline in order to prevent drying , as previously described ( holtmaat et al . at the end of each electrophysiological experiment the mouse was perfused and the brain was placed in fixative solution for 1 to 2 hr in order to stain the neurobiotin - filled neurons ( same procedures and solution described above ) . in order to control for the efficacy of the d1 receptor expression described above , we stained slices from d2 egfp mice , showing that d2 expressing neurons were not stained by the antibody ( figure s5 ) .

the single biggest threat to human kind dominance on the planet is the virus - ( joshua lederberg , nobel laureate 1958 ) . as seen in history , when influenza pandemic occurs , many millions of people around the world become ill and a proportion die . depending on the virulence of the ( re)emerged influenza virus , the susceptibility of the population and the effectiveness of countermeasures , up to half the population could have developed illness . in the absence of early or effective interventions , society is also likely to face much wider social and economic disruption , significant threats to the continuity of essential services , lower production levels , shortages and distribution difficulties . difficulties in maintaining business and service continuity will be exacerbated if the virus affects those of working age more than other groups , and fear of infection , illness , and care providing responsibilities , stress , bereavement and potential travel disruption are likely to lead to higher levels of absence . high levels of public and political concern , general scrutiny and demands for advice and information are also inevitable at all stages of an influenza pandemic . for example ; in uk , one illustrative assessment suggests that illness - related absence from work of 25% of employees over the course of pandemic ( only half of what may be expected in a widespread pandemic ) could reduce the year s gross domestic product ( gdp ) by between 3 billion and 7 billion . additional premature deaths could cause a further reduction of between 1 billion and 7 billion . in the longer term the impact of premature deaths could reduce future lifetime earnings by between 21 billion and 26 billion at a low case fatality rate and by between 145 billion and 172 billion at a high case fatality rate ( 2 ) . past pandemics have varied in scale , severity and consequence , although in general their impact has been much than that of even the most severe winter epidemic . it is not possible to forecast the precise characteristics , spread and impact of a new influenza virus strain . modelling suggests that from the time it begins in the country of origin it may take as little as two to four weeks to build from a few to around 1,000 cases . within one to two weeks , it is likely to spread to all major population centres , with its peak possibly only 50 days from initial entry . vaccination or mass treatment with antiviral medicines can be expected to modify this profile of pandemic ( 2 ) . in order to prepare a health care system for potential influenza pandemic , all un member countries got guidelines from who that contain all objectives and activities that should be done in every stage of influenza pandemic(5 ) . it should be done by preparing the preparedness and contingency plans for each of countries separately . preparedness plan contains all the instruction on which measures should be taken during pandemic influenza , while contingency plan should acknowledge more detailed implementation of the measures form preparedness plan . its major characteristics are : openness ( interaction between community , population and environment ) , complexity ( made of more subsystems ) and dynamics ( continuous process of ensuring and using healthcare services ) ( 3 ) . on june 11 , 2009 , the world health organization ( who ) signalled that a global pandemic of novel influenza a ( h1n1 ) was underway by raising the worldwide pandemic alert level to phase 6 ( 4 ) . at the time , more than 70 countries had reported cases of novel influenza a ( h1n1 ) infection and there were ongoing community level outbreaks of novel h1n1 in multiple parts of the world . since the who declaration of a pandemic , the new h1n1 virus has continued to spread ; with the number of countries reporting cases of novel h1n1 nearly doubled . official statistics account up to 360 persons with confirmed diagnosis of new influence infection died till the first half of january 2010 in uk . this study has following objectives : to determine if bosnia and herzegovina healthcare system is prepared for influenza pandemic;to indicate strengths and weaknesses in planed resolution of pandemic influenza in bosnia and herzegovina healthcare system . to determine if bosnia and herzegovina healthcare system is prepared for influenza pandemic ; to indicate strengths and weaknesses in planed resolution of pandemic influenza in bosnia and herzegovina healthcare system . on june 11 , 2009 , the world health organization ( who ) signalled that a global pandemic of novel influenza a ( h1n1 ) was underway by raising the worldwide pandemic alert level to phase 6 ( 4 ) . at the time , more than 70 countries had reported cases of novel influenza a ( h1n1 ) infection and there were ongoing community level outbreaks of novel h1n1 in multiple parts of the world . since the who declaration of a pandemic , the new h1n1 virus has continued to spread ; with the number of countries reporting cases of novel h1n1 nearly doubled . official statistics account up to 360 persons with confirmed diagnosis of new influence infection died till the first half of january 2010 in uk . this study has following objectives : to determine if bosnia and herzegovina healthcare system is prepared for influenza pandemic;to indicate strengths and weaknesses in planed resolution of pandemic influenza in bosnia and herzegovina healthcare system . to determine if bosnia and herzegovina healthcare system is prepared for influenza pandemic ; to indicate strengths and weaknesses in planed resolution of pandemic influenza in bosnia and herzegovina healthcare system . the applied methodology is based on qualitative systematic review , comparing established elements of healthcare systems with who s guidelines on pandemic preparedness . based on this comparison ( primarily through contingency and preparedness plans ) it will be possible to acknowledge level of preparedness of bosnia and herzegovina healthcare system . starting point is : whether healthcare system of a country ( bosnia and herzegovina ) is prepared for pandemic influenza ? in order to obtain objectives it is necessary to critically evaluate available findings on preparedness of healthcare system of bosnia and herzegovina ( b&h ) compared in details to preparedness of healthcare system of united kingdom ( uk ) but in certain elements with some other european countries . to conduct this systematic review , preparedness plans of the two mentioned countries have been reviewed in details as uk plan has been the most extensive preparedness system as stated by the who and b&h with its formal preparation system . in 2005 , who presented new global influenza preparedness plan ; it updates , significantly revises and replaces the influenza pandemic plan . the role of who and guidelines for national and regional planning published by who in 1999 . this plan redefines the phases of increasing public health risk associated with the emergence of a new influenza virus subtype that may pose a pandemic threat . redefinition of the phases was needed to address the public health risks of influenza infection in animals , link phase changes more directly with changes in public health response , and focus on early events during a pandemic alert period when rapid , coordinated global and national actions might help to possibly contain or delay the spread of a new human influenza strain ( 5 ) . in order to accomplish the public health goals for each phase , the specific objectives and actions to be taken by who , and those recommended for national authorities , are divided into five categories : planning and coordinationsituation monitoring and assessmentprevention and containmenthealth system responsecommunications ( 5 ) . planning and coordination situation monitoring and assessment prevention and containment health system response in october of 2007 government of uk passed new national framework for respond on pandemic influenza . it established main goal ( to take every practical step to assure highest possible level of preparedness of uk for pandemic influenza ) , as well as strategic objectives based on which main goal will be accomplished . both contingency and preparedness plan were made based on who s guidelines ( 2 ) . there are 11 strategic objectives covering all aspects of citizens protection , risk management , organization of health and social care system to adept to provide treatment and support for large numbers of likely to suffer from influenza or its complications and other relevant issues for an adequate preparedness strategy ( 2 ) . achieving these strategic objectives will require the development , maintenance , testing and when necessary implementation of operational response arrangements ( 2 ) . an influenza pandemic requires flexible plans based on a combination of strategies to develop an effective and sustainable response . detailed medical or pharmaceutical countermeasures , combined with public health and personal infection control initiatives , and possible application of additional measures to reduce social mixing , form the basis of the uk s mitigation strategy are given in the section covering contingency measures ( 2 ) . as an influenza pandemic will result in intense and sustained pressure on all parts of the health and social care system , it is expected to limit the scope for mutual aid and threatening the overwhelm services at its peak ( 2 ) . section on health care system respond contains in details instructions on surveillance to detect the emergence of a novel virus strain , monitoring its spread and health impact with providing prompt access to reliable diagnostics tests , as well as normal patient pathways and service delivery arrangement and all other issues related to this ( 2 ) . emphasise the significance of cooperation of all authorities involved in influenza pandemic preparedness ( governmental , public , business , non - governmental , voluntary and individuals ) this part main aims among others are to improve general awareness and understanding of influenza amongst the population and promote good hygiene and other general precautionary measures ; prepare the country for the probable emergence of a new or re - emerging influenza virus ; mobilise the population as partners at the response phase ; etc . ( 2 ) b&h shares international borders with croatia to the north , south and west , and serbia and montenegro to the east ; consists of two entities and one district : the federation of bosnia and herzegovina ( fb&h ) , republic of srpska ( rs ) and the independently administered brcko district . health care finance , management and organization in b&h is the responsibility of each entity , and brcko district runs a health care system under its own authority . one for republic of srpska , one for brcko district , and within fb&h : one for fb&h and ten cantonal ministries ( one for each canton ) . acknowledging the threat that pandemic influenza represents on both global and national level and because of request from who to all member countries , council of ministers , based on clause 17 of law of council of ministries ( official gazette b&h , number 30/03 , 42/03 ) , and on suggestion from ministry of civil affairs , passed preparedness and control plan for pandemic influenza in bosnia and herzegovina ( 6 ) . surveillance system for human seasonal influenza in b&h is part of routine passive surveillance of communicable diseases . sporadic cases are registered individually , and during the epidemics they are registered collectively weekly ( 6 ) . since there is no sentinel surveillance in b&h , by the preparedness and control plan routine surveillance of acute respiratory infections ( ari ) and influenzalike illness ( ili ) will be strengthen in city centers with corresponding network of passive surveillance . also , information exchange and common active surveillance between veterinary and human sector will intensify ( 6 ) . as of 2012 , when this paper is finalized , there is no sentinel surveillance for influenza established in any part of b&h . to limit spreading of influenza there are pharmaceutical and non - pharmaceutical measures . in lack of specific vaccines and antiviral medicines , important part in contingency have non - pharmaceutical measures ( veterinary contingency measures , limitation of human travel in affected areas , use of personal preventive means , general and personal hygiene etc ) ( 6 ) . since it is not part of mandatory vaccination there is no constant secure founding for procurement of seasonal vaccines ( estimated need for 500.000 vaccines ) . average annual usage of seasonal vaccines is 150.000 doses ( 6 ) . until the moment that this article is being written , b&h remains only country from this region that does not have contingency plan . since contingency plan represents guide for implementation of preparedness plan , the lack of one , decreases general preparedness of b&h s healthcare system . but economy is probably not to blame , since all the neighboring countries , that are not economically much more advanced have one . for example serbia ( 7 ) , croatia ( 8) and fyr of macedonia ( 9 ) have detailed and well prepared contingency plans , with clear instruction of how to implement measures from preparedness plan . so most likely , that the reason is actually because of specifics of organization of b&h s healthcare system . since contingency plan should be made on national level , but healthcare decisions are being made on entity level , it is very difficult to make unique contingency plan that needs great deal of practical data . also majority of the other european countries have preparedness plans as well as detailed contingency plans france ( 10 ) , austria ( 11 ) , hungary ( 12 ) italy ( 13 ) . detailed analysis of the preparedness plans of several european countries health care systems and bosnia and herzegovina by the who guidelines elements by phases is presented in table 2 . b&h included -covered all elements of influenza pandemic preparedness in their preparedness plans ; and all countries but b&h have had contingency plans . in 2005 , who presented new global influenza preparedness plan ; it updates , significantly revises and replaces the influenza pandemic plan . the role of who and guidelines for national and regional planning published by who in 1999 . this plan redefines the phases of increasing public health risk associated with the emergence of a new influenza virus subtype that may pose a pandemic threat . redefinition of the phases was needed to address the public health risks of influenza infection in animals , link phase changes more directly with changes in public health response , and focus on early events during a pandemic alert period when rapid , coordinated global and national actions might help to possibly contain or delay the spread of a new human influenza strain ( 5 ) . in order to accomplish the public health goals for each phase , the specific objectives and actions to be taken by who , and those recommended for national authorities , are divided into five categories : planning and coordinationsituation monitoring and assessmentprevention and containmenthealth system responsecommunications ( 5 ) . in october of 2007 government of uk passed new national framework for respond on pandemic influenza . it established main goal ( to take every practical step to assure highest possible level of preparedness of uk for pandemic influenza ) , as well as strategic objectives based on which main goal will be accomplished . both contingency and preparedness plan were made based on who s guidelines ( 2 ) . there are 11 strategic objectives covering all aspects of citizens protection , risk management , organization of health and social care system to adept to provide treatment and support for large numbers of likely to suffer from influenza or its complications and other relevant issues for an adequate preparedness strategy ( 2 ) . achieving these strategic objectives will require the development , maintenance , testing and when necessary implementation of operational response arrangements ( 2 ) . an influenza pandemic requires flexible plans based on a combination of strategies to develop an effective and sustainable response . detailed medical or pharmaceutical countermeasures , combined with public health and personal infection control initiatives , and possible application of additional measures to reduce social mixing , form the basis of the uk s mitigation strategy are given in the section covering contingency measures ( 2 ) . as an influenza pandemic will result in intense and sustained pressure on all parts of the health and social care system , it is expected to limit the scope for mutual aid and threatening the overwhelm services at its peak ( 2 ) . section on health care system respond contains in details instructions on surveillance to detect the emergence of a novel virus strain , monitoring its spread and health impact with providing prompt access to reliable diagnostics tests , as well as normal patient pathways and service delivery arrangement and all other issues related to this ( 2 ) . emphasise the significance of cooperation of all authorities involved in influenza pandemic preparedness ( governmental , public , business , non - governmental , voluntary and individuals ) this part main aims among others are to improve general awareness and understanding of influenza amongst the population and promote good hygiene and other general precautionary measures ; prepare the country for the probable emergence of a new or re - emerging influenza virus ; mobilise the population as partners at the response phase ; etc . emphasise the significance of cooperation of all authorities involved in influenza pandemic preparedness ( governmental , public , business , non - governmental , voluntary and individuals ) this part main aims among others are to improve general awareness and understanding of influenza amongst the population and promote good hygiene and other general precautionary measures ; prepare the country for the probable emergence of a new or re - emerging influenza virus ; mobilise the population as partners at the response phase ; etc . b&h shares international borders with croatia to the north , south and west , and serbia and montenegro to the east ; consists of two entities and one district : the federation of bosnia and herzegovina ( fb&h ) , republic of srpska ( rs ) and the independently administered brcko district . health care finance , management and organization in b&h is the responsibility of each entity , and brcko district runs a health care system under its own authority . one for republic of srpska , one for brcko district , and within fb&h : one for fb&h and ten cantonal ministries ( one for each canton ) . acknowledging the threat that pandemic influenza represents on both global and national level and because of request from who to all member countries , council of ministers , based on clause 17 of law of council of ministries ( official gazette b&h , number 30/03 , 42/03 ) , and on suggestion from ministry of civil affairs , passed preparedness and control plan for pandemic influenza in bosnia and herzegovina ( 6 ) . surveillance system for human seasonal influenza in b&h is part of routine passive surveillance of communicable diseases . sporadic cases are registered individually , and during the epidemics they are registered collectively weekly ( 6 ) . since there is no sentinel surveillance in b&h , by the preparedness and control plan routine surveillance of acute respiratory infections ( ari ) and influenzalike illness ( ili ) will be strengthen in city centers with corresponding network of passive surveillance . also , information exchange and common active surveillance between veterinary and human sector will intensify ( 6 ) . as of 2012 , when this paper is finalized , there is no sentinel surveillance for influenza established in any part of b&h . to limit spreading of influenza there are pharmaceutical and non - pharmaceutical measures . in lack of specific vaccines and antiviral medicines , important part in contingency have non - pharmaceutical measures ( veterinary contingency measures , limitation of human travel in affected areas , use of personal preventive means , general and personal hygiene etc ) ( 6 ) . since it is not part of mandatory vaccination there is no constant secure founding for procurement of seasonal vaccines ( estimated need for 500.000 vaccines ) . average annual usage of seasonal vaccines is 150.000 doses ( 6 ) . until the moment that this article is being written , b&h remains only country from this region that does not have contingency plan . since contingency plan represents guide for implementation of preparedness plan , the lack of one , decreases general preparedness of b&h s healthcare system . but economy is probably not to blame , since all the neighboring countries , that are not economically much more advanced have one . for example serbia ( 7 ) , croatia ( 8) and fyr of macedonia ( 9 ) have detailed and well prepared contingency plans , with clear instruction of how to implement measures from preparedness plan . so most likely , that the reason is actually because of specifics of organization of b&h s healthcare system . since contingency plan should be made on national level , but healthcare decisions are being made on entity level , it is very difficult to make unique contingency plan that needs great deal of practical data . also majority of the other european countries have preparedness plans as well as detailed contingency plans france ( 10 ) , austria ( 11 ) , hungary ( 12 ) italy ( 13 ) detailed analysis of the preparedness plans of several european countries health care systems and bosnia and herzegovina by the who guidelines elements by phases is presented in table 2 . b&h included -covered all elements of influenza pandemic preparedness in their preparedness plans ; and all countries but b&h have had contingency plans . to limit spreading of influenza there are pharmaceutical and non - pharmaceutical measures . in lack of specific vaccines and antiviral medicines , important part in contingency have non - pharmaceutical measures ( veterinary contingency measures , limitation of human travel in affected areas , use of personal preventive means , general and personal hygiene etc ) ( 6 ) . since it is not part of mandatory vaccination there is no constant secure founding for procurement of seasonal vaccines ( estimated need for 500.000 vaccines ) . average annual usage of seasonal vaccines is 150.000 doses ( 6 ) . until the moment that this article is being written , b&h remains only country from this region that does not have contingency plan . since contingency plan represents guide for implementation of preparedness plan , the lack of one , decreases general preparedness of b&h s healthcare system . but economy is probably not to blame , since all the neighboring countries , that are not economically much more advanced have one . for example serbia ( 7 ) , croatia ( 8) and fyr of macedonia ( 9 ) have detailed and well prepared contingency plans , with clear instruction of how to implement measures from preparedness plan . so most likely , that the reason is actually because of specifics of organization of b&h s healthcare system . since contingency plan should be made on national level , but healthcare decisions are being made on entity level , it is very difficult to make unique contingency plan that needs great deal of practical data . also majority of the other european countries have preparedness plans as well as detailed contingency plans france ( 10 ) , austria ( 11 ) , hungary ( 12 ) italy ( 13 ) . detailed analysis of the preparedness plans of several european countries health care systems and bosnia and herzegovina by the who guidelines elements by phases is presented in table 2 . b&h included -covered all elements of influenza pandemic preparedness in their preparedness plans ; and all countries but b&h have had contingency plans . after the presented analysis , it is possible to conclude that disregarding the obstacles in b&h health care system policy bosnia and herzegovina has preparedness plans , that are made based on who s guidelines . while other analysed countries besides preparedness has also contingency plan , b&h still has to work on making one . based on that fact , one can says that uk s , france , austria , hungary , italy , croatia , serbia and montenegro healthcare systems are better prepared for pandemic influenza , than b&h s . weaknesses of b&h s healthcare system are late forming of preparedness plan , and lack of contingency plan . implementation of activities stated to be establish by the preparedness plan in 2009 has never being done ( e.g. sentinel surveillance , vaccination policy etc ) although preparedness plan exists it is in need for constant urging on fulfilling of activities and objectives in all categories ( planning , monitoring , contingency , respond and communication ) in every stage of pandemic .

objectives : to determine if bosnia and herzegovina healthcare system is prepared for influenza pandemic ; and to indicate strengths and weaknesses in planed resolution of pandemic influenza in bosnia and herzegovina healthcare system.methodology:qualitative systematic review , comparing established elements of healthcare systems with who s guidelines on pandemic preparedness . critical evaluations of available findings on preparedness of healthcare system of bosnia and herzegovina ( b&h ) compared in details to preparedness of healthcare system of united kingdom ( uk ) but in certain elements with some other european countries.results and discussion : analysis of preparedness plans of b&h and uk are presented in details , with comparison of b&h with eight other countries by who guidelines categories and phases of pandemic preparedness and contingency plans.conclusions:disregarding the obstacles in b&h health care system policy bosnia and herzegovina has preparedness plans , that are made based on who s guidelines but unlike all other analyzed countries does not have contingency plan . this can be seen as strength while weaknesses of b&h s healthcare system are : late forming of preparedness plan with poor implementation of set activities , and lack of contingency plan .

INTRODUCTION Pandemic influenza type A (H1N1) 2009 METHODOLOGY RESULTS AND DISCUSSION WHO Guidelines A National Framework for responding to an influenza pandemic of United Kingdom (UK) Communication and public engagement Preparedness and control plan for pandemic influenza in Bosnia and Herzegovina Contingency measures: CONCLUSIONS Conflict of interest

as seen in history , when influenza pandemic occurs , many millions of people around the world become ill and a proportion die . difficulties in maintaining business and service continuity will be exacerbated if the virus affects those of working age more than other groups , and fear of infection , illness , and care providing responsibilities , stress , bereavement and potential travel disruption are likely to lead to higher levels of absence . high levels of public and political concern , general scrutiny and demands for advice and information are also inevitable at all stages of an influenza pandemic . for example ; in uk , one illustrative assessment suggests that illness - related absence from work of 25% of employees over the course of pandemic ( only half of what may be expected in a widespread pandemic ) could reduce the year s gross domestic product ( gdp ) by between 3 billion and 7 billion . within one to two weeks , it is likely to spread to all major population centres , with its peak possibly only 50 days from initial entry . vaccination or mass treatment with antiviral medicines can be expected to modify this profile of pandemic ( 2 ) . in order to prepare a health care system for potential influenza pandemic , all un member countries got guidelines from who that contain all objectives and activities that should be done in every stage of influenza pandemic(5 ) . it should be done by preparing the preparedness and contingency plans for each of countries separately . preparedness plan contains all the instruction on which measures should be taken during pandemic influenza , while contingency plan should acknowledge more detailed implementation of the measures form preparedness plan . its major characteristics are : openness ( interaction between community , population and environment ) , complexity ( made of more subsystems ) and dynamics ( continuous process of ensuring and using healthcare services ) ( 3 ) . this study has following objectives : to determine if bosnia and herzegovina healthcare system is prepared for influenza pandemic;to indicate strengths and weaknesses in planed resolution of pandemic influenza in bosnia and herzegovina healthcare system . to determine if bosnia and herzegovina healthcare system is prepared for influenza pandemic ; to indicate strengths and weaknesses in planed resolution of pandemic influenza in bosnia and herzegovina healthcare system . this study has following objectives : to determine if bosnia and herzegovina healthcare system is prepared for influenza pandemic;to indicate strengths and weaknesses in planed resolution of pandemic influenza in bosnia and herzegovina healthcare system . to determine if bosnia and herzegovina healthcare system is prepared for influenza pandemic ; to indicate strengths and weaknesses in planed resolution of pandemic influenza in bosnia and herzegovina healthcare system . the applied methodology is based on qualitative systematic review , comparing established elements of healthcare systems with who s guidelines on pandemic preparedness . based on this comparison ( primarily through contingency and preparedness plans ) it will be possible to acknowledge level of preparedness of bosnia and herzegovina healthcare system . starting point is : whether healthcare system of a country ( bosnia and herzegovina ) is prepared for pandemic influenza ? in order to obtain objectives it is necessary to critically evaluate available findings on preparedness of healthcare system of bosnia and herzegovina ( b&h ) compared in details to preparedness of healthcare system of united kingdom ( uk ) but in certain elements with some other european countries . to conduct this systematic review , preparedness plans of the two mentioned countries have been reviewed in details as uk plan has been the most extensive preparedness system as stated by the who and b&h with its formal preparation system . in 2005 , who presented new global influenza preparedness plan ; it updates , significantly revises and replaces the influenza pandemic plan . the role of who and guidelines for national and regional planning published by who in 1999 . this plan redefines the phases of increasing public health risk associated with the emergence of a new influenza virus subtype that may pose a pandemic threat . redefinition of the phases was needed to address the public health risks of influenza infection in animals , link phase changes more directly with changes in public health response , and focus on early events during a pandemic alert period when rapid , coordinated global and national actions might help to possibly contain or delay the spread of a new human influenza strain ( 5 ) . in order to accomplish the public health goals for each phase , the specific objectives and actions to be taken by who , and those recommended for national authorities , are divided into five categories : planning and coordinationsituation monitoring and assessmentprevention and containmenthealth system responsecommunications ( 5 ) . planning and coordination situation monitoring and assessment prevention and containment health system response in october of 2007 government of uk passed new national framework for respond on pandemic influenza . it established main goal ( to take every practical step to assure highest possible level of preparedness of uk for pandemic influenza ) , as well as strategic objectives based on which main goal will be accomplished . both contingency and preparedness plan were made based on who s guidelines ( 2 ) . there are 11 strategic objectives covering all aspects of citizens protection , risk management , organization of health and social care system to adept to provide treatment and support for large numbers of likely to suffer from influenza or its complications and other relevant issues for an adequate preparedness strategy ( 2 ) . achieving these strategic objectives will require the development , maintenance , testing and when necessary implementation of operational response arrangements ( 2 ) . an influenza pandemic requires flexible plans based on a combination of strategies to develop an effective and sustainable response . detailed medical or pharmaceutical countermeasures , combined with public health and personal infection control initiatives , and possible application of additional measures to reduce social mixing , form the basis of the uk s mitigation strategy are given in the section covering contingency measures ( 2 ) . as an influenza pandemic will result in intense and sustained pressure on all parts of the health and social care system , it is expected to limit the scope for mutual aid and threatening the overwhelm services at its peak ( 2 ) . section on health care system respond contains in details instructions on surveillance to detect the emergence of a novel virus strain , monitoring its spread and health impact with providing prompt access to reliable diagnostics tests , as well as normal patient pathways and service delivery arrangement and all other issues related to this ( 2 ) . emphasise the significance of cooperation of all authorities involved in influenza pandemic preparedness ( governmental , public , business , non - governmental , voluntary and individuals ) this part main aims among others are to improve general awareness and understanding of influenza amongst the population and promote good hygiene and other general precautionary measures ; prepare the country for the probable emergence of a new or re - emerging influenza virus ; mobilise the population as partners at the response phase ; etc . ( 2 ) b&h shares international borders with croatia to the north , south and west , and serbia and montenegro to the east ; consists of two entities and one district : the federation of bosnia and herzegovina ( fb&h ) , republic of srpska ( rs ) and the independently administered brcko district . health care finance , management and organization in b&h is the responsibility of each entity , and brcko district runs a health care system under its own authority . one for republic of srpska , one for brcko district , and within fb&h : one for fb&h and ten cantonal ministries ( one for each canton ) . acknowledging the threat that pandemic influenza represents on both global and national level and because of request from who to all member countries , council of ministers , based on clause 17 of law of council of ministries ( official gazette b&h , number 30/03 , 42/03 ) , and on suggestion from ministry of civil affairs , passed preparedness and control plan for pandemic influenza in bosnia and herzegovina ( 6 ) . surveillance system for human seasonal influenza in b&h is part of routine passive surveillance of communicable diseases . sporadic cases are registered individually , and during the epidemics they are registered collectively weekly ( 6 ) . since there is no sentinel surveillance in b&h , by the preparedness and control plan routine surveillance of acute respiratory infections ( ari ) and influenzalike illness ( ili ) will be strengthen in city centers with corresponding network of passive surveillance . as of 2012 , when this paper is finalized , there is no sentinel surveillance for influenza established in any part of b&h . in lack of specific vaccines and antiviral medicines , important part in contingency have non - pharmaceutical measures ( veterinary contingency measures , limitation of human travel in affected areas , use of personal preventive means , general and personal hygiene etc ) ( 6 ) . until the moment that this article is being written , b&h remains only country from this region that does not have contingency plan . since contingency plan represents guide for implementation of preparedness plan , the lack of one , decreases general preparedness of b&h s healthcare system . but economy is probably not to blame , since all the neighboring countries , that are not economically much more advanced have one . for example serbia ( 7 ) , croatia ( 8) and fyr of macedonia ( 9 ) have detailed and well prepared contingency plans , with clear instruction of how to implement measures from preparedness plan . so most likely , that the reason is actually because of specifics of organization of b&h s healthcare system . since contingency plan should be made on national level , but healthcare decisions are being made on entity level , it is very difficult to make unique contingency plan that needs great deal of practical data . also majority of the other european countries have preparedness plans as well as detailed contingency plans france ( 10 ) , austria ( 11 ) , hungary ( 12 ) italy ( 13 ) . detailed analysis of the preparedness plans of several european countries health care systems and bosnia and herzegovina by the who guidelines elements by phases is presented in table 2 . b&h included -covered all elements of influenza pandemic preparedness in their preparedness plans ; and all countries but b&h have had contingency plans . in 2005 , who presented new global influenza preparedness plan ; it updates , significantly revises and replaces the influenza pandemic plan . the role of who and guidelines for national and regional planning published by who in 1999 . this plan redefines the phases of increasing public health risk associated with the emergence of a new influenza virus subtype that may pose a pandemic threat . redefinition of the phases was needed to address the public health risks of influenza infection in animals , link phase changes more directly with changes in public health response , and focus on early events during a pandemic alert period when rapid , coordinated global and national actions might help to possibly contain or delay the spread of a new human influenza strain ( 5 ) . in order to accomplish the public health goals for each phase , the specific objectives and actions to be taken by who , and those recommended for national authorities , are divided into five categories : planning and coordinationsituation monitoring and assessmentprevention and containmenthealth system responsecommunications ( 5 ) . in october of 2007 government of uk passed new national framework for respond on pandemic influenza . it established main goal ( to take every practical step to assure highest possible level of preparedness of uk for pandemic influenza ) , as well as strategic objectives based on which main goal will be accomplished . both contingency and preparedness plan were made based on who s guidelines ( 2 ) . there are 11 strategic objectives covering all aspects of citizens protection , risk management , organization of health and social care system to adept to provide treatment and support for large numbers of likely to suffer from influenza or its complications and other relevant issues for an adequate preparedness strategy ( 2 ) . achieving these strategic objectives will require the development , maintenance , testing and when necessary implementation of operational response arrangements ( 2 ) . an influenza pandemic requires flexible plans based on a combination of strategies to develop an effective and sustainable response . detailed medical or pharmaceutical countermeasures , combined with public health and personal infection control initiatives , and possible application of additional measures to reduce social mixing , form the basis of the uk s mitigation strategy are given in the section covering contingency measures ( 2 ) . as an influenza pandemic will result in intense and sustained pressure on all parts of the health and social care system , it is expected to limit the scope for mutual aid and threatening the overwhelm services at its peak ( 2 ) . section on health care system respond contains in details instructions on surveillance to detect the emergence of a novel virus strain , monitoring its spread and health impact with providing prompt access to reliable diagnostics tests , as well as normal patient pathways and service delivery arrangement and all other issues related to this ( 2 ) . emphasise the significance of cooperation of all authorities involved in influenza pandemic preparedness ( governmental , public , business , non - governmental , voluntary and individuals ) this part main aims among others are to improve general awareness and understanding of influenza amongst the population and promote good hygiene and other general precautionary measures ; prepare the country for the probable emergence of a new or re - emerging influenza virus ; mobilise the population as partners at the response phase ; etc . emphasise the significance of cooperation of all authorities involved in influenza pandemic preparedness ( governmental , public , business , non - governmental , voluntary and individuals ) this part main aims among others are to improve general awareness and understanding of influenza amongst the population and promote good hygiene and other general precautionary measures ; prepare the country for the probable emergence of a new or re - emerging influenza virus ; mobilise the population as partners at the response phase ; etc . b&h shares international borders with croatia to the north , south and west , and serbia and montenegro to the east ; consists of two entities and one district : the federation of bosnia and herzegovina ( fb&h ) , republic of srpska ( rs ) and the independently administered brcko district . health care finance , management and organization in b&h is the responsibility of each entity , and brcko district runs a health care system under its own authority . one for republic of srpska , one for brcko district , and within fb&h : one for fb&h and ten cantonal ministries ( one for each canton ) . acknowledging the threat that pandemic influenza represents on both global and national level and because of request from who to all member countries , council of ministers , based on clause 17 of law of council of ministries ( official gazette b&h , number 30/03 , 42/03 ) , and on suggestion from ministry of civil affairs , passed preparedness and control plan for pandemic influenza in bosnia and herzegovina ( 6 ) . surveillance system for human seasonal influenza in b&h is part of routine passive surveillance of communicable diseases . sporadic cases are registered individually , and during the epidemics they are registered collectively weekly ( 6 ) . since there is no sentinel surveillance in b&h , by the preparedness and control plan routine surveillance of acute respiratory infections ( ari ) and influenzalike illness ( ili ) will be strengthen in city centers with corresponding network of passive surveillance . as of 2012 , when this paper is finalized , there is no sentinel surveillance for influenza established in any part of b&h . in lack of specific vaccines and antiviral medicines , important part in contingency have non - pharmaceutical measures ( veterinary contingency measures , limitation of human travel in affected areas , use of personal preventive means , general and personal hygiene etc ) ( 6 ) . until the moment that this article is being written , b&h remains only country from this region that does not have contingency plan . since contingency plan represents guide for implementation of preparedness plan , the lack of one , decreases general preparedness of b&h s healthcare system . but economy is probably not to blame , since all the neighboring countries , that are not economically much more advanced have one . for example serbia ( 7 ) , croatia ( 8) and fyr of macedonia ( 9 ) have detailed and well prepared contingency plans , with clear instruction of how to implement measures from preparedness plan . so most likely , that the reason is actually because of specifics of organization of b&h s healthcare system . since contingency plan should be made on national level , but healthcare decisions are being made on entity level , it is very difficult to make unique contingency plan that needs great deal of practical data . also majority of the other european countries have preparedness plans as well as detailed contingency plans france ( 10 ) , austria ( 11 ) , hungary ( 12 ) italy ( 13 ) detailed analysis of the preparedness plans of several european countries health care systems and bosnia and herzegovina by the who guidelines elements by phases is presented in table 2 . b&h included -covered all elements of influenza pandemic preparedness in their preparedness plans ; and all countries but b&h have had contingency plans . in lack of specific vaccines and antiviral medicines , important part in contingency have non - pharmaceutical measures ( veterinary contingency measures , limitation of human travel in affected areas , use of personal preventive means , general and personal hygiene etc ) ( 6 ) . until the moment that this article is being written , b&h remains only country from this region that does not have contingency plan . since contingency plan represents guide for implementation of preparedness plan , the lack of one , decreases general preparedness of b&h s healthcare system . but economy is probably not to blame , since all the neighboring countries , that are not economically much more advanced have one . for example serbia ( 7 ) , croatia ( 8) and fyr of macedonia ( 9 ) have detailed and well prepared contingency plans , with clear instruction of how to implement measures from preparedness plan . so most likely , that the reason is actually because of specifics of organization of b&h s healthcare system . since contingency plan should be made on national level , but healthcare decisions are being made on entity level , it is very difficult to make unique contingency plan that needs great deal of practical data . also majority of the other european countries have preparedness plans as well as detailed contingency plans france ( 10 ) , austria ( 11 ) , hungary ( 12 ) italy ( 13 ) . detailed analysis of the preparedness plans of several european countries health care systems and bosnia and herzegovina by the who guidelines elements by phases is presented in table 2 . b&h included -covered all elements of influenza pandemic preparedness in their preparedness plans ; and all countries but b&h have had contingency plans . after the presented analysis , it is possible to conclude that disregarding the obstacles in b&h health care system policy bosnia and herzegovina has preparedness plans , that are made based on who s guidelines . while other analysed countries besides preparedness has also contingency plan , b&h still has to work on making one . based on that fact , one can says that uk s , france , austria , hungary , italy , croatia , serbia and montenegro healthcare systems are better prepared for pandemic influenza , than b&h s . weaknesses of b&h s healthcare system are late forming of preparedness plan , and lack of contingency plan . implementation of activities stated to be establish by the preparedness plan in 2009 has never being done ( e.g. sentinel surveillance , vaccination policy etc ) although preparedness plan exists it is in need for constant urging on fulfilling of activities and objectives in all categories ( planning , monitoring , contingency , respond and communication ) in every stage of pandemic .

neurological disease is one of the major burdens to healthcare , and in the case of neurodegenerative disorders , the incidence will undoubtedly rise with increased life expectancy . consequently , there has been a concerted effort to understand the genetic basis of neurological disease , particularly in the post - genome era in which large population studies are highlighting new susceptibility loci for further study ( 1,2 ) . the mouse has played a key role in discerning the normal function of genes in the nervous system but also the consequences of their disruption in human disease ( 3,4 ) . mice have a range of advantages as a model system in neuroscience , including their ability to perform behavioural tasks and respond to pharmacological challenges that can be extrapolated directly to human phenotypic traits ( 5,6 ) . the generation of mouse knockouts has been a commonly used approach to study gene disruption in the context of a whole organism . recent years have seen the initiation of an internationally coordinated programme to create es cells carrying conditional null alleles for all protein - coding genes , and this will be a valuable resource for the research community ( 7,8 ) . however , in the majority of neurological disorders , a homozygous loss - of - function mutation does not occur and therefore studying a gene knockout may produce a far more severe phenotype or initiate compensatory mechanisms that are not wholly relevant to the disease in question ( 9 ) . indeed , a large proportion of human disease is caused by mutations that influence protein structure , binding affinity or function in far subtler manner than complete inactivation ( 10,11 ) . in addition , it is becoming apparent that genetic variants such as single nucleotide polymorphisms ( snps ) outside of protein - coding regions are likely to play an important role in disease ( 1,2 ) . alternative mouse models are therefore vital , and random mutagenesis has become a well - established tool to elucidate both gene function and characterize novel disease pathways ( 12 ) . several large - scale mutagenesis programs were established in the late 1990s as a systematic approach to generating new mouse models of human disease ( 1315 ) . the various strategies are reviewed extensively elsewhere ( 16,17 ) , but briefly , male mice are treated with a controlled dose of n - ethyl - n - nitrosourea ( enu ) that causes random point mutations to occur in their germline . mutagenized males are then bred to wild - type females over one or two generations to generate mutant animals for analysis ( 16 ) . in a phenotype - driven approach , the progeny is screened initially for abnormalities using a simple yet quantitative assessment of physiology and behaviour ( 18,19 ) . this is followed by further analysis of specific traits or body systems of interest , as discussed in more detail below ( 14,15,20 ) . inheritance testing of the selected phenotype is then confirmed prior to positional cloning of the mutation itself . one advantage of this random mutagenesis approach is it can create a range of mutations , including hypomorphic ( reduced amount of gene product ) , hypermorphic ( increased amount of gene product ) and neomorphic ( altered function ) alleles , in addition to those that are loss of function , such as the replacement of a coding amino acid with a premature stop codon . the mutation may also be specific for a particular isoform of the gene , which may have important functional consequences . furthermore , the unbiased nature of the phenotyping process and the pleiotropic nature of mutations mean that novel functions can be assigned for genes that had previously only been studied using a restricted set of experiments in the knockout mouse ( see below ) . the major bottle - neck of the phenotype - driven approach used to be the identification of the causative mutation based on genetic mapping followed by sequencing of candidate genes . with the advent of affordable next - generation sequencing technology , this notoriously laborious process can now be completed far more efficiently ( 2123 ) . phenotype - driven screening will not detect every potentially interesting mutant and is limited by the sensitivity of the screens themselves ( 24,25 ) . neuropathological analysis , although a key feature of the phenotyping process , may also be limited to specific tissues or histopathological methods ( 26 ) . nevertheless , cryopreservation of tissues from each mutant line allows retrospective screens to be carried out for mutations in a gene of interest ; advances in the rapid detection of mutations have made it practical to use this gene - driven approach ( 27,28 ) . although there is no guarantee of any measureable phenotype in the resulting mutant , it has been calculated from pilot studies that 5000 dna samples are sufficient to identify at least two alleles with 90% confidence ( 29 ) , and enu archives much larger than this are now available for screening ( 28,30 ) . furthermore , the fact that many thousands of mutants are generated means that multiple individual mice may contain mutations in the same gene , an allelic series ( 31 ) . phenotype correlations to be carried out on mice of the same genetic background which is an important consideration , particularly in behavioural neuroscience ( 6,32 ) . a number of approaches have been taken to quantify behaviour in phenotype - driven mutagenesis screens , and the major large - scale enu programmes have focussed on a range of neurobehavioural domains for their primary screening protocols ( 14,15,20 ) . typically , high - throughput tests such as the open - field are employed as they are both rapid and electronically quantifiable . advantageously , this test provides preliminary data on a number of parameters , including general activity in a novel environment as well as a measure of anxiety based on thigmotaxis ( 33 ) . yet even such deceptively simple tests rely on consistency between those handling the animals and data interpretation to generate scientifically meaningful results ( 34,35 ) . this is paramount when the performance of a single animal compared with a control data set may define a new mutation of importance ; thus , care must be taken to define standardized protocols ( 32,3638 ) . nonetheless , even tests that are used to model aspects of neuropsychiatric disorders can be amenable to a high - throughput screen , such as sensorimotor gating or pre - pulse inhibition ( ppi ) . this particular paradigm quantifies the ability to respond to and filter auditory information , and is one parameter that can be analysed in mice as an endophenotype ; the approximation of a similar response known to be abnormal in schizophrenia ( 5 ) . consequently , ppi and open field , along with rotarod testing for motor function and hotplate for nociception , make up the primary screen of several phenotyping pipelines ( 25,37,39,40 ) . an outlier identified from a high - throughput screen is usually taken through a more focussed and/or complementary series of secondary behavioural tests to more accurately define the phenotype of potential interest . for example , inactivity in the open field may be a consequence of anxiety or simply due to motor dysfunction , arousal or sensory defects ( 6 ) . additional physiological parameters outside of the nervous system must also be taken into account when interpreting such data ; an overtly obese mouse , or one with cardiovascular defects may struggle to run on a rotarod , for instance ( 41,42 ) . another issue to consider is the statistical power of the primary screen used to isolate outliers for follow - up studies ( 20 ) . recent work based on the open - field test has demonstrated that subtler phenotypes can be reliably identified by screening an additional second generation ( g2 ) of mutant animals . although this entails the additional time and cost of more breeding , it was argued that the false discovery rate was dramatically reduced ( 43 ) . despite these caveats , a considerable number of neurological enu mutants have been successfully mapped and cloned , revealing important new insights into gene function in the nervous system ( 16,44 ) . the inherent complexity of the molecular mechanisms that define the function of the brain means that many enu mutants with abnormal neurological phenotypes are yet to be characterized . importantly , primary screening data are usually publically available from the major centres allowing searches to be made for phenotypes of interest [ see box 1 in gondo et al . table 1 contains a selection of the more recently identified neurological enu mutants , and some of those that have been studied in some detail are discussed below , focussing on three key areas of neurological disease research . table 1.examples of neurobehavioural enu mouse mutants ( 2007)mutant gene namemutation(s ) studiedphenotypic effectsreferencesaf4v280a/+ataxia , cataracts , t cell development defects , adult - onset purkinje cell death(52,55,57)arcn1i422t / i422tcoat colour dilution , ataxia , purkinje cell death(128)atp13i810n/+small size , seizures , elevated exploratory locomotion , sleep abnormalities(85,88)cacna1ar1255l/+ataxia , cerebellar atrophy(129)cacna1ar1255l / r1255lsevere ataxia , premature deathdisc1l100p/+ppi , latent inhibition defects(71)disc1l100p / l100pppi , working memory , latent inhibition defects , hyperactivitydisc1q31l/+ppi defectsdisc1q31l / q31lppi , working memory , latent inhibition defects , depressive - like behaviourdisc1q31l / l100pppi defectsfbxl3c358s/+lengthened circadian period(102,109)c358/c358lengthened circadian period , reduced anxiety , depression - like behaviourfoxp2r552h/+normal development , defects in motor learning(130)foxp2r552h / r552hsevere motor impairment , developmental delay , cerebellar foliation defects , 34-week survivalfoxp2s321x / s321xsevere motor impairment , developmental delay , cerebellar foliation defects , 34-week survivalfoxp2n549k / n549klate - onset cerebellar foliation defects , 35-month survivalfoxc1f107l / f107lcortical and skull abnormalities(131)garsc201r/+reduced grip strength , reduction in axonal diameter(63)garsc201r / c201rneurodeveopmental delay , 3-week survivalgrin1c844r/+hyperactivity , increased novelty seeking(132)npc1d1005g/+abnormal cholesterol metabolism , purkinje cell loss , late - onset ataxia(126)ror - alphac257x / c257xataxia , severe cerebellar atrophy(133)rtn4r189h / r189hgreater social preference , subtle cognitive defects(134)snap-25bi67t/+subtle ataxia , ppi defects , advanced circadian phase(76,106)trpc3t635a/+ataxia , late - onset purkinje cell death(59)jenna / tuba1as140g/+hyperactivity , increased acoustic startle , neuronal migration defects(135137)tuba1ad85g/+hyperactivity , neuronal migration defects(138 ) examples of neurobehavioural enu mouse mutants ( 2007 ) historically , spontaneous mouse mutants were identified during the maintenance of large breeding colonies . as a consequence , phenotypes such as ataxia represent many of the first lines to be cloned as such characteristics are particularly conspicuous ( 45,46 ) . the same is true to some extent of phenotype - driven mutagenesis , where there will often be a bias towards movement defects that are both early onset and easy to recognize . for example , three independent enu mutants in the peripheral myelin protein 22 ( pmp22 ) gene were some of the first to be cloned and characterized from the mutagenesis screen at mrc harwell ( 14 ) . initially identified due to their resting tremor , these models of human peripheral neuropathy provided an excellent example of the range of phenotypic and pathological severity that can be generated by an allelic series of point mutants ( 4749 ) . ataxia or defects in motor co - ordination are features of many neurological disorders and are typically characterized by neurodegeneration in the cerebellum , the structure in the brain necessary for controlling motor function . although the genetic cause for a considerable proportion of inherited ataxias is known , the pathogenic mechanisms involved are still unclear ( 50,51 ) . as part of a screen to identify new models of human ataxia , the dominant enu mutant robotic ( rob ) was selected due to its jerky , ataxic gait that is apparent from as early as 3 weeks of age . pathological analysis revealed a progressive yet highly specific loss of cerebellar purkinje cells that begin several weeks after the onset of the ataxic gait ( 52 ) . the causative mutation was subsequently identified in the gene af4 ( all fused from chromosome 4 , also known as aff1 ) that at that time was known to be a putative transcriptional activator implicated in leukaemia ( 53 ) . the mouse knockout of af4 has defects in b- and t - cell development consistent with a role in leukaemogensis , but no neurological abnormalities were reported ( 54 ) ; consequently , the rob mouse revealed an important new function for af4 in the cns that was not apparent from the null allele . biochemical data demonstrated that the mutant protein accumulated in rob purkinje cells due to the loss of a conserved ubiqutin ligase - binding site , and that this increase in af4 protein levels was detrimental to purkinje cell survival ( 55 ) . understanding the proposed gain - of - function mechanism for the rob mutation required detailed examination of the role of af4 in transcriptional regulation . first , it was discovered that af4 and related proteins are part of a large complex co - ordinating rna polymerase ii processivity and chromatin remodelling via histone methylation ( 56 ) . subsequently , transcriptional profiling of purkinje cells from wild - type and rob mice identified igf-1 as target of the af4-containing complex and revealed a pathogenic pathway in the mutant cerebellum ( 57 ) . significantly , dysfunction of the igf-1 pathway is a recurrent finding in mouse ataxic mutants and human ataxia patients and thus represents a common neuropathological mechanism that may be targeted for treatment ( 58 ) . moonwaker ( mwk ) , a second dominant ataxic mutant characterized from the same enu screen , also displays gait defects around weaning age , although purkinje cell loss in this case is later in onset from around 6 months of age ( 59 ) . the causative mutation was identified in the trpc3 gene , encoding the transient receptor potential cation channel c3 ( trpc3 ) . the point mutation occurs in a highly conserved cytoplasmic linker region of trpc3 , resulting in altered gating and increased activity of the channel as a gain - of - function mechanism ( 59 ) . significantly , purkinje cell morphology is impaired in the mwk mouse , which is consistent with the highly specific expression of trpc3 in purkinje cells during their dendritic development . genetic ablation of this channel in the mouse also causes gait defects ( 60 ) ; however , the mwk enu mutant defines a novel role for trpc3 in both the development and survival of purkinje cells . although no proven causative mutations have been described to date ( 61 ) , a polymorphism influencing the methylation of trpc3 was reported in idiopathic ataxia , although its frequency did not reach the significance in the population analysed ( 62 ) . for example , a mouse originally identified due to its reduced grip strength contains a mutation in glycyl t - rna synthetase or gars ( 63 ) . gars heterozygous mutants show a reduction in the axon diameter of peripheral nerves and some muscle denervation , but no axonal degeneration . gars homozygous mutants , however , only survive for a few weeks with major defects in the cns observed . importantly , this gene is mutated in charcot - marie - tooth type 2d ( cmt2d ) disease , a form of dominantly inherited motor neuropathy . the relatively mild phenotype in gars mice differs from the more severe gars dominant mutant that also contains a point mutation ( p279k ) in the same gene ( 64 ) . these mice display an early - onset loss of peripheral nerve axons and abnormal morphology of the neuromuscular junction . interestingly , a gene - trap insertional mutant that is essentially a complete gene knockout shows no dominant phenotype , suggesting that haploinsufficiency is not the cause of neuropathy in the two - point mutants ( 64 ) . to investigate this further , both point mutants were crossed with transgenic lines over - expressing either wild - type or mutant forms of gars . these studies demonstrated that the dominant neuropathy phenotype observed in gars and gars mice is caused predominantly by a dose - dependent gain of function that is not alleviated by over - expression of the wild - type protein ( 65 ) . considering that dominant mutations in three other trna synthetase genes cause neuropathies similar in pathology to cmt2d , these mouse lines will continue to be valuable models of the heterogenous nature of these disorders . the significant genetic component of psychiatric disorders is well - documented and many susceptibility genes are conserved in rodents , although it is accepted that examining specific symptoms related to emotions or psychotic behaviour can not be realistically therefore , research has relied on the aforementioned endophenotypes to analyse specific pathophysiological features of psychiatric disease by studying mutations in genes directly associated with a particular disorder , relevant neurobiological processes such as neurotransmission , or behavioural responses to pharmacological agents known to modulate particular symptoms ( 6,6769 ) . perhaps a reflection of the complex aetiology of psychiatric disease and interpretation of behavioural assays in mice , relatively few enu mutants have been characterized in this area ; however , some important examples have been identified . of all the genes implicated in schizophrenia , disrupted - in - schizophrenia 1 ( disc1 ) is one of the most prominent , with both the disc1 locus and polymorphic variants in the disc1 gene associated with the disorder ( 70 ) . this gene was therefore selected for gene - driven screening in a mouse enu mutant archive , in which two missense mutations were identified ( 71 ) . interestingly , both lines showed a range of behavioural deficits although few were shared ; the disc1-q31l homozygous mutant displayed depressive - like behaviour , whereas the disc1-l100p homozygous line showed abnormalities related to schizophrenia , such as ppi and cognitive defects . these schizophrenic endophenotypes were reversible with antipsychotic treatment ( 71 ) as well as an inhibitor of gsk3 , a known signalling molecule that interacts with disc1 during neurodevelopment ( 72 ) . detailed pathological studies have since identified morphological defects in the dendrites of l100p cortical neurons ( 73 ) . these two mutants show how point mutations in a single gene can cause a heterogeneous behavioural profile . as a caveat to this work , an independent group has been unable to reproduce either the depressive or sensorimotor gating abnormalities in these mutants ( 74 ) ; this could be explained by the additional generations of backcrossing to the wild - type ( c57bl/6 ) strain carried out prior to testing or differences in the behavioural protocols , for example ( 75 ) . the dominant blind - drunk ( bdr ) mutant was originally identified due to its subtle gait disturbance and positional cloning isolated a missense mutation in the neuronal isoform of snap-25 , part of the core snare complex essential for exocytosis ( 76 ) . the mutation causes increased binding affinities within the snare complex and disrupted exocytotic vesicle recycling in vivo , limiting neurotransmission under continued stimulation ( 76 ) . behavioural testing of these mice showed no overt cognitive deficits , but an impaired ppi response . importantly , the study of snare proteins has been limited by the neonatal lethality of knockout lines ( 77,78 ) ; bdr therefore provides valuable new insights into the role of the snare complex and aberrant neurotransmission relevant to psychiatric disease due to the relatively subtle nature of the mutation . furthermore , snap-25 itself has been implicated in schizophrenia from a range of genetic ( 79 ) , pathological ( 80,81 ) and functional ( 82 ) studies of the synapse . bipolar disorder shares some overlapping symptoms with schizophrenia as part of a spectrum of psychotic disorders , although it is associated more readily with both manic and depressive episodes . many genes have been implicated in bipolar disorder , although similar to schizophrenia , it is unlikely that a single genetic factor will be causative ( 83,84 ) . in a phenotype - driven screen , the myshkin ( myk ) dominant mutant was selected due to its small size in addition to the presence of spontaneous , recurrent convulsive seizures and was originally presented as a model of epilepsy ( 85 ) . a missense mutation in the na , k - atpase ( nka ) 3 isoform ( atp1a3 ) caused an approximate 40% reduction of the enzyme activity in the myk brain ; this gene has been implicated both in bipolar disorder and rapid - onset dystonia parkinsonism ( 86,87 ) . when examined in more detail , the behavioural phenotype in myk mice also included highly elevated exploratory locomotion and sleep abnormalities in addition to enhanced sensitivity to d - amphetamine ; significantly , these mania - related behaviours could be attenuated with mood stabilizers such as lithium ( 88 ) . studies of primary neurons suggested that enhanced calcium - dependent signal - transduction pathways downstream of nka may underlie the behavioural phenotypes and are potential therapeutic targets ( 88 ) . myk mice therefore provide important insights into neuronal signalling in mania , particularly as epilepsy and bipolar disorder can be co - morbid in humans ( 89 ) . non - genetic influences are undoubtedly a major risk factor in psychiatric disease , ranging from prenatal stress to adverse life events during adolescence ( 90,91 ) . in recent years , increasing attention has been paid to how best to incorporate such elements into behavioural experiments in mice to understand the synergy between genes and the environment while potentially improving the construct validity of such models ( 9294 ) . building on work in wild - type rodents , there is now a range of gene environment paradigms being applied to mutant mice , and enu lines have provided useful examples of these methods . to examine the significance of early life events combined with a synaptic mutation , a variable prenatal stress protocol was applied to the bdr snap-25 mutant and significantly , the sensorimotor gating ( ppi ) deficits observed in non - stressed mutants were markedly enhanced by stress in utero and social interaction abnormalities were observed only in bdr animals from stressed dams ( 95 ) . these results showed for the first time that combining a synaptic mouse point mutant with a prenatal stressor paradigm produces both modified and previously unseen phenotypes relevant to the study of psychiatric disorders . disc1-l100p and -q31l mutants have also been further studied in the context of a postnatal stress paradigm . chronic social defeat ( csd ) , a correlate of psychosocial stress in humans , was applied to both heterozygous mutant lines and , interestingly , each responded differently to the csd stressor ( 96 ) . measures of anxiety were influenced by csd in the q31l line , whereas social interaction was affected in the l100p mutant ( 96 ) . the exact pathophysiological mechanisms that underlie such gene environment interactions are still unclear , but these studies serve to illustrate that enu mutants with subtle behavioural defects are highly suited for assessing this phenomenon . circadian rhythms are of vital importance to mammalian physiology , and as a consequence , disruption of these daily cycles can have profound effects on human health . indeed , studies are beginning to define a direct association between disrupted rest / activity patterns and an increasing number of neurological disorders , including psychiatric and neurodegenerative disease ( 9799 ) . in the mouse , there are well - established methods for analysing aspects of circadian behaviour , predominantly based around quantifying wheel - running activity under differential lighting conditions ( 100 ) . screening enu mutants using these methods , although not as high - throughput as other behavioural screens , has been particularly successful ; a considerable number of lines displaying abnormally long or short active periods or aberrant responses to shifts in the light / dark cycle have been identified ( 101 ) . of those that have been characterized at a mechanistic level , the afterhours ( afh ) mutant displayed considerably lengthened activity profiles under constant lighting conditions , with a free - running period of approximately 27 h ( 102,103 ) . afh contains a loss - of - function mutation in the e3 ubiquitin ligase fbxl3 , causing a reduction in the normal degradation of cryptochrome ( cry ) proteins . a post - translational feedback loop containing cry and period ( per ) is essential for normal pacemaking in the suprachiasmatic nucleus ( scn ) , and it was shown that the imbalance of normal cry levels resulted in an extended period of the core clock in these mutants ( 102,103 ) . by crossing the afh line to a casein kinase ( csnk1e ) mutant that causes destabilization of per , it was recently shown that the circadian period is finely controlled by both cry and per proteins independently as opposed to the mutual stabilization model predicted by in vitro experiments ( 104 ) . although the mechanisms that link disturbed sleep / wake profiles and psychiatric disease are unclear , it is intuitive that the multiple brain regions disrupted in conditions such as schizophrenia are likely to overlap with those involved in the complex processes of sleep and the maintenance of circadian rhythms ( 105 ) . in view of the schizophrenic endophenotypes in the bdr mouse ( see above ) , this mutant line was also screened for circadian activity ( 106 ) . the behavioural data showed an advanced phase of activity under light / dark cycles , but also fragmentation of the profiles , somewhat reminiscent of the disturbed sleep / wake patterns observed in schizophrenia ( 107 ) . interestingly , the timing of activity was normal under constant light or dark , suggesting that the core clock in bdr was functioning correctly . this was confirmed by molecular analysis of the scn , although expression of the signalling neuropeptide arginine vasopressin ( avp ) was phase advanced in line with the activity data . these results suggest that disrupted neurotransmission in the scn due to the bdr snap-25 mutation influences important hypothalamic outputs , potentially causing desynchrony between the brain and the periphery ( 106 ) . due to the undoubted anatomical overlap between complex behavioural domains , it is also becoming clear that mouse mutants originally identified from circadian screens are likely to show additional behavioural abnormalities ( 108 ) ; indeed , homozygous afh mutants also show hyperactivity , reduced anxiety and depression - like behaviour , suggesting this mutant may also model some aspects of mania related to disrupted circadian rhythms ( 109 ) . the examples above illustrate the complex and varied behavioural phenotypes that can be generated by either phenotype- or genotype - driven enu mutagenesis . another practical application of enu mutants is for identifying new loci that either suppress or enhance a disease phenotype of interest , also known as a modifier screen . successfully utilized in lower organisms ( 110 ) this strategy may be particularly suited to neurodegenerative disease models , where performance in a simple motor - coordination task would provide the baseline data and used to identify outliers in a genetic cross with a population of random mutants ( 16 ) . in particular , the identification of a suppressor mutation may help identify new therapeutic targets or disease mechanisms . a prime example of this phenomenon using a single enu mutant line is the legs - at - odd - angles ( loa ) mouse that contains a mutation in the cytoplasmic dyenin heavy chain ( dync1h1 ) ( 112 ) . while crossing loa mice to model of huntington 's disease enhances the pathological phenotype by slowing clearance of huntingtin protein ( 113 ) , the neurodegenerative phenotype of the sodg93a model of amyotrophic lateral sclerosis ( als ) is significantly suppressed ( 114 ) . preliminary data from an enu screen for modifiers of k3 mice , a model of alzheimer 's disease ( ad ) , have recently been described ( 115 ) . this model expresses mutant tau protein and has been well studied for pathological and behavioural abnormalities ( 116 ) ; consequently , a simple battery of motor tests could be used to define the k3 phenotype . a number of mutant pedigrees , including one that partially rescued the tremor observed in k3 mice , were described , although the identification of this potential suppressor mutation or the analysis of additional cognitive phenotypes was not reported ( 115 ) . such studies therefore rely on a robust and quantifiable phenotypic assay for the disease model in the first instance ; however , this is a potentially very valuable and unbiased approach to understanding how in the interaction between genes can influence pathogenic pathways . while detailed optimization and standardization of enu mutant phenotyping is undoubtedly important , due to obvious practical and financial constraints , primary screening is typically carried out on adult mice between 8 and 18 weeks of age ( 25,37 ) . as a consequence , with particular reference to age - related disorders such as neurodegeneration , potentially valuable mouse models that display late - onset phenotypes or pathology would not be detected . to address this issue , a small number of ageing enu screens have been established , with the aim of testing cohorts of mutants at several time points up to 18 months of age or beyond [ for examples , see http://www.har.mrc.ac.uk/research/functional-genomics-and-disease-mechanisms-section/disease-model-discovery-and-translatio-2 and johnson ( 117 ) ] . it is well - established that the behavioural response in ageing mice can be quite different from younger animals ( 118 ) , and issues of general health and weight must also be taken into account ( 119 ) . however , these challenging and time - consuming studies are a very important way of identify genes and pathways involved in age - related disease . finally , looking further ahead , advances in sequencing technology and data analysis will have an important role to play in the exploitation of enu mutants , not just at the positional cloning stage . gene - driven screens are currently limited to screening small numbers of loci for an allelic series of mutations ; however , it will become financially feasible in the coming years to sequence the entire genome of all archived mutants . another approach would be to focus on the mouse exome or even the transcriptome from archived tissue ( 120 ) . although this would dramatically reduce the amount of data to generate and analyse , with the realization that non - protein - coding sequences also play such a key role in the function of the cns ( 121,122 ) , a genome - wide database of mutants would maximize the potential of the enu mutagenesis resource that numbers over 50 000 individually archived lines . indeed , enu mutations in regulatory non - coding regions causing distinct phenotypes have already been described ( 123,124 ) . as more is revealed regarding the complexity and multi - factorial nature of human neurological disease , access to models that recapitulate aspects of the behavioural and/or pathological abnormalities associated with these disorders is essential . with the ability to carry out longitudinal studies to identify early biomarkers and by focussing on specific endophenotypes , the mouse will continue to be a vital tool to identify disease pathways in neurodegenerative and psychiatric disease research . enu mutagenesis has already generated a significant number of new clinically relevant mouse models , and technological improvements in phenotyping and sequencing strategies will only help improve the efficiency of their identification . access to point mutants will also complement gene knockout studies to model the heterogeneous range of human disease phenotypes . for example , the well - studied mouse model of the lysosomal storage disorder niemann - pick type c ( npc ) does not express any npc protein and serves as a model for the severe infantile forms of this disease ( npc1 ) ( 125 ) ; however , a recently described enu mutant ( npc1 ) provides a more practical model of the more common yet later - onset npc pathology that is caused by missense mutations ( 126 ) . furthermore , the number of potential genetic variants that lead to disease susceptibility is ever - expanding , including novel snps or de novo copy number variants that will drive future mouse modelling experiments ( 127 ) as well as epigenetic and environmental factors . therefore , in combination with transgenics and conditional knockouts , enu mutagenesis will continue to play an important role in the identification and characterization of mutations that underlie human neurological disease .

identifying genes involved in behavioural disorders in man is a challenge as the cause is often multigenic and the phenotype is modulated by environmental cues . mouse mutants are a valuable tool for identifying novel pathways underlying specific neurological phenotypes and exploring the influence both genetic and non - genetic factors . many human variants causing behavioural disorders are not gene deletions but changes in levels of expression or activity of a gene product ; consequently , large - scale mouse enu mutagenesis has the advantage over the study of null mutants in that it generates a range of point mutations that frequently mirror the subtlety and heterogeneity of human genetic lesions . enu mutants have provided novel and clinically relevant functional information on genes that influence many aspects of mammalian behaviour , from neuropsychiatric endophenotypes to circadian rhythms . this review will highlight some of the most important findings that have been made using this method in several key areas of neurological disease research .

INTRODUCTION BEHAVIOURAL SCREENING MOVEMENT DISORDERS AND NEURODEGENERATION NEUROPSYCHIATRIC DISEASE CIRCADIAN BEHAVIOUR FUTURE PROSPECTS CONCLUSION FUNDING

neurological disease is one of the major burdens to healthcare , and in the case of neurodegenerative disorders , the incidence will undoubtedly rise with increased life expectancy . consequently , there has been a concerted effort to understand the genetic basis of neurological disease , particularly in the post - genome era in which large population studies are highlighting new susceptibility loci for further study ( 1,2 ) . mice have a range of advantages as a model system in neuroscience , including their ability to perform behavioural tasks and respond to pharmacological challenges that can be extrapolated directly to human phenotypic traits ( 5,6 ) . the generation of mouse knockouts has been a commonly used approach to study gene disruption in the context of a whole organism . however , in the majority of neurological disorders , a homozygous loss - of - function mutation does not occur and therefore studying a gene knockout may produce a far more severe phenotype or initiate compensatory mechanisms that are not wholly relevant to the disease in question ( 9 ) . indeed , a large proportion of human disease is caused by mutations that influence protein structure , binding affinity or function in far subtler manner than complete inactivation ( 10,11 ) . alternative mouse models are therefore vital , and random mutagenesis has become a well - established tool to elucidate both gene function and characterize novel disease pathways ( 12 ) . several large - scale mutagenesis programs were established in the late 1990s as a systematic approach to generating new mouse models of human disease ( 1315 ) . inheritance testing of the selected phenotype is then confirmed prior to positional cloning of the mutation itself . one advantage of this random mutagenesis approach is it can create a range of mutations , including hypomorphic ( reduced amount of gene product ) , hypermorphic ( increased amount of gene product ) and neomorphic ( altered function ) alleles , in addition to those that are loss of function , such as the replacement of a coding amino acid with a premature stop codon . the mutation may also be specific for a particular isoform of the gene , which may have important functional consequences . furthermore , the unbiased nature of the phenotyping process and the pleiotropic nature of mutations mean that novel functions can be assigned for genes that had previously only been studied using a restricted set of experiments in the knockout mouse ( see below ) . the major bottle - neck of the phenotype - driven approach used to be the identification of the causative mutation based on genetic mapping followed by sequencing of candidate genes . phenotype - driven screening will not detect every potentially interesting mutant and is limited by the sensitivity of the screens themselves ( 24,25 ) . nevertheless , cryopreservation of tissues from each mutant line allows retrospective screens to be carried out for mutations in a gene of interest ; advances in the rapid detection of mutations have made it practical to use this gene - driven approach ( 27,28 ) . furthermore , the fact that many thousands of mutants are generated means that multiple individual mice may contain mutations in the same gene , an allelic series ( 31 ) . phenotype correlations to be carried out on mice of the same genetic background which is an important consideration , particularly in behavioural neuroscience ( 6,32 ) . a number of approaches have been taken to quantify behaviour in phenotype - driven mutagenesis screens , and the major large - scale enu programmes have focussed on a range of neurobehavioural domains for their primary screening protocols ( 14,15,20 ) . consequently , ppi and open field , along with rotarod testing for motor function and hotplate for nociception , make up the primary screen of several phenotyping pipelines ( 25,37,39,40 ) . an outlier identified from a high - throughput screen is usually taken through a more focussed and/or complementary series of secondary behavioural tests to more accurately define the phenotype of potential interest . additional physiological parameters outside of the nervous system must also be taken into account when interpreting such data ; an overtly obese mouse , or one with cardiovascular defects may struggle to run on a rotarod , for instance ( 41,42 ) . another issue to consider is the statistical power of the primary screen used to isolate outliers for follow - up studies ( 20 ) . despite these caveats , a considerable number of neurological enu mutants have been successfully mapped and cloned , revealing important new insights into gene function in the nervous system ( 16,44 ) . the inherent complexity of the molecular mechanisms that define the function of the brain means that many enu mutants with abnormal neurological phenotypes are yet to be characterized . table 1 contains a selection of the more recently identified neurological enu mutants , and some of those that have been studied in some detail are discussed below , focussing on three key areas of neurological disease research . table 1.examples of neurobehavioural enu mouse mutants ( 2007)mutant gene namemutation(s ) studiedphenotypic effectsreferencesaf4v280a/+ataxia , cataracts , t cell development defects , adult - onset purkinje cell death(52,55,57)arcn1i422t / i422tcoat colour dilution , ataxia , purkinje cell death(128)atp13i810n/+small size , seizures , elevated exploratory locomotion , sleep abnormalities(85,88)cacna1ar1255l/+ataxia , cerebellar atrophy(129)cacna1ar1255l / r1255lsevere ataxia , premature deathdisc1l100p/+ppi , latent inhibition defects(71)disc1l100p / l100pppi , working memory , latent inhibition defects , hyperactivitydisc1q31l/+ppi defectsdisc1q31l / q31lppi , working memory , latent inhibition defects , depressive - like behaviourdisc1q31l / l100pppi defectsfbxl3c358s/+lengthened circadian period(102,109)c358/c358lengthened circadian period , reduced anxiety , depression - like behaviourfoxp2r552h/+normal development , defects in motor learning(130)foxp2r552h / r552hsevere motor impairment , developmental delay , cerebellar foliation defects , 34-week survivalfoxp2s321x / s321xsevere motor impairment , developmental delay , cerebellar foliation defects , 34-week survivalfoxp2n549k / n549klate - onset cerebellar foliation defects , 35-month survivalfoxc1f107l / f107lcortical and skull abnormalities(131)garsc201r/+reduced grip strength , reduction in axonal diameter(63)garsc201r / c201rneurodeveopmental delay , 3-week survivalgrin1c844r/+hyperactivity , increased novelty seeking(132)npc1d1005g/+abnormal cholesterol metabolism , purkinje cell loss , late - onset ataxia(126)ror - alphac257x / c257xataxia , severe cerebellar atrophy(133)rtn4r189h / r189hgreater social preference , subtle cognitive defects(134)snap-25bi67t/+subtle ataxia , ppi defects , advanced circadian phase(76,106)trpc3t635a/+ataxia , late - onset purkinje cell death(59)jenna / tuba1as140g/+hyperactivity , increased acoustic startle , neuronal migration defects(135137)tuba1ad85g/+hyperactivity , neuronal migration defects(138 ) examples of neurobehavioural enu mouse mutants ( 2007 ) historically , spontaneous mouse mutants were identified during the maintenance of large breeding colonies . as a consequence , phenotypes such as ataxia represent many of the first lines to be cloned as such characteristics are particularly conspicuous ( 45,46 ) . for example , three independent enu mutants in the peripheral myelin protein 22 ( pmp22 ) gene were some of the first to be cloned and characterized from the mutagenesis screen at mrc harwell ( 14 ) . initially identified due to their resting tremor , these models of human peripheral neuropathy provided an excellent example of the range of phenotypic and pathological severity that can be generated by an allelic series of point mutants ( 4749 ) . as part of a screen to identify new models of human ataxia , the dominant enu mutant robotic ( rob ) was selected due to its jerky , ataxic gait that is apparent from as early as 3 weeks of age . pathological analysis revealed a progressive yet highly specific loss of cerebellar purkinje cells that begin several weeks after the onset of the ataxic gait ( 52 ) . the mouse knockout of af4 has defects in b- and t - cell development consistent with a role in leukaemogensis , but no neurological abnormalities were reported ( 54 ) ; consequently , the rob mouse revealed an important new function for af4 in the cns that was not apparent from the null allele . biochemical data demonstrated that the mutant protein accumulated in rob purkinje cells due to the loss of a conserved ubiqutin ligase - binding site , and that this increase in af4 protein levels was detrimental to purkinje cell survival ( 55 ) . first , it was discovered that af4 and related proteins are part of a large complex co - ordinating rna polymerase ii processivity and chromatin remodelling via histone methylation ( 56 ) . subsequently , transcriptional profiling of purkinje cells from wild - type and rob mice identified igf-1 as target of the af4-containing complex and revealed a pathogenic pathway in the mutant cerebellum ( 57 ) . significantly , dysfunction of the igf-1 pathway is a recurrent finding in mouse ataxic mutants and human ataxia patients and thus represents a common neuropathological mechanism that may be targeted for treatment ( 58 ) . the point mutation occurs in a highly conserved cytoplasmic linker region of trpc3 , resulting in altered gating and increased activity of the channel as a gain - of - function mechanism ( 59 ) . these mice display an early - onset loss of peripheral nerve axons and abnormal morphology of the neuromuscular junction . interestingly , a gene - trap insertional mutant that is essentially a complete gene knockout shows no dominant phenotype , suggesting that haploinsufficiency is not the cause of neuropathy in the two - point mutants ( 64 ) . considering that dominant mutations in three other trna synthetase genes cause neuropathies similar in pathology to cmt2d , these mouse lines will continue to be valuable models of the heterogenous nature of these disorders . perhaps a reflection of the complex aetiology of psychiatric disease and interpretation of behavioural assays in mice , relatively few enu mutants have been characterized in this area ; however , some important examples have been identified . of all the genes implicated in schizophrenia , disrupted - in - schizophrenia 1 ( disc1 ) is one of the most prominent , with both the disc1 locus and polymorphic variants in the disc1 gene associated with the disorder ( 70 ) . this gene was therefore selected for gene - driven screening in a mouse enu mutant archive , in which two missense mutations were identified ( 71 ) . interestingly , both lines showed a range of behavioural deficits although few were shared ; the disc1-q31l homozygous mutant displayed depressive - like behaviour , whereas the disc1-l100p homozygous line showed abnormalities related to schizophrenia , such as ppi and cognitive defects . these two mutants show how point mutations in a single gene can cause a heterogeneous behavioural profile . the dominant blind - drunk ( bdr ) mutant was originally identified due to its subtle gait disturbance and positional cloning isolated a missense mutation in the neuronal isoform of snap-25 , part of the core snare complex essential for exocytosis ( 76 ) . importantly , the study of snare proteins has been limited by the neonatal lethality of knockout lines ( 77,78 ) ; bdr therefore provides valuable new insights into the role of the snare complex and aberrant neurotransmission relevant to psychiatric disease due to the relatively subtle nature of the mutation . furthermore , snap-25 itself has been implicated in schizophrenia from a range of genetic ( 79 ) , pathological ( 80,81 ) and functional ( 82 ) studies of the synapse . bipolar disorder shares some overlapping symptoms with schizophrenia as part of a spectrum of psychotic disorders , although it is associated more readily with both manic and depressive episodes . many genes have been implicated in bipolar disorder , although similar to schizophrenia , it is unlikely that a single genetic factor will be causative ( 83,84 ) . a missense mutation in the na , k - atpase ( nka ) 3 isoform ( atp1a3 ) caused an approximate 40% reduction of the enzyme activity in the myk brain ; this gene has been implicated both in bipolar disorder and rapid - onset dystonia parkinsonism ( 86,87 ) . studies of primary neurons suggested that enhanced calcium - dependent signal - transduction pathways downstream of nka may underlie the behavioural phenotypes and are potential therapeutic targets ( 88 ) . non - genetic influences are undoubtedly a major risk factor in psychiatric disease , ranging from prenatal stress to adverse life events during adolescence ( 90,91 ) . in recent years , increasing attention has been paid to how best to incorporate such elements into behavioural experiments in mice to understand the synergy between genes and the environment while potentially improving the construct validity of such models ( 9294 ) . building on work in wild - type rodents , there is now a range of gene environment paradigms being applied to mutant mice , and enu lines have provided useful examples of these methods . to examine the significance of early life events combined with a synaptic mutation , a variable prenatal stress protocol was applied to the bdr snap-25 mutant and significantly , the sensorimotor gating ( ppi ) deficits observed in non - stressed mutants were markedly enhanced by stress in utero and social interaction abnormalities were observed only in bdr animals from stressed dams ( 95 ) . these results showed for the first time that combining a synaptic mouse point mutant with a prenatal stressor paradigm produces both modified and previously unseen phenotypes relevant to the study of psychiatric disorders . disc1-l100p and -q31l mutants have also been further studied in the context of a postnatal stress paradigm . the exact pathophysiological mechanisms that underlie such gene environment interactions are still unclear , but these studies serve to illustrate that enu mutants with subtle behavioural defects are highly suited for assessing this phenomenon . indeed , studies are beginning to define a direct association between disrupted rest / activity patterns and an increasing number of neurological disorders , including psychiatric and neurodegenerative disease ( 9799 ) . in the mouse , there are well - established methods for analysing aspects of circadian behaviour , predominantly based around quantifying wheel - running activity under differential lighting conditions ( 100 ) . screening enu mutants using these methods , although not as high - throughput as other behavioural screens , has been particularly successful ; a considerable number of lines displaying abnormally long or short active periods or aberrant responses to shifts in the light / dark cycle have been identified ( 101 ) . of those that have been characterized at a mechanistic level , the afterhours ( afh ) mutant displayed considerably lengthened activity profiles under constant lighting conditions , with a free - running period of approximately 27 h ( 102,103 ) . although the mechanisms that link disturbed sleep / wake profiles and psychiatric disease are unclear , it is intuitive that the multiple brain regions disrupted in conditions such as schizophrenia are likely to overlap with those involved in the complex processes of sleep and the maintenance of circadian rhythms ( 105 ) . in view of the schizophrenic endophenotypes in the bdr mouse ( see above ) , this mutant line was also screened for circadian activity ( 106 ) . this was confirmed by molecular analysis of the scn , although expression of the signalling neuropeptide arginine vasopressin ( avp ) was phase advanced in line with the activity data . these results suggest that disrupted neurotransmission in the scn due to the bdr snap-25 mutation influences important hypothalamic outputs , potentially causing desynchrony between the brain and the periphery ( 106 ) . due to the undoubted anatomical overlap between complex behavioural domains , it is also becoming clear that mouse mutants originally identified from circadian screens are likely to show additional behavioural abnormalities ( 108 ) ; indeed , homozygous afh mutants also show hyperactivity , reduced anxiety and depression - like behaviour , suggesting this mutant may also model some aspects of mania related to disrupted circadian rhythms ( 109 ) . the examples above illustrate the complex and varied behavioural phenotypes that can be generated by either phenotype- or genotype - driven enu mutagenesis . another practical application of enu mutants is for identifying new loci that either suppress or enhance a disease phenotype of interest , also known as a modifier screen . in particular , the identification of a suppressor mutation may help identify new therapeutic targets or disease mechanisms . this model expresses mutant tau protein and has been well studied for pathological and behavioural abnormalities ( 116 ) ; consequently , a simple battery of motor tests could be used to define the k3 phenotype . such studies therefore rely on a robust and quantifiable phenotypic assay for the disease model in the first instance ; however , this is a potentially very valuable and unbiased approach to understanding how in the interaction between genes can influence pathogenic pathways . however , these challenging and time - consuming studies are a very important way of identify genes and pathways involved in age - related disease . finally , looking further ahead , advances in sequencing technology and data analysis will have an important role to play in the exploitation of enu mutants , not just at the positional cloning stage . although this would dramatically reduce the amount of data to generate and analyse , with the realization that non - protein - coding sequences also play such a key role in the function of the cns ( 121,122 ) , a genome - wide database of mutants would maximize the potential of the enu mutagenesis resource that numbers over 50 000 individually archived lines . indeed , enu mutations in regulatory non - coding regions causing distinct phenotypes have already been described ( 123,124 ) . as more is revealed regarding the complexity and multi - factorial nature of human neurological disease , access to models that recapitulate aspects of the behavioural and/or pathological abnormalities associated with these disorders is essential . with the ability to carry out longitudinal studies to identify early biomarkers and by focussing on specific endophenotypes , the mouse will continue to be a vital tool to identify disease pathways in neurodegenerative and psychiatric disease research . enu mutagenesis has already generated a significant number of new clinically relevant mouse models , and technological improvements in phenotyping and sequencing strategies will only help improve the efficiency of their identification . access to point mutants will also complement gene knockout studies to model the heterogeneous range of human disease phenotypes . for example , the well - studied mouse model of the lysosomal storage disorder niemann - pick type c ( npc ) does not express any npc protein and serves as a model for the severe infantile forms of this disease ( npc1 ) ( 125 ) ; however , a recently described enu mutant ( npc1 ) provides a more practical model of the more common yet later - onset npc pathology that is caused by missense mutations ( 126 ) . therefore , in combination with transgenics and conditional knockouts , enu mutagenesis will continue to play an important role in the identification and characterization of mutations that underlie human neurological disease .

the canadian population is aging , a trend that is expected to continue for the next several decades due to a reduced fertility rate , an increase in life expectancy , and the aging of the baby boom cohort . in 2011 , an estimated 5 million canadians were 65 years of age or older , a number that is expected to double in the next 25 years to reach approximately 10.4 million older adults by 2036 . aging is associated with an increased risk of chronic illness including cardiovascular disease , cancer , arthritis , cataracts , osteoporosis , type 2 diabetes , hypertension , and dementia . in addition , while it is well - documented that aging is associated with senescent cognitive changes , some older adults will experience more significant cognitive impairment related to the aforementioned conditions . dementia is an umbrella term for a syndrome that includes cognitive impairment in several domains ( e.g. , memory , attention , language , executive function , and visuospatial abilities ) and functional impairment as measured through the assessment of activities of daily living ( adl ; i.e. , eating , bathing , dressing , toileting , transferring , and/or continence ) and/or the instrumental activities of daily living ( iadl ; i.e. , housework , shopping , managing finances , taking medications as prescribed , and/or driving a vehicle ) . due to this functional impairment in individuals with mild dementia estimates of the prevalence of dementia demonstrate a wide range , wherein in individuals over the age of 65 years there is a prevalence of 610% whereas in individuals over the age of 85 years there is a prevalence of 3050% . in addition , studies have demonstrated that dementia is more common in industrialized countries for various reasons possibly including longer life spans and exposure to more environmental pollutants . there are various types of dementia , the most common types being alzheimer 's disease ( ad ) , dementia with lewy bodies ( dlb ) , and vascular dementia ( vad ) . several medical conditions can also lead to the development of dementia , including huntington 's disease , multiple sclerosis , hiv , parkinson 's disease , pick 's disease , and progressive supranuclear palsy . however , when examining the dementia population , ad and dlb are two of the most common types of dementia , accounting for over 60% of all dementia diagnoses . the prevalence estimates change radically depending on the sample ; for example , about 5% of people between the ages of 65 and 74 have ad , whereas nearly half the people over the age of 85 have ad . the average duration is approximately 10 years but includes a range of anywhere from 3 to 20 years . ad is most commonly characterized by deficits in memory , attention , executive functioning , and language . even in mild ad , marked deficits in episodic memory are present ; significant impairment across the attentional domain along with moderate deficits in language and executive functioning are also prevalent . dlb is a neurodegenerative disease that may develop in old age , producing a combination of dementia , parkinsonism , and mental disturbances in the form of hallucinations . the average age of onset of dlb is 67 years and the average duration of the illness is nine years . according to autopsy reports , dlb accounts for 1520% of all dementias . in terms of cognitive deficits , individuals with dlb memory deficits are not marked in the early stages of the disease but as the disease progresses it also becomes impaired . early presenting symptoms of dlb there is an increased interest in examining the driving abilities of older adults diagnosed with dementia , especially those in early stages of the disease when they are most likely to be still driving actively ( e.g. , ) . certainly there are many benefits associated with driving an automobile , including better control of transportation timing , widespread accessibility of locations , access to employment and essential needs , increased social participation , and a sense of autonomy and independence . in fact , carp draws an important connection between mobility and quality of life , stating that well - being depends on success in meeting life - maintenance and higher - order needs . satisfaction of any need depends on congruence between the need and the resources for meeting it . mobility is a key factor in determining congruence , because community services and facilities are irrelevant if they are inaccessible . thus , even though mobility is often measured in the number of trips an individual completes , the concept may be more related to the ability to access services and social interaction . due to the link between quality of life and mobility through the use of a personal automobile there is interest in maintaining mobility in the mild dementia population so long as an individual can continue to drive safely . driving is considered to be a complex and dynamic task involving primarily cognitive ( e.g. , attention ) , perceptual ( e.g. , visual perception ) , and psychomotor processes ( e.g. , reaction time ) . research has demonstrated that deficits in cognitive domains such as attention , global functioning , and visuospatial abilities are linked to impaired driving in driver 's with mild dementia [ 14 , 15 ] . however , a diagnosis of mild dementia does not necessarily signify the inability to drive safely or the necessity to revoke driving privileges , since individuals with a mild dementia diagnosis continue to drive for an average of 4 years following their diagnosis ( e.g. , ) . eby et al . , for example , used in - vehicle technology to explore the on - road driving behaviours of individuals with mild dementia and compared this behaviour to individuals without cognitive impairment . while the mild dementia group was found to significantly restrict their driving , they were found to drive as safely as the control group . our previous work using a driving simulator showed that drivers with mild dementia had significantly more errors and crashes during a standardized simulator assessment course [ 14 , 15 ] . in addition , we found measures of global cognition , attention , and visuospatial processing were significantly related to simulator performance among individuals with mild dementia ; however , the associations depended on dementia type . numerous other studies have examined the relationship between neuropsychological test performance and indicators of driving ability among individuals with dementia ; however , the results are mixed . this may be attributed to the fact that driving is a complex task and that mild dementia patients are a rather heterogeneous population . currently , there is no evidence to support using an individual cognitive or neuropsychological test to determine driving fitness . although examining the specific cognitive impairments associated with impaired driving is an important task , what has yet to be examined is the specific cognitive profile of licensed drivers with a mild dementia diagnosis . given that ad and dlb are the most common types of dementia , the purpose of this paper is to describe the cognitive profile of licensed drivers with mild ad and mild dlb . we hypothesized that , in comparison to healthy controls , mild ad drivers will exhibit impairments in attention , while mild dlb will demonstrate deficits in attention and visuospatial skills . the cognitive profiles of licensed drivers with mild ad and mild dlb will be compared to each other and to neurologically healthy older adult controls . there were three participant groups ( n = 56 ) including a group of healthy older adult controls and two groups of individuals diagnosed with early stage dementia ( dlb and ad ) . all participants were over the age of 65 years , were english speaking , and held a valid driver 's license . the mean age of the control group was 77.00 years ( sd = 5.86 ) with a range of 68 to 86 years , the mean years of education were 13.14 ( sd = 3.18 ) , and the group was comprised of 52.4% women and 47.6% men . the mean age of the early ad group was 78.50 years ( sd = 7.22 ) with a range of 66 to 90 years , the mean years of education were 13.05 ( sd = 3.94 ) , and the group was comprised of 45% women and 55% men . finally , the early dlb group had a mean age of 76.40 ( sd = 6.59 ) with a range of 68 to 88 years , the mean years of education were 14.20 ( sd = 4.55 ) , and the group was comprised of 40% women and 60% men . participants were matched for age and years of education . a convenience sample of healthy older adult controls ( n = 21 ) was obtained through advertisements in a community newspaper . these participants completed a 20-minute screening call in order to determine if they qualified to participate in this study . the exclusion criteria included any serious visual or hearing impairments left uncorrected , serious health problems , any medications that could alter cognitive abilities , any history of substance abuse , and any history of learning disabilities . for control participants , abnormal mini - mental state exam ( mmse ) scores ( < 25 ) was also grounds for exclusion ; however , in practice no participants were excluded for this reason . participants diagnosed with mild dementia were a convenience sample recruited from a tertiary care facility in ottawa . participants who had a diagnosis of probable early dlb or ad at the memory clinic were contacted in order to determine their willingness to participate in the study . participants were assessed for severity , using the global deterioration rating scale and only participants in the mild stages of dementia were included in this study ( i.e. , stages 3 and 4 ) . the same exclusion criteria were used with the exception of medications , since the majority of participants with mild dementia were taking psychoactive medications , such as acetylcholinesterase inhibitors . participants in the mild dementia were grouped in one of the two dementia groups depending on the diagnosis ( i.e. , ad or dlb ) . in this study , all participants with dementia were diagnosed by the supervising neurologist at the memory clinic . all diagnoses of dementia were accomplished using a multimodal approach to diagnosis of dementia which greatly reduces diagnostic error . additionally , diagnosis of specific dementia group ( ad and dlb ) was accomplished using the current gold standards in diagnosis of dementia . dlb was diagnosed using the diagnostic criteria outlined by the first symposium on dlb , which has good predictive validity . ad was diagnosed using the diagnostic criteria outlined by the national institute of neurological and communicative diseases and stroke / alzheimer 's disease and related disorders association , which has excellent predictive validity ( nincds - adrda ) . using this method of diagnosis ensured that diagnostic groups had a good level of reliability . the mmse is a brief cognitive screening tool that is based on a 30-point scale where 30 indicates the best performance . typically , scores less than 24 indicate a cognitive abnormality or probable dementia . a score of 27 or greater is usually used to identify normal healthy adults . in the aging population , scores above 25 are used to identify normal healthy older adults , as such , only healthy controls with scores above and including 25 participated in this study . the mmse has often been recommended as a screening tool for cognitive impairments in community dwelling older adults , as it has good sensitivity ( 80% ) and high specificity ( 98% ) . the mmse correlates ( r = .79 ) with the cognitive and self - contained part of the cambridge examination for mental disorders of the elderly ( camcog ) , the gold standard screening tool . the drs-2 is a measure of global neuropsychological functioning for older adults with suspected dementia ; it assesses attention , memory , visuospatial construction , conceptualization , and initiation / perseveration [ 2325 ] . typically , higher functioning older adults can complete the battery in about 10 minutes , whereas participants with cognitive impairment may take approximately 45 minutes . the drs-2 consists of 36 tasks and 32 stimuli , which yields five subscale scores and an assessment of the participant 's overall level of cognitive functioning . a test - retest reliability correlation coefficient was .97 with subscale correlation coefficients ranging from .61 to .94 . the drs was administered twice with a 1-week interval between administrations to a group of 30 participants diagnosed with dementia of the alzheimer 's type . a split - half reliability coefficient was .90 , utilizing a sample of 25 participants aged 65 to 94 years who received diagnoses of either organic brain syndrome or senile dementia . the alpha coefficients were attention ( .95 ) , initiation - perseveration ( .87 ) , conceptualization ( .95 ) , and memory ( .75 ) . the drs-2 was compared with the mini - mental state examination ( mmse ) , which displayed a significant correlation ( r = .82 ) with the drs-2 showing a greater sensitivity to change than the mmse . in addition , correlations with the wechsler adult intelligence scale indicated a correlation of .75 between the wais full scale and the drs-2 total score . the drs-2 and its alternate version were administered to all participants , with one version being administered in the first session and the second version in the second session . this was done in order to measure cognitive fluctuations between testing sessions , which are well - documented in individuals with dlb . the tea is a measure of attention that specifically assesses various types of attention in an ecologically valid manner as the tests are related to everyday tasks . there are eight subtests : ( 1 ) map search task , ( 2 ) elevator counting task , ( 3 ) elevator counting with distraction , ( 4 ) visual elevator task , ( 5 ) auditory elevator with reversal , ( 6 ) telephone search , ( 7 ) telephone search with dual task , and ( 8) lottery . the ufov is a measure of attention that is composed of three subtests : processing speed , divided attention , and selective attention . test performance on the ufov has been related to performance on functional activities such as driving ( e.g. , ) . ufov scores among individuals with dementia have been shown to be more predictive of driving behaviour than dementia severity . the vosp is a measure of visuoperceptual and spatial abilities that assesses object and space perception . the vosp contains 8 subtests : shape detection , incomplete letters , silhouettes , object decision , dot counting , progressive silhouettes , position discrimination , number allocation , and cube analysis . in addition , a vosp object perception composite score was calculated by adding the first four subtests and a vosp space perception composite score was calculated by adding the last four subtests ; these calculations were extracted from the user manual . the bnt is a test of word finding ability that consists of 60 large ink drawings that are presented in order of increasing difficulty . participants were asked to identify the picture correctly . in dementia research , it is common to provide cues to individuals who can not name the picture . at first a semantic cue is given ( e.g. , for a pelican , a semantic cue would be it 's a bird ) , if the individual still does not respond a phonetic cue is provided ( e.g. , for a pelican , pe ) . normative data indicates that any score below 45 is abnormal . in this study , the total number of correctly identified drawings , whether the participant was cued or not , was used as the participant 's score on this test ( maximum score = 60 ) . potential control participants were contacted by telephone and were asked to complete a brief questionnaire to collect their demographic information . patients who indicated interest in participating were contacted by telephone in order to verify that they were willing to participate and that they met the exclusion criteria . when a participant agreed to participate they were asked if they had time to answer a short questionnaire to collect their demographic information . in the event that they were unable to answer the demographic information on their own , a date was scheduled to have a phone interview with the participant and their primary caregiver . the recruitment of each participant was followed by a brief conversation with the supervising neurologists about the potential participant 's ability to provide consent and to review the diagnosis and dementia stage . all participants who met the inclusion criteria at the end of the demographic information questionnaire were asked for their availability . at this point all participants began the first testing session by completing the consent form . during the first testing session , participants with ad or dlb were asked questions pertaining to the global deterioration rating scale ( gds ) in order to verify if they were in fact in the early stages of dementia ( stages 3 and 4 ) ; participants whose disease was at other stages were excluded . participants that met all the criteria participated in a first session of testing immediately following the screening . this session lasted approximately two and a half hours and participants were offered as many breaks as needed during the testing . all participants underwent a neuropsychological and computerized assessment including a test of general cognitive functioning ( drs-2 ) , visuospatial / perceptual abilities ( vosp ) , word finding ( bnt ) , attention ( tea ) , and processing speed ( ufov ) . participants also underwent a simulated drive of approximately 20 minutes ; the results of the simulated driving component are detailed elsewhere and a brief summary is included as part of the introduction of this article [ 14 , 15 ] . all neuropsychological and computerized testing was completed according to the protocol specified by each measure . the neuropsychological and computerized testing was administered in the presence of the participant and the investigator only . once the first session of testing was completed ( i.e. , consent signed , gds completed for participants with ad and dlb , gds depression , mmse , drs-2 , the bnt , and the vosp ) another testing session was booked . during this second session , the drs-2 was administered a second time , the tea was completed , and the ufov and the simulated driving task were administered . the drs-2 and its alternate form were used and the order from sessions 1 to 2 was counterbalanced between participants . in order to compare the cognitive performance between the three groups , a series of between subjects anovas were executed where group had three levels ( i.e. , ad , dlb , and control ) and each of the neuropsychological measures was treated as dependent variables . statistically significant omnibus anovas were subsequently examined through tukey 's lsd test , a post hoc test that corrects for multiple comparisons . the analysis involving the mmse and the drs total indicated a statistically significant effect of group f(2,53 ) = 19.99 and p < .001 . post hoc analysis showed that , for both the mmse and drs measures , ad and dlb participants scored significantly poorer than healthy controls ( see table 2 ) . measures of attention ( i.e. , ufov and tea ) showed a statistically significant effect of group on all measures . when comparing dementia groups , the results showed dlb participants performed worse than ad participants on the tea subtest 1 ( i.e. , map search 1 minute and 2 minutes ) as well as the tea subtest 2 ( i.e. , auditory elevator counting ) . results of the post hoc analysis showed that ufov - processing speed was significantly different between all three groups with dlb participants exhibiting the slowest scores and healthy controls showing the highest scores ( see table 3 ) . ufov - divided attention and ufov - sustained attention showed a similar pattern of results where both ad and dlb participants scored significantly poorer than healthy controls . there were not statistically significant differences between ad and dlb participants in terms of divided attention and sustained attention . a statistically significant effect of group was noted for the majority of vosp subtests as well as the vosp object , space , and total composite scores . through examining the post hoc contrasts , it was observed that ad and dlb participants performed poorer than controls based on the vosp object composite scores ( see table 4 ) . however , only the dlb group performed poorer than controls based on the vosp space and total composite scores . results from the analysis including the bnt as the dependent variable showed a statistically significant omnibus anova , f(2,53 ) = 7.16 and p = .002 . multiple comparisons found that both dementia groups performed worse than healthy controls ( see table 5 ) . cognitive fluctuations were assessed based on changes in participants ' drs scores over a one - week period . a statistically significant effect of group was noted when including the change in the drs total score , f(2,53 ) = 3.24 and p = .047 . through inspection of the multiple comparison results , the data showed that only ad participants exhibited statistically significant cognitive fluctuations in comparison to healthy controls ( p = .014 ; see table 6 ) . the purpose of this manuscript was to present the cognitive profile of licensed drivers with mild ad and mild dlb and contrast their performance with group of healthy controls . we administered a number of relevant neuropsychological measures within the domains of general cognition , attention , visuospatial / perception , language , andcognitive fluctuations . our results indicated differences between healthy controls and demented participants on almost all neuropsychological measures . these striking differences are surprising given that all demented participants were in the mild stages of the disease ( i.e. , stages 3 and 4 ) . typically , this population is able to function autonomously and maintain their activities of daily living ( adls ) . in light of these sweeping differences in cognitive function and considering that all individuals in this sample were licensed drivers , it is important for clinicians to consider that statistically significant differences between demented and healthy participants may not translate into functional impairments . the results also showed that dlb drivers exhibited poorer attentional and visuospatial abilities in comparison , not only to controls , but also with reference to ad participants . indeed , the clinical presentation for dlb includes visuospatial and attentional impairments whereas a hallmark for ad is memory impairment . thus , these differences in neuropsychological test performance between dementia groups are consistent with the clinical and research literature . the analysis presented here contributes to the body of knowledge used by clinicians and researchers who work with dementia populations . in particular , the norms we present were based on data collected from an educated sample of licensed drivers in an urban canadian setting . when comparing these norms to individual patient performance an important inclusion criterion for this particular study was the possession of a valid driver 's license and all participants reported being active drivers at the time of data collection . a limitation to this study was that we did not collect data regarding on - road driving behaviour . such data might include self- or police - reported collisions , self - reported driving behaviour , or an on - road evaluation . the collection of these additional measures would allow for the determination of whether the differences in neuropsychological performance noted here correspond to indices of driving safety . future research should examine the value of the measures we administered in predicting driving safety among demented drivers using a large sample of participants . driving is multifactorial and complex behaviour that supports mobility and has been related to quality of life . many healthcare professionals are tasked with making recommendations regarding fitness to drive and it is thus important for them to weigh both mobility needs and safety concerns when making this recommendation . clinicians should triangulate multiple sources of information including subjective complaints , family reports , neuropsychological test performance , neurological assessment , and indicators of driving safety ( i.e. , on - road or simulator assessment ) . the norms presented in this paper may be useful to clinicians who are looking for comparative data when assessing patients . research should continue to examine the predictive value of individual sources of information to support the mobility and safety needs of older adults with cognitive impairment . estimates suggest that ad and dlb are the most common forms of dementia among older adults . driving is primary means of mobility among older adults and has been shown to contribute to quality of life . researchers and clinicians suggest that individuals in the mild stages of cognitive impairment can often drive safely ( e.g. , eby et al . ) . the norms presented here suggest statistically significant differences between demented and healthy participants on almost all neuropsychological tests that were administered . however , in the absence of indices of driving behaviour , it is difficult to discern whether these significant differences result in meaningful impacts in terms of activities of daily living , including driving ability .

purpose . alzheimer 's disease ( ad ) and dementia with lewy bodies ( dlb ) constitute two of the most common forms of dementia in north america . driving is a primary means of mobility among older adults and the risk of dementia increases with advanced age . the purpose of this paper is to describe the cognitive profile of licensed drivers with mild ad and mild dlb . method . licensed drivers with mild ad , mild dlb , and healthy controls completed neuropsychological tests measuring general cognition , attention , visuospatial / perception , language , and cognitive fluctuations . results . the results showed differences between healthy controls and demented participants on almost all neuropsychological measures . participants with early dlb were found to perform significantly worse on some measures of attention and visuospatial functioning in comparison with early ad . discussion . future research should examine the relationship between neuropsychological measures and driving outcomes among individuals with mild ad and mild dlb .

1. Introduction 2. Method 3. Results 4. Discussion 5. Conclusion

the canadian population is aging , a trend that is expected to continue for the next several decades due to a reduced fertility rate , an increase in life expectancy , and the aging of the baby boom cohort . in 2011 , an estimated 5 million canadians were 65 years of age or older , a number that is expected to double in the next 25 years to reach approximately 10.4 million older adults by 2036 . aging is associated with an increased risk of chronic illness including cardiovascular disease , cancer , arthritis , cataracts , osteoporosis , type 2 diabetes , hypertension , and dementia . in addition , while it is well - documented that aging is associated with senescent cognitive changes , some older adults will experience more significant cognitive impairment related to the aforementioned conditions . dementia is an umbrella term for a syndrome that includes cognitive impairment in several domains ( e.g. , memory , attention , language , executive function , and visuospatial abilities ) and functional impairment as measured through the assessment of activities of daily living ( adl ; i.e. , housework , shopping , managing finances , taking medications as prescribed , and/or driving a vehicle ) . due to this functional impairment in individuals with mild dementia estimates of the prevalence of dementia demonstrate a wide range , wherein in individuals over the age of 65 years there is a prevalence of 610% whereas in individuals over the age of 85 years there is a prevalence of 3050% . in addition , studies have demonstrated that dementia is more common in industrialized countries for various reasons possibly including longer life spans and exposure to more environmental pollutants . there are various types of dementia , the most common types being alzheimer 's disease ( ad ) , dementia with lewy bodies ( dlb ) , and vascular dementia ( vad ) . several medical conditions can also lead to the development of dementia , including huntington 's disease , multiple sclerosis , hiv , parkinson 's disease , pick 's disease , and progressive supranuclear palsy . however , when examining the dementia population , ad and dlb are two of the most common types of dementia , accounting for over 60% of all dementia diagnoses . the prevalence estimates change radically depending on the sample ; for example , about 5% of people between the ages of 65 and 74 have ad , whereas nearly half the people over the age of 85 have ad . the average duration is approximately 10 years but includes a range of anywhere from 3 to 20 years . ad is most commonly characterized by deficits in memory , attention , executive functioning , and language . even in mild ad , marked deficits in episodic memory are present ; significant impairment across the attentional domain along with moderate deficits in language and executive functioning are also prevalent . dlb is a neurodegenerative disease that may develop in old age , producing a combination of dementia , parkinsonism , and mental disturbances in the form of hallucinations . the average age of onset of dlb is 67 years and the average duration of the illness is nine years . in terms of cognitive deficits , individuals with dlb memory deficits are not marked in the early stages of the disease but as the disease progresses it also becomes impaired . early presenting symptoms of dlb there is an increased interest in examining the driving abilities of older adults diagnosed with dementia , especially those in early stages of the disease when they are most likely to be still driving actively ( e.g. , ) . certainly there are many benefits associated with driving an automobile , including better control of transportation timing , widespread accessibility of locations , access to employment and essential needs , increased social participation , and a sense of autonomy and independence . satisfaction of any need depends on congruence between the need and the resources for meeting it . mobility is a key factor in determining congruence , because community services and facilities are irrelevant if they are inaccessible . due to the link between quality of life and mobility through the use of a personal automobile there is interest in maintaining mobility in the mild dementia population so long as an individual can continue to drive safely . driving is considered to be a complex and dynamic task involving primarily cognitive ( e.g. , attention ) , perceptual ( e.g. , visual perception ) , and psychomotor processes ( e.g. research has demonstrated that deficits in cognitive domains such as attention , global functioning , and visuospatial abilities are linked to impaired driving in driver 's with mild dementia [ 14 , 15 ] . however , a diagnosis of mild dementia does not necessarily signify the inability to drive safely or the necessity to revoke driving privileges , since individuals with a mild dementia diagnosis continue to drive for an average of 4 years following their diagnosis ( e.g. , for example , used in - vehicle technology to explore the on - road driving behaviours of individuals with mild dementia and compared this behaviour to individuals without cognitive impairment . while the mild dementia group was found to significantly restrict their driving , they were found to drive as safely as the control group . our previous work using a driving simulator showed that drivers with mild dementia had significantly more errors and crashes during a standardized simulator assessment course [ 14 , 15 ] . in addition , we found measures of global cognition , attention , and visuospatial processing were significantly related to simulator performance among individuals with mild dementia ; however , the associations depended on dementia type . numerous other studies have examined the relationship between neuropsychological test performance and indicators of driving ability among individuals with dementia ; however , the results are mixed . this may be attributed to the fact that driving is a complex task and that mild dementia patients are a rather heterogeneous population . although examining the specific cognitive impairments associated with impaired driving is an important task , what has yet to be examined is the specific cognitive profile of licensed drivers with a mild dementia diagnosis . given that ad and dlb are the most common types of dementia , the purpose of this paper is to describe the cognitive profile of licensed drivers with mild ad and mild dlb . we hypothesized that , in comparison to healthy controls , mild ad drivers will exhibit impairments in attention , while mild dlb will demonstrate deficits in attention and visuospatial skills . the cognitive profiles of licensed drivers with mild ad and mild dlb will be compared to each other and to neurologically healthy older adult controls . there were three participant groups ( n = 56 ) including a group of healthy older adult controls and two groups of individuals diagnosed with early stage dementia ( dlb and ad ) . all participants were over the age of 65 years , were english speaking , and held a valid driver 's license . the mean age of the control group was 77.00 years ( sd = 5.86 ) with a range of 68 to 86 years , the mean years of education were 13.14 ( sd = 3.18 ) , and the group was comprised of 52.4% women and 47.6% men . the mean age of the early ad group was 78.50 years ( sd = 7.22 ) with a range of 66 to 90 years , the mean years of education were 13.05 ( sd = 3.94 ) , and the group was comprised of 45% women and 55% men . finally , the early dlb group had a mean age of 76.40 ( sd = 6.59 ) with a range of 68 to 88 years , the mean years of education were 14.20 ( sd = 4.55 ) , and the group was comprised of 40% women and 60% men . a convenience sample of healthy older adult controls ( n = 21 ) was obtained through advertisements in a community newspaper . the exclusion criteria included any serious visual or hearing impairments left uncorrected , serious health problems , any medications that could alter cognitive abilities , any history of substance abuse , and any history of learning disabilities . participants diagnosed with mild dementia were a convenience sample recruited from a tertiary care facility in ottawa . participants who had a diagnosis of probable early dlb or ad at the memory clinic were contacted in order to determine their willingness to participate in the study . participants were assessed for severity , using the global deterioration rating scale and only participants in the mild stages of dementia were included in this study ( i.e. the same exclusion criteria were used with the exception of medications , since the majority of participants with mild dementia were taking psychoactive medications , such as acetylcholinesterase inhibitors . participants in the mild dementia were grouped in one of the two dementia groups depending on the diagnosis ( i.e. , ad or dlb ) . in this study , all participants with dementia were diagnosed by the supervising neurologist at the memory clinic . all diagnoses of dementia were accomplished using a multimodal approach to diagnosis of dementia which greatly reduces diagnostic error . additionally , diagnosis of specific dementia group ( ad and dlb ) was accomplished using the current gold standards in diagnosis of dementia . dlb was diagnosed using the diagnostic criteria outlined by the first symposium on dlb , which has good predictive validity . ad was diagnosed using the diagnostic criteria outlined by the national institute of neurological and communicative diseases and stroke / alzheimer 's disease and related disorders association , which has excellent predictive validity ( nincds - adrda ) . the mmse is a brief cognitive screening tool that is based on a 30-point scale where 30 indicates the best performance . a score of 27 or greater is usually used to identify normal healthy adults . in the aging population , scores above 25 are used to identify normal healthy older adults , as such , only healthy controls with scores above and including 25 participated in this study . the mmse has often been recommended as a screening tool for cognitive impairments in community dwelling older adults , as it has good sensitivity ( 80% ) and high specificity ( 98% ) . the mmse correlates ( r = .79 ) with the cognitive and self - contained part of the cambridge examination for mental disorders of the elderly ( camcog ) , the gold standard screening tool . the drs-2 is a measure of global neuropsychological functioning for older adults with suspected dementia ; it assesses attention , memory , visuospatial construction , conceptualization , and initiation / perseveration [ 2325 ] . typically , higher functioning older adults can complete the battery in about 10 minutes , whereas participants with cognitive impairment may take approximately 45 minutes . the drs-2 consists of 36 tasks and 32 stimuli , which yields five subscale scores and an assessment of the participant 's overall level of cognitive functioning . a test - retest reliability correlation coefficient was .97 with subscale correlation coefficients ranging from .61 to .94 . the drs was administered twice with a 1-week interval between administrations to a group of 30 participants diagnosed with dementia of the alzheimer 's type . a split - half reliability coefficient was .90 , utilizing a sample of 25 participants aged 65 to 94 years who received diagnoses of either organic brain syndrome or senile dementia . the alpha coefficients were attention ( .95 ) , initiation - perseveration ( .87 ) , conceptualization ( .95 ) , and memory ( .75 ) . in addition , correlations with the wechsler adult intelligence scale indicated a correlation of .75 between the wais full scale and the drs-2 total score . the drs-2 and its alternate version were administered to all participants , with one version being administered in the first session and the second version in the second session . this was done in order to measure cognitive fluctuations between testing sessions , which are well - documented in individuals with dlb . the tea is a measure of attention that specifically assesses various types of attention in an ecologically valid manner as the tests are related to everyday tasks . there are eight subtests : ( 1 ) map search task , ( 2 ) elevator counting task , ( 3 ) elevator counting with distraction , ( 4 ) visual elevator task , ( 5 ) auditory elevator with reversal , ( 6 ) telephone search , ( 7 ) telephone search with dual task , and ( 8) lottery . the ufov is a measure of attention that is composed of three subtests : processing speed , divided attention , and selective attention . ufov scores among individuals with dementia have been shown to be more predictive of driving behaviour than dementia severity . the vosp is a measure of visuoperceptual and spatial abilities that assesses object and space perception . the vosp contains 8 subtests : shape detection , incomplete letters , silhouettes , object decision , dot counting , progressive silhouettes , position discrimination , number allocation , and cube analysis . in addition , a vosp object perception composite score was calculated by adding the first four subtests and a vosp space perception composite score was calculated by adding the last four subtests ; these calculations were extracted from the user manual . the bnt is a test of word finding ability that consists of 60 large ink drawings that are presented in order of increasing difficulty . at first a semantic cue is given ( e.g. , for a pelican , a semantic cue would be it 's a bird ) , if the individual still does not respond a phonetic cue is provided ( e.g. in this study , the total number of correctly identified drawings , whether the participant was cued or not , was used as the participant 's score on this test ( maximum score = 60 ) . the recruitment of each participant was followed by a brief conversation with the supervising neurologists about the potential participant 's ability to provide consent and to review the diagnosis and dementia stage . all participants who met the inclusion criteria at the end of the demographic information questionnaire were asked for their availability . during the first testing session , participants with ad or dlb were asked questions pertaining to the global deterioration rating scale ( gds ) in order to verify if they were in fact in the early stages of dementia ( stages 3 and 4 ) ; participants whose disease was at other stages were excluded . participants that met all the criteria participated in a first session of testing immediately following the screening . this session lasted approximately two and a half hours and participants were offered as many breaks as needed during the testing . all participants underwent a neuropsychological and computerized assessment including a test of general cognitive functioning ( drs-2 ) , visuospatial / perceptual abilities ( vosp ) , word finding ( bnt ) , attention ( tea ) , and processing speed ( ufov ) . participants also underwent a simulated drive of approximately 20 minutes ; the results of the simulated driving component are detailed elsewhere and a brief summary is included as part of the introduction of this article [ 14 , 15 ] . all neuropsychological and computerized testing was completed according to the protocol specified by each measure . the neuropsychological and computerized testing was administered in the presence of the participant and the investigator only . once the first session of testing was completed ( i.e. , consent signed , gds completed for participants with ad and dlb , gds depression , mmse , drs-2 , the bnt , and the vosp ) another testing session was booked . during this second session , the drs-2 was administered a second time , the tea was completed , and the ufov and the simulated driving task were administered . the drs-2 and its alternate form were used and the order from sessions 1 to 2 was counterbalanced between participants . in order to compare the cognitive performance between the three groups , a series of between subjects anovas were executed where group had three levels ( i.e. , ad , dlb , and control ) and each of the neuropsychological measures was treated as dependent variables . statistically significant omnibus anovas were subsequently examined through tukey 's lsd test , a post hoc test that corrects for multiple comparisons . the analysis involving the mmse and the drs total indicated a statistically significant effect of group f(2,53 ) = 19.99 and p < .001 . post hoc analysis showed that , for both the mmse and drs measures , ad and dlb participants scored significantly poorer than healthy controls ( see table 2 ) . measures of attention ( i.e. when comparing dementia groups , the results showed dlb participants performed worse than ad participants on the tea subtest 1 ( i.e. , auditory elevator counting ) . results of the post hoc analysis showed that ufov - processing speed was significantly different between all three groups with dlb participants exhibiting the slowest scores and healthy controls showing the highest scores ( see table 3 ) . ufov - divided attention and ufov - sustained attention showed a similar pattern of results where both ad and dlb participants scored significantly poorer than healthy controls . there were not statistically significant differences between ad and dlb participants in terms of divided attention and sustained attention . a statistically significant effect of group was noted for the majority of vosp subtests as well as the vosp object , space , and total composite scores . through examining the post hoc contrasts , it was observed that ad and dlb participants performed poorer than controls based on the vosp object composite scores ( see table 4 ) . multiple comparisons found that both dementia groups performed worse than healthy controls ( see table 5 ) . cognitive fluctuations were assessed based on changes in participants ' drs scores over a one - week period . through inspection of the multiple comparison results , the data showed that only ad participants exhibited statistically significant cognitive fluctuations in comparison to healthy controls ( p = .014 ; see table 6 ) . the purpose of this manuscript was to present the cognitive profile of licensed drivers with mild ad and mild dlb and contrast their performance with group of healthy controls . we administered a number of relevant neuropsychological measures within the domains of general cognition , attention , visuospatial / perception , language , andcognitive fluctuations . our results indicated differences between healthy controls and demented participants on almost all neuropsychological measures . these striking differences are surprising given that all demented participants were in the mild stages of the disease ( i.e. , stages 3 and 4 ) . in light of these sweeping differences in cognitive function and considering that all individuals in this sample were licensed drivers , it is important for clinicians to consider that statistically significant differences between demented and healthy participants may not translate into functional impairments . the results also showed that dlb drivers exhibited poorer attentional and visuospatial abilities in comparison , not only to controls , but also with reference to ad participants . thus , these differences in neuropsychological test performance between dementia groups are consistent with the clinical and research literature . in particular , the norms we present were based on data collected from an educated sample of licensed drivers in an urban canadian setting . when comparing these norms to individual patient performance an important inclusion criterion for this particular study was the possession of a valid driver 's license and all participants reported being active drivers at the time of data collection . a limitation to this study was that we did not collect data regarding on - road driving behaviour . such data might include self- or police - reported collisions , self - reported driving behaviour , or an on - road evaluation . the collection of these additional measures would allow for the determination of whether the differences in neuropsychological performance noted here correspond to indices of driving safety . future research should examine the value of the measures we administered in predicting driving safety among demented drivers using a large sample of participants . driving is multifactorial and complex behaviour that supports mobility and has been related to quality of life . clinicians should triangulate multiple sources of information including subjective complaints , family reports , neuropsychological test performance , neurological assessment , and indicators of driving safety ( i.e. the norms presented in this paper may be useful to clinicians who are looking for comparative data when assessing patients . research should continue to examine the predictive value of individual sources of information to support the mobility and safety needs of older adults with cognitive impairment . estimates suggest that ad and dlb are the most common forms of dementia among older adults . driving is primary means of mobility among older adults and has been shown to contribute to quality of life . . the norms presented here suggest statistically significant differences between demented and healthy participants on almost all neuropsychological tests that were administered .

plasmids and protein purification for all experiments , the cytoplasmic domain of the indicated syt isoform was used . 28 , and cdna encoding the cytoplasmic domains of syt xiii and xv were provided by m. craxton ( laboratory of molecular biology , cambridge , uk ) . the amino acid residues encoding these cytoplasmic domains ( c2a - c2b ) are as follows : i , 96 - 421 ; ii , 139 - 423 ; iii , 290 - 569 ; iv , 152 - 425 ; v , 218 - 491 ; vi , 143 - 426 ; vii , 134 - 403 ; viii , 97 - 395 ; ix , 104 - 386 ; x , 223 - 501 ; xi , 150 - 430 ; xii , 114 - 421 ; xiii , 158 - 427 ; xv , 139 - 421 . each isoform was subcloned into either ptrc - hisa ( invitrogen ) or pet28a ( novagen ) . syts were expressed in escherichia coli and purified as previously described ( 25 ) . briefly , bacterial pellets were resuspended in 25 mm hepes - koh , 400 mm kcl , 20 mm imidazole , and 5 mm -mercaptoethanol , and then lysed by sonication followed by addition of triton x-100 ( final 2% v / v ) . bacteria were pelleted , and extracts were mixed with ni - nitrilotriacetic acid - agarose ( qiagen ) for 2 h at 4 c . beads were washed two times in wash buffer ( 25 mm hepes - koh , 400 mm kcl , 20 mm imidazole , and 5 mm -mercaptoethanol , 1 mm mgcl2 ) plus 10 g / ml dnase and rnase ( roche applied science ) to remove any bound rna / dna . proteins were eluted from beads in the resuspension buffer with 500 mm imidazole and 10% glycerol ( w / v ) and dialyzed overnight against 25 mm hepes ph 7.4 , 200 mm kcl , 10% glycerol ( w / v ) , 1 mm dithiothreitol ( buffer a ) . because of technical difficulties , we were unable to express and purify appropriate quantities of recombinant syt xiv and xvi from e. coli . plasmids to generate recombinant full - length synaptobrevin 2 and the full - length t - snare heterodimer ( syntaxin 1a and snap-25b ) , were provided by j. e. rothman ( columbia university ) , and proteins were expressed and purified as described ( 3 , 24 ) . cdna encoding full - length rat syntaxin 1a and syntaxin 4 were provided by r. h. scheller ( genentech , san francisco , ca ) and j. m. edwardson ( cambridge , uk ) , respectively . cdna encoding full - length snap-25b was provided by m. c. wilson ( university of new mexico ) . cdna encoding snap-23 and snap-29 were provided by r. jahn ( max planck institute , gottingen , germany ) . the following t - snare heterodimer pairs were subcloned into prsf - duet ( novagen ) and expressed as above : syntaxin1a / snap-23 , syntaxin1a / snap-29 , syntaxin4/snap-23 , and syntaxin4/snap-25b . primary references for syb 2 , snap-25b , and syntaxin 1a can be found in ref . reconstitution of v - snare and t - snare vesicles was carried out as previously described ( 3 , 24 ) . v - snares were reconstituted using a lipid mix composed of 30% 1-palmitoyl , 2-oleoyl phosphatidylethanolamine ( pe ) , 52% 1-palmitoyl , 2-oleoyl phosphatidylcholine ( pc ) , 15% 1,2-dioleoyl phosphatidylserine ( ps ) , 1.5% n-(7-nitro-2 - 1,3-benzoxadiazol-4-yl)-1,2-dipalmitoyl phosphatidylethanolamine ( nbd - pe , donor ) , and 1.5% n-(lissamine rhodamine b sulfonyl)-1,2-dipalmitoyl phosphatidylethanolamine ( rhodamine - pe , acceptor ) . t - snares were reconstituted in 30% pe , 55% pc , and 15% ps ( mol mol ) . when ps was omitted , pc was increased to 70% for the t - snare vesicles . v - snare and t - snare vesicles were reconstituted to give 100 copies or 80 copies per vesicle , respectively , as described ( 24 ) . at 80 copies / vesicle , the t - snare concentration in the fusion assay is 3 m . protein - free ( pf ) vesicles were prepared as described previously ( 24 ) . flotation assays flotation assays were carried out as described previously with modifications ( 24 , 25 ) . briefly , pf vesicles with ( 30% pe/15% ps/55% pc ) or without ( 30% pe/70% pc ) ps , or t - snare vesicles ( 30% pe/70% pc ) , were mixed with syt ( 10 m ) . all binding reactions were carried out in a total volume of 100 l of buffer a with either 0.2 mm egta or 1 mm ca . samples were incubated with shaking for 1 h at room temperature and were then mixed with an equal volume of 80% accudenz medium , transferred to a beckman ultracentrifuge tube and layered with 150 l each of 35 and 30% accudenz media in buffer a. finally , samples were layered with 20 l of buffer a lacking glycerol and centrifuged at 280,000 g for 2.5 h at 4 c . all buffers / media contained either 0.2 mm egta or 1 mm ca.40 l of vesicles from the 0%/30% interface were collected from each tube , and one - third of the collected sample was resolved by sds - page and stained with coomassie blue . all gels contain standards ( stds ) that serve as references for the electrophoretic mobility of the proteins used in the flotation assays . 6 , the indicated concentrations of syt iv , viii , and xii were included in reactions along with 10 m syt i. all coomassie - stained gels are representative examples from 3 trials . figure 1.conservation of the putative ca ligands among different isoforms of syt . a , model depicting the structure of syt . the image was generated using the crystal structure of the cytoplasmic domain of syt iii ( 37 ) ; remaining segments were added with a drawing program . b , schematic diagram showing the five ca - coordinating residues in the c2a and c2b domains of syt i. the diagram is modified and reproduced with permission from the authors of ref . 51 . c , conservation among the ca ligands in the c2a and c2b domains of syt i - xiii and xv . numbers correspond to the order of the five aspartate residues that function as ca ligands in the c2a domain of syt i ( d172 , 178 , 230 , 232 , 238 ) and c2b ( d303 , 309 , 363 , 365 , 371 ) ( 6 ) . single letter codes indicate substitutions of conserved aspartate residues ; ( + ) denotes conservation of an aspartate , and ( - ) indicates a gap . based on conservation of the ca ligands , we predicted which syts might ( + ) and might not ( - ) stimulate in vitro snare - mediated fusion in response to ca . conservation of the putative ca ligands among different isoforms of syt . a , model depicting the structure of syt . the image was generated using the crystal structure of the cytoplasmic domain of syt iii ( 37 ) ; remaining segments were added with a drawing program . b , schematic diagram showing the five ca - coordinating residues in the c2a and c2b domains of syt i. the diagram is modified and reproduced with permission from the authors of ref . c , conservation among the ca ligands in the c2a and c2b domains of syt i - xiii and xv . numbers correspond to the order of the five aspartate residues that function as ca ligands in the c2a domain of syt i ( d172 , 178 , 230 , 232 , 238 ) and c2b ( d303 , 309 , 363 , 365 , 371 ) ( 6 ) . single letter codes indicate substitutions of conserved aspartate residues ; ( + ) denotes conservation of an aspartate , and ( - ) indicates a gap . based on conservation of the ca ligands , we predicted which syts might ( + ) and might not ( - ) stimulate in vitro snare - mediated fusion in response to ca . fusion assays each reaction consisted of 45 l of purified t - snare vesicles ( 30% pe/15% ps/55% pc ) and 5 l of purified , v - snare vesicles ( 30% pe/1.5% nbd - pe/1.5% rhodamine - pe/15% ps/52% pc ) plus 0.2 mm egta or 1 mm ca in a total volume of 75 l of buffer a. 10 m syt was added where indicated . for competition experiments in fig . 5 , the indicated concentrations of syt iv , viii , and xii were included in reactions along with 10 m syt i. fusion reactions were carried out in 96-well plates , and nbd fluorescence was monitored at 37 c in a plate reader ( biotek instruments ; excitation 460 nm , emission 538 nm ) . after 2 h , 0.5% n - dodecylmaltoside ( w / v , roche applied science ) was added to maximally dequench the nbd fluorescence . raw fluorescence was normalized to obtain % maximum fluorescence as described ( 24 , 34 ) . ( eq.1)\documentclass[10pt]{article } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{pmc } \usepackage[euler]{upgreek } \pagestyle{empty } \oddsidemargin -1.0 in \begin{document } \begin{equation*}\;\;\%\hspace{1em}{\mathrm{stim\;\;in}}\hspace{1em}{\mathrm{ca}}^{2+}=\frac{({\mathrm{fusion\;\;in}}\hspace{1em}{\mathrm{ca}}^{2+})-({\mathrm{fusion\;\;in}}\hspace{1em}{\mathrm{egta}})}{{\mathrm{fusion\;\;in}}\hspace{1em}{\mathrm{egta}}}{\times}100\;\;\end{equation*}\end{document } percent stimulation over ( -)syt was calculated by equation 2 . ( eq.2)\documentclass[10pt]{article } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{pmc } \usepackage[euler]{upgreek } \pagestyle{empty } \oddsidemargin -1.0 in \begin{document } \begin{equation*}\;\;\%\hspace{1em}{\mathrm{stim\;over}}(-){\mathrm{syt}}=\frac{({\mathrm{fusion\;\;by}}\hspace{1em}{\mathrm{ca}}^{2+}{\cdot}{\mathrm{syt}})-({\mathrm{fusion\;\;by}}(-){\mathrm{syt}})}{{\mathrm{fusion\;\;by}}(-){\mathrm{syt}}}{\times}100\;\;\end{equation*}\end{document } conservation of the ca ligands in the c2a and c2b domains of syt to date , seventeen isoforms of syt have been identified in vertebrates ( 26 ) . all members of this protein family share similar overall domain structures ( shown in fig . 1a ) ; the cytoplasmic domains are comprised of tandem c2 domains ( c2a and c2b ) that are tethered together by a short linker ( fig . c2 domains , first identified in protein kinase c , are now found in a variety of proteins where they often , but not always , mediate binding to ca and phospholipids ( 38 ) . in syt i , both c2 domains mediate ca - regulated interactions with anionic phospholipids and t - snares ( 6 ) . however , the five aspartic acid residues that coordinate ca ions in each c2 domain of syt i ( fig . the strict conservation of these ca ligands in syt i - vii and ix - x suggest that these isoforms bind ca and therefore might be able to stimulate fusion mediated by neuronal snare proteins . in contrast , syt viii , xi - xiii , and xv harbor substitutions that are expected to disrupt ca binding activity in both c2 domains , suggesting that these isoforms would be incapable of coupling ca to fusion . divergent activities of syt isoforms during neuronal snare - mediated membrane fusion in vitro studies revealed that in response to ca , syt i stimulates snare - catalyzed membrane fusion ( 12 , 24 ) , providing direct evidence that syt i can act as a ca sensor for exocytosis in vivo ( 4 - 6 ) . more recently , this observation was extended to two other syt family members , syt vii and ix ( 25 ) . in contrast , syt iv failed to stimulate membrane fusion in reconstituted fusion assays ( 12 ) , consistent with its putative role as a negative regulator of secretion from neuroendocrine cells ( 31 - 33 ) . however , the ability of the other thirteen isoforms of syt to regulate membrane fusion remained unknown . a , illustration depicting the in vitro fusion assay used in this study . fusion of v - snare ( syb ; v ) vesicles , containing a donor ( d ) and acceptor ( a ) fret pair , with unlabeled t - snare vesicles ( syntaxin 1a / snap-25b ; t ) , results in dilution of the fret pair and an increase in donor fluorescence . b , syt isoforms differentially regulate fusion between v- and t - snare vesicles . fusion was monitored for 120 min at 37 c in the absence ( -syt ) or presence ( + syt , 10 m ) of different syt isoforms , normalized to the maximum donor fluorescence signal ( % max fl ) , and plotted as a function of time . normalized fusion data from fourteen isoforms of syt . a , illustration depicting the in vitro fusion assay used in this study . fusion of v - snare ( syb ; v ) vesicles , containing a donor ( d ) and acceptor ( a ) fret pair , with unlabeled t - snare vesicles ( syntaxin 1a / snap-25b ; t ) , results in dilution of the fret pair and an increase in donor fluorescence . b , syt isoforms differentially regulate fusion between v- and t - snare vesicles . fusion was monitored for 120 min at 37 c in the absence ( -syt ) or presence ( + syt , 10 m ) of different syt isoforms , normalized to the maximum donor fluorescence signal ( % max fl ) , and plotted as a function of time . representative traces for the different syt isoforms are shown from 3 independent trials . using the in vitro membrane fusion assay , we screened fourteen syt isoforms for their abilities to regulate fusion between v - snare vesicles ( v ) , that harbor synaptobrevin 2 , and t - snare vesicles ( t ) , that harbor syntaxin 1a / snap-25b heterodimers ( fig . 2b ; only a subset of syt isoforms stimulated snare - catalyzed fusion in the presence of ca : syt i - iii , v - vii , and ix - x . this group of syts corresponded to the isoforms that were predicted to serve as ca sensors ( fig . 1c ) , with the exception of syt iv , which has one asp to ser substitution in the c2a domain but harbors an intact set of putative ca ligands in c2b . 3a , where the average extent of fusion ( % maximum fluorescence intensity ) , from all trials was plotted for each syt isoform . to better visualize the impact of syt isoforms on fusion and to highlight differences that might have been obscured by averaging across all trials ( because of sample - to - sample differences in the overall extent of fusion ) , we compared the extent of fusion obtained in ca to the extent of fusion obtained in egta within each individual trial ( fig . 3b , equation 1 under ; experimental procedures ) , then averaged the data . in addition , we compared the extent of fusion obtained by casyt to fusion mediated by v- and t - snares in the absence of syt ( fig . 3c , equation 2 under ; experimental procedures ) , again within each individual trial , and then calculated the average values . the former plot shows the range of stimulation mediated by syts under our experimental conditions , whereas the latter plot indicates the extent to which each syt isoform was able to stimulate or suppress fusion mediated by snares themselves . a key finding was that syts iv , viii , xi , and xii were unable to respond to ca , but were able to inhibit fusion mediated by snares alone ( fig . stimulation of membrane fusion is related to the ability of syts to engage ps and t - snares in response to ca in response to ca , the tandem c2 domains of syt i engage t - snares and the anionic phospholipid ps , and previous studies indicate that these interactions are critical for the regulation of membrane fusion ( 15 , 16 , 18 , 24 , 25 ) . however , others have concluded that syt i operates independently of its ca - promoted interactions with snares ( 20 , 21 ) , and that syt ix functions without binding snares at all ( 22 , 23 ) . to further explore the mechanism of action of syts , we carried out experiments to relate their activity in the fusion assay with their abilities to engage both ps and t - snares . 24 ) to determine which of the syt isoforms that had been screened in the fusion assay interact with full - length , membrane - embedded , syntaxin 1a / snap-25b heterodimers , and ps . all of the syt isoforms tested exhibited some degree of t - snare and ps binding activity in the absence of ca ( fig . 4 , b , c , and e ) . however , only the stimulatory syts i - iii , v - vii , and ix - x bound t - snares and ps in a ca - promoted manner ( fig . the syts that failed to stimulate fusion , isoforms iv , viii , xi , xii , xiii , and xv , also bound t - snares and ps , but these interactions were not enhanced by ca . the t - snare binding data are in agreement with previously observed results for syts i , vii , and ix ( 25 ) ( but see also ref . 22 ) , and the ps binding data are in agreement with a study focused on syts i - xii ( 28 ) . protein - free pc / pe vesicles were used as a negative control ; no binding of any isoform of syt to these vesicles was detected , indicating sytlipid interactions are specific for ps under our experimental conditions . a , average extent of fusion ( % max fi ) at t = 120 min , in either 0.2 mm egta ( open bars ) or 1 mm ca ( filled bars ) , was plotted for each syt isoform . ( -)syt indicates fusion between v- and t - snare vesicles in the absence of syt . b , average % stimulation in ca ( fusion in ca compared with fusion in egta within each individual trial and then averaged ; equation 1 in ; experimental procedures ) was plotted for v- and t - snare - mediated fusion and for each syt isoform . c , average % stimulation over ( -)syt ( fusion by casyt compared with fusion mediated by snares in the absence of syt within each individual trial and then averaged ; equation 2 in ; experimental procedures ) was plotted for each syt isoform . a , average extent of fusion ( % max fi ) at t = 120 min , in either 0.2 mm egta ( open bars ) or 1 mm ca ( filled bars ) , was plotted for each syt isoform . ( -)syt indicates fusion between v- and t - snare vesicles in the absence of syt . b , average % stimulation in ca ( fusion in ca compared with fusion in egta within each individual trial and then averaged ; equation 1 in ; experimental procedures ) was plotted for v- and t - snare - mediated fusion and for each syt isoform . c , average % stimulation over ( -)syt ( fusion by casyt compared with fusion mediated by snares in the absence of syt within each individual trial and then averaged ; equation 2 in ; experimental procedures ) was plotted for each syt isoform . ( n 3 ) . the extent to which syt associated with t - snare vesicles in egta or ca was quantified using densitometry and normalized to the syntaxin band in the corresponding lane ; the % change in t - snare binding in response to ca was plotted ( fig . 4c ) . the amount of syt that bound to ps - containing vesicles in the presence of ca was also plotted ( fig . 4 , c and e ) exhibited similar patterns ; syts that bind ps in response to ca also bind t - snares in response to ca , and vice versa . these findings agree with the notion that syt acts by engaging membranes and snares at the same time and that these interactions are coupled ( 10 , 16 ) . we then plotted the effect syts had on snare - mediated fusion ( % stimulation over ( -)syt from fig . 3c ) against the % increase in ca - promoted binding to t - snares ( fig . 4d ) and against the extent of ps binding in the presence of ca ( fig . these plots illustrate a strong correlation between the ability of a syt isoform to bind both of these effectors in a ca - dependent manner with their abilities to stimulate snare - catalyzed fusion in response to ca . similarly , there is a strong correlation between syts that interact with t - snares and ps in a ca - independent manner with their abilities to inhibit snare - catalyzed fusion . in no case did a syt isoform stimulate fusion without binding to ps and t - snares in a ca - promoted manner , supporting a model in which syts stimulate fusion through ca - dependent interactions with both of these effectors . syt isoforms can modulate the activity of each other in the course of screening syts i - xiii and xv , we identified a class of syts ( iv , viii , xi , and xii ) that failed to respond to ca in our biochemical assays and that inhibited snare - catalyzed fusion ( fig . these findings prompted the idea that these inhibitory syts might antagonize the action of syt isoforms that stimulate fusion in response to ca ( e.g. see ref . , we asked whether a subset of inhibitory syts could affect the ability of syt i to stimulate snare - catalyzed membrane fusion . we fixed the amount of syt i in the fusion assay and titrated increasing amounts of syts iv ( fig . 5 , c and d ) , and xii ( fig . 5 , syt iv and viii inhibited syt - stimulated fusion in a dose - dependent manner , whereas syt xii had no appreciable effect over the range of concentrations tested . we note that higher concentrations of syt iv , viii , and xii could not be tested because of low protein yields and volume constraints of the fusion assay . syt xi , another inhibitory syt , behaved in a similar manner to syt iv and viii , i.e. it inhibited syt i - stimulated fusion in a dose - dependent manner.5 because syt i and iv have been co - localized to large dense core vesicles ( ldcvs ) in pc12 cells ( 29 , 33 ) , these findings support the idea that different syt isoforms on the same secretory organelle might serve to fine - tune the efficiency of exocytosis . next , we found that syts iv and viii significantly reduced ca - promoted binding of syt i to t - snares ( fig . in contrast , syt xii , the inhibitory syt that failed to suppress syt i - stimulated fusion , significantly reduced syt it - snare interactions only when added to the assay in a 3-fold molar excess of syt i ( fig . thus , it appears that inhibitory isoforms of syt can impact fusion in two ways : first , they directly inhibit snare - mediated fusion , and second , they displace syt i from the fusion complex . these properties are clearly apparent for syt iv and viii ; syt xii differs to some extent as this isoform exhibits less inhibitory activity in terms of both shutting down snare function and displacing syt i. moreover , these experiments provide further support for the idea that syts act , at least in part , via direct interactions with t - snares to regulate fusion . specificity of functional pairing between syts and t - snares during membrane fusion at present , approximately thirty - six snare proteins have been identified in humans ( 2 ) . different snares are targeted to distinct subcellular compartments where they catalyze many , and perhaps all , intracellular membrane fusion events ( 40 ) . it was proposed that pairing between specific v- and t - snares encodes the specificity of intracellular membrane fusion reactions ( 41 ) . while snare pairing clearly plays a central role in the specificity of fusion , it is now established that other mechanisms also come into play ( 42 ) . a , diagram of the flotation assay used to monitor binding of the different syt isoforms to t - snare vesicles ( t ) , or protein free vesicles , with ( pf(+ps ) ) or without ps ( pf ) . syt will float to the top of the gradient only if it interacts with vesicles . after centrifugation , samples are collected from the top of the gradient and analyzed by sds - page ; proteins are visualized by staining with coomassie blue . b , flotation assays were carried out as depicted in panel a. binding was monitored in either 0.2 mm egta ( - ) or 1 mm ca(+ ) . for the syt iv gel only , a line was added between the t - snare and syt iv standards to indicate lanes that were originally spaced further apart on the same gel and were combined for this figure . c , amount of syt that co - floated with t - snare vesicles in 0.2 mm egta or 1 mm ca from panel b was quantified using densitometry and normalized to the syntaxin band in each lane . the average % increase in t - snare binding in the presence of ca versus egta was calculated using normalized data and plotted for each isoform . c - f , red is used to denote the inhibitory syts indicated in fig . d , for each syt isoform , % stimulation over syt ( from fig . 3c ) was plotted against the average % increase in t - snare binding for each syt isoform from panel c. e , the amount of syt bound to ps - harboring vesicles in 1 mmca from panel b was quantified using densitometry . the average total optical density is plotted for each isoform ( because there is no other band in each lane against which the syt signal can be normalized as in panel c ) . 3c ) was plotted against the average amount of syt bound to ps - harboring vesicles in 1 mm ca from panel e. syts differ in their ability to bind ps and t - snares . a , diagram of the flotation assay used to monitor binding of the different syt isoforms to t - snare vesicles ( t ) , or protein free vesicles , with ( pf(+ps ) ) or without ps ( pf ) . syt will float to the top of the gradient only if it interacts with vesicles . after centrifugation , samples are collected from the top of the gradient and analyzed by sds - page ; proteins are visualized by staining with coomassie blue . b , flotation assays were carried out as depicted in panel a. binding was monitored in either 0.2 mm egta ( - ) or 1 mm ca(+ ) . for the syt iv gel only , a line was added between the t - snare and syt iv standards to indicate lanes that were originally spaced further apart on the same gel and were combined for this figure . c , amount of syt that co - floated with t - snare vesicles in 0.2 mm egta or 1 mm ca from panel b was quantified using densitometry and normalized to the syntaxin band in each lane . the average % increase in t - snare binding in the presence of ca versus egta was calculated using normalized data and plotted for each isoform . c - f , red is used to denote the inhibitory syts indicated in fig . d , for each syt isoform , % stimulation over syt ( from fig . 3c ) was plotted against the average % increase in t - snare binding for each syt isoform from panel c. e , the amount of syt bound to ps - harboring vesicles in 1 mmca from panel b was quantified using densitometry . the average total optical density is plotted for each isoform ( because there is no other band in each lane against which the syt signal can be normalized as in panel c ) . 3c ) was plotted against the average amount of syt bound to ps - harboring vesicles in 1 mm ca from panel e. utilizing a subset of the plasma membrane snares ( snap-25b , snap-23 , syntaxin 1a , and syntaxin 4 ) and a ubiquitously expressed snare ( snap-29 ) , we asked whether sytt - snare pairing might also contribute to the specificity of membrane fusion reactions . first , we observed that the extent of fusion between syb 2 vesicles and target vesicles that harbored distinct sets of t - snares differed for some t - snare pairs . namely , syntaxin 1a / snap-29 and syntaxin 4/snap-25b did not fuse as efficiently as did syntaxin 1a / snap-25b , syntaxin 1a / snap-23 , or syntaxin 4/snap-23 vesicles , all of which exhibited similar levels of fusion ( fig . 7 , red and green traces ) . we then tested the ability of syt i to stimulate fusion mediated by these different snare pairs . we found that syt i stimulated fusion whenever syntaxin 1a or snap-25b were present on the t - snare vesicle , but failed to efficiently stimulate fusion when both syntaxin 1a and snap-25b had been replaced with syntaxin 4 and snap-23 ( see also ref . in addition , casyt was able to activate otherwise weak snare - mediated fusion reactions ( e.g. syntaxin 1a / snap-29 and syntaxin 4/snap-25b t - snare vesicles ) by severalfold ( fig . 7 ) . moreover , syt i had only a slight effect on fusion between syb 2 vesicles and syntaxin 4/snap-23 vesicles even though this particular snare pair catalyzed fusion in a manner that was similar to reactions mediated by syntaxin 1a / snap-25b and syntaxin 1a / snap-23 . thus , syt i does not stimulate all snare - mediated fusion reactions equally , but rather , discriminates between t - snares , potentially adding a level of specificity to snare - mediated fusion reactions . previous studies characterized the effect of syts i , iv , vii , and ix on fusion mediated by neuronal snares ( 12 , 24 , 25 ) . in the current study we extended this analysis to include fourteen isoforms of syt . most of these isoforms inhibited fusion , to a limited extent under our experimental conditions , in the absence of ca ( e.g. syt i ; see also ref . we note that all isoforms of syt tested bound t - snares to some extent in egta . these ca - independent syt - t - snare interactions may function by halting snare complex assembly and inhibiting fusion in the absence of ca . v - vii , ix , and x stimulated fusion in response to ca , whereas syts iv , viii , xi - xiii , and xv failed to facilitate fusion in the presence or absence of ca . furthermore , some of the syts that failed to stimulate fusion actually inhibited fusion ( figs . 2 and 3 ) . without exception , stimulatory syts bound to ps and t - snares in response to ca whereas inhibitory syts did not ( fig . 4 ) . moreover , a subset of inhibitory syts reduced the extent of syt i - stimulated fusion . these data indicate that the presence of different isoforms of syts on the same secretory organelle might fine - tune the efficiency ( fig . 5 ) , ca - sensitivity ( 25 ) , and mode of exocytosis ( 31 ) by competing for interactions with effectors . below , we relate these in vitro findings with studies aimed at understanding the function of syt isoforms in cells . figure 5.syts iv and viii , but not xii , inhibit syt i - mediated stimulation of membrane fusion . syt i ( 10 m ) stimulates fusion between v- and t - snare vesicles in response to ca ( panels a , c , and e , red trace ) . a , increasing concentrations of syt iv were added to fusion reactions containing 10 m syt i and 1 mm ca . fusion was monitored for 120 min at 37 c and plotted as % max fl over time . b , average extent of fusion from the data in panel a ( % max fl ) at t = 120 min was plotted as a function of [ syt iv ] . c , experiments were carried out as in panel a except syt viii was used in place of syt iv . d , data from panel c were plotted as a function of [ syt viii ] . e , experiments were carried out as in panel a except syt xii was used in place of syt iv . f , data from e were plotted as a function of [ syt xii ] . in all plots , syts iv and viii , but not xii , inhibit syt i - mediated stimulation of membrane fusion . syt i ( 10 m ) stimulates fusion between v- and t - snare vesicles in response to ca ( panels a , c , and e , red trace ) . a , increasing concentrations of syt iv were added to fusion reactions containing 10 m syt i and 1 mm ca . fusion was monitored for 120 min at 37 c and plotted as % max fl over time . b , average extent of fusion from the data in panel a ( % max fl ) at t = 120 min was plotted as a function of [ syt iv ] . c , experiments were carried out as in panel a except syt viii was used in place of syt iv . d , data from panel c were plotted as a function of [ syt viii ] . e , experiments were carried out as in panel a except syt xii was used in place of syt iv . f , data from e were plotted as a function of [ syt xii ] . in all plots , a , left , binding of syt i and iv to t - snare ( t ) vesicles was carried out in the absence ( - ) or presence ( + ) of ca . syts were assayed individually and in the indicated combinations using 10 m each syt , or 10 m syt i and 30 m syt iv ( representing a 1:1 and 1:3 molar ratio of syt i : competitor syt , respectively ) . one - third of each sample was subjected to sds - page ; gels were stained with coomassie blue . right , the amount of syt i bound to t - snares was quantified by densitometry , normalized to the snap-25 band in each lane , and plotted as a percentage of snap-25 . data represent the average and s.e . from n b , experiments were carried out as in panel a , except syt viii was used in place of syt iv . c , experiments were carried out as in panel a , except syt xii was used in place of syt iv . statistical analysis was carried out using a student 's t test ; , p < 0.05 ; , p < 0.01 ; n.s . a only , the line on the gel ( left panel ) separates groups of lanes that were originally spaced further apart on the same gel and were combined for the figure . a , left , binding of syt i and iv to t - snare ( t ) vesicles was carried out in the absence ( - ) or presence ( + ) of ca . syts were assayed individually and in the indicated combinations using 10 m each syt , or 10 m syt i and 30 m syt iv ( representing a 1:1 and 1:3 molar ratio of syt i : competitor syt , respectively ) . one - third of each sample was subjected to sds - page ; gels were stained with coomassie blue . right , the amount of syt i bound to t - snares was quantified by densitometry , normalized to the snap-25 band in each lane , and plotted as a percentage of snap-25 . b , experiments were carried out as in panel a , except syt viii was used in place of syt iv . c , experiments were carried out as in panel a , except syt xii was used in place of syt iv . statistical analysis was carried out using a student 's t test ; , p < 0.05 ; , p < 0.01 ; n.s . ( not significant ) a only , the line on the gel ( left panel ) separates groups of lanes that were originally spaced further apart on the same gel and were combined for the figure . 2a using t - snare vesicles that harbored the indicated t - snare pairs and v - snare vesicles than harbored syb 2 . fusion was monitored in the absence ( -syt ) or presence ( + syt ) of 10 m syt i in either 0.2 mm egta or 1 mm ca and plotted as the % max fluorescence intensity over time . 2a using t - snare vesicles that harbored the indicated t - snare pairs and v - snare vesicles than harbored syb 2 . fusion was monitored in the absence ( -syt ) or presence ( + syt ) of 10 m syt i in either 0.2 mm egta or 1 mm ca and plotted as the % max fluorescence intensity over time . a recent flurry of reports addressed the role of syt ii , which is highly homologous to syt i , during exocytosis at synapses . in specific subsets of neurons , syt ii plays a critical role in ca - triggered neurotransmitter release ( 23 , 44 - 46 ) and can restore rapid release when expressed in syt i knock - out cortical neurons ( 23 ) . however , the mechanism by which syt ii functions had not been explored in detail . syt ii binds ps in response to ca and here we show that this isoform also interacts with the t - snares syntaxin 1a and snap-25b in a ca - promoted manner , and stimulates snare - catalyzed fusion . so , as expected , syt ii likely shares the same mechanism of action as syt i ( 27 , 46 ) . it was previously reported that syt ix does not bind to neuronal t - snares ( 22 ) but is able to rescue fast neurotransmitter release in syt i knock - out neurons ( 23 ) . together , these studies would seem to rule out both ca - independent and ca - dependent syt - snare interactions during exocytosis . however , another study employing three different kinds of binding assays demonstrated that syt ix does in fact bind neuronal t - snares in a ca - promoted manner ( 25 ) . here , we confirm these observations , as well as the fact that syt ix can directly stimulate snare - catalyzed fusion in response to ca . so , analogous to syts i and ii , syt ix is likely to regulate membrane fusion via ca - promoted interactions with both ps and t - snares . syt iv , a seizure - induced isoform with a single asp to ser substitution of one of the ca ligands in the c2a domain ( 29 ) is unable to bind ps and t - snares in a ca - dependent manner ( fig . 4 , b , c , and e and refs . 30 , 31 ) . i and iv are co - localized to large dense core vesicles in pc12 cells , where up - regulation of syt iv was shown to decrease the frequency of large dense core vesicle membrane fusion events and to destabilize fusion pores ( 31 , 33 ) . syt iv also favored the formation of tiny fusion pores , with one - fifth the transmitter flux of full - sized kiss - and - run fusion pores , that always close without dilating ( 31 , 33 ) . so , syt iv negatively regulates the rate of exocytosis and also affects fusion pore structure and dynamics . activity - induced up - regulation of syt iv negatively regulates the release of neurotrophic factors from dense core vesicles , thereby regulating synaptic plasticity . up - regulation of syt iv might act homeostatically to dampen overactive circuits.6 here , we demonstrate that syt iv itself inhibits snare - catalyzed membrane fusion ( figs . 2 and 3 ; see also ref . we also show that syt iv down - regulates casyt i - stimulated membrane fusion in our reduced assay system ( fig . 5 , a and b ) , in part by competing with syt i for interactions with t - snares ( fig . 6a ) . therefore , using a reconstituted system , we have recapitulated the negative regulatory role syt iv plays during dense core vesicle exocytosis . it should be noted that syt iv might play a positive role during other kinds of fusion events . for example , syt iv appears to be required for homotypic fusion of immature secretory granules ( isgs ) in pc12 cells ( 47 ) , perhaps by operating on a distinct set of snares . syt iv was also reported to play a positive role in secretion from glial cells ( 48 ) . syt iv ( 29 ) and syt x ( 49 ) are of particular interest because their expression levels are induced by activity . interestingly , these two isoforms had opposite effects in the in vitro fusion assay ; syt iv inhibited snare - catalyzed fusion , whereas syt x stimulated fusion ( figs . for example , in situ hybridization experiments in mouse brain have shown that the syt iv and x mrna signals are very strong in the hippocampus and dentate gyrus , respectively , but show little overlap within these regions ( allen brain atlas ) . on the other hand , the syt i mrna signal overlaps significantly with both the syt iv and syt x mrna signals . a detailed analysis of the tissue distribution and subcellular localization of syt iv and x will provide insight into why these two activity - induced isoforms have such different effects on fusion mediated by neuronal snare proteins . one possibility is that syt x is preferentially expressed in inhibitory neurons and facilitates inhibitory synaptic transmission . in this scenario , finally , it was reported that syt xii was expressed on synaptic vesicles but failed to co - immunoprecipitate with neuronal t - snares ( 50 ) . in contrast , our co - flotation assays clearly demonstrate significant levels of ca - independent binding of syt xii to membrane - embedded t - snares ( fig . ( 50 ) could be because of differences in experimental conditions ; i.e. using purified components as opposed to co - immunoprecipitation from brain extracts where the low abundance of syt xii might fall below the limits of detection . if syt i and xii are indeed expressed on the same synaptic vesicles at similar levels , our results indicate that syt xii might not be able to modulate the function of syt i during synaptic transmission . in conclusion , we have shown that the ability of syts to mediate ca - triggered membrane fusion is strictly related to their abilities to engage ps and t - snares in a ca - dependent manner . we also identified a subset of inhibitory syts that antagonized syt i function during fusion by competing for sytsnare interactions , further establishing t - snares as critical syt effectors . finally , we further confirm that syt i is selective for certain snare pairs over others ( fig . 7 and refs . 16 , 43 ) , supporting the hypothesis that during intracellular membrane transport and exocytosis , the action of syts on specific t - snares might contribute to the specificity of snare - mediated membrane fusion events . whether the syt isoforms that are inhibitory in our fusion assay screen ( using neuronal snares ) stimulate fusion mediated by other snare pairs is an interesting question that remains to be addressed . elucidation of the expression pattern and subcellular localization of each isoform of syt is needed to determine which t - snares a given syt isoform will encounter in vivo .

ca2 + -triggered exocytosis in neurons and neuroendocrine cells is regulated by the ca2 + -binding protein synaptotagmin ( syt ) i. sixteen additional isoforms of syt have been identified , but little is known concerning their biochemical or functional properties . here , we assessed the abilities of fourteen syt isoforms to directly regulate snare ( soluble n - ethylmaleimide - sensitive factor ( nsf ) attachment protein receptor)-catalyzed membrane fusion . one group of isoforms stimulated neuronal snare - mediated fusion in response to ca2 + , while another set inhibited snare catalyzed fusion in both the absence and presence of ca2 + . biochemical analysis revealed a strong correlation between the ability of syt isoforms to bind 1,2-dioleoyl phosphatidylserine ( ps ) and t - snares in a ca2 + -promoted manner with their abilities to enhance fusion , further establishing ps and snares as critical effectors for syt action . the ability of syt i to efficiently stimulate fusion was specific for certain snare pairs , suggesting that syts might contribute to the specificity of intracellular membrane fusion reactions . finally , a subset of inhibitory syts down - regulated the ability of syt i to activate fusion , demonstrating that syt isoforms can modulate the function of each other .

EXPERIMENTAL PROCEDURES RESULTS DISCUSSION

v - snares were reconstituted using a lipid mix composed of 30% 1-palmitoyl , 2-oleoyl phosphatidylethanolamine ( pe ) , 52% 1-palmitoyl , 2-oleoyl phosphatidylcholine ( pc ) , 15% 1,2-dioleoyl phosphatidylserine ( ps ) , 1.5% n-(7-nitro-2 - 1,3-benzoxadiazol-4-yl)-1,2-dipalmitoyl phosphatidylethanolamine ( nbd - pe , donor ) , and 1.5% n-(lissamine rhodamine b sulfonyl)-1,2-dipalmitoyl phosphatidylethanolamine ( rhodamine - pe , acceptor ) . numbers correspond to the order of the five aspartate residues that function as ca ligands in the c2a domain of syt i ( d172 , 178 , 230 , 232 , 238 ) and c2b ( d303 , 309 , 363 , 365 , 371 ) ( 6 ) . based on conservation of the ca ligands , we predicted which syts might ( + ) and might not ( - ) stimulate in vitro snare - mediated fusion in response to ca . numbers correspond to the order of the five aspartate residues that function as ca ligands in the c2a domain of syt i ( d172 , 178 , 230 , 232 , 238 ) and c2b ( d303 , 309 , 363 , 365 , 371 ) ( 6 ) . based on conservation of the ca ligands , we predicted which syts might ( + ) and might not ( - ) stimulate in vitro snare - mediated fusion in response to ca . ( eq.2)\documentclass[10pt]{article } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{pmc } \usepackage[euler]{upgreek } \pagestyle{empty } \oddsidemargin -1.0 in \begin{document } \begin{equation*}\;\;\%\hspace{1em}{\mathrm{stim\;over}}(-){\mathrm{syt}}=\frac{({\mathrm{fusion\;\;by}}\hspace{1em}{\mathrm{ca}}^{2+}{\cdot}{\mathrm{syt}})-({\mathrm{fusion\;\;by}}(-){\mathrm{syt}})}{{\mathrm{fusion\;\;by}}(-){\mathrm{syt}}}{\times}100\;\;\end{equation*}\end{document } conservation of the ca ligands in the c2a and c2b domains of syt to date , seventeen isoforms of syt have been identified in vertebrates ( 26 ) . in syt i , both c2 domains mediate ca - regulated interactions with anionic phospholipids and t - snares ( 6 ) . divergent activities of syt isoforms during neuronal snare - mediated membrane fusion in vitro studies revealed that in response to ca , syt i stimulates snare - catalyzed membrane fusion ( 12 , 24 ) , providing direct evidence that syt i can act as a ca sensor for exocytosis in vivo ( 4 - 6 ) . however , the ability of the other thirteen isoforms of syt to regulate membrane fusion remained unknown . fusion was monitored for 120 min at 37 c in the absence ( -syt ) or presence ( + syt , 10 m ) of different syt isoforms , normalized to the maximum donor fluorescence signal ( % max fl ) , and plotted as a function of time . fusion was monitored for 120 min at 37 c in the absence ( -syt ) or presence ( + syt , 10 m ) of different syt isoforms , normalized to the maximum donor fluorescence signal ( % max fl ) , and plotted as a function of time . using the in vitro membrane fusion assay , we screened fourteen syt isoforms for their abilities to regulate fusion between v - snare vesicles ( v ) , that harbor synaptobrevin 2 , and t - snare vesicles ( t ) , that harbor syntaxin 1a / snap-25b heterodimers ( fig . 2b ; only a subset of syt isoforms stimulated snare - catalyzed fusion in the presence of ca : syt i - iii , v - vii , and ix - x . to better visualize the impact of syt isoforms on fusion and to highlight differences that might have been obscured by averaging across all trials ( because of sample - to - sample differences in the overall extent of fusion ) , we compared the extent of fusion obtained in ca to the extent of fusion obtained in egta within each individual trial ( fig . in addition , we compared the extent of fusion obtained by casyt to fusion mediated by v- and t - snares in the absence of syt ( fig . stimulation of membrane fusion is related to the ability of syts to engage ps and t - snares in response to ca in response to ca , the tandem c2 domains of syt i engage t - snares and the anionic phospholipid ps , and previous studies indicate that these interactions are critical for the regulation of membrane fusion ( 15 , 16 , 18 , 24 , 25 ) . to further explore the mechanism of action of syts , we carried out experiments to relate their activity in the fusion assay with their abilities to engage both ps and t - snares . the syts that failed to stimulate fusion , isoforms iv , viii , xi , xii , xiii , and xv , also bound t - snares and ps , but these interactions were not enhanced by ca . b , average % stimulation in ca ( fusion in ca compared with fusion in egta within each individual trial and then averaged ; equation 1 in ; experimental procedures ) was plotted for v- and t - snare - mediated fusion and for each syt isoform . ( -)syt indicates fusion between v- and t - snare vesicles in the absence of syt . b , average % stimulation in ca ( fusion in ca compared with fusion in egta within each individual trial and then averaged ; equation 1 in ; experimental procedures ) was plotted for v- and t - snare - mediated fusion and for each syt isoform . the extent to which syt associated with t - snare vesicles in egta or ca was quantified using densitometry and normalized to the syntaxin band in the corresponding lane ; the % change in t - snare binding in response to ca was plotted ( fig . 4 , c and e ) exhibited similar patterns ; syts that bind ps in response to ca also bind t - snares in response to ca , and vice versa . these plots illustrate a strong correlation between the ability of a syt isoform to bind both of these effectors in a ca - dependent manner with their abilities to stimulate snare - catalyzed fusion in response to ca . similarly , there is a strong correlation between syts that interact with t - snares and ps in a ca - independent manner with their abilities to inhibit snare - catalyzed fusion . in no case did a syt isoform stimulate fusion without binding to ps and t - snares in a ca - promoted manner , supporting a model in which syts stimulate fusion through ca - dependent interactions with both of these effectors . syt isoforms can modulate the activity of each other in the course of screening syts i - xiii and xv , we identified a class of syts ( iv , viii , xi , and xii ) that failed to respond to ca in our biochemical assays and that inhibited snare - catalyzed fusion ( fig . these findings prompted the idea that these inhibitory syts might antagonize the action of syt isoforms that stimulate fusion in response to ca ( e.g. , we asked whether a subset of inhibitory syts could affect the ability of syt i to stimulate snare - catalyzed membrane fusion . it inhibited syt i - stimulated fusion in a dose - dependent manner.5 because syt i and iv have been co - localized to large dense core vesicles ( ldcvs ) in pc12 cells ( 29 , 33 ) , these findings support the idea that different syt isoforms on the same secretory organelle might serve to fine - tune the efficiency of exocytosis . next , we found that syts iv and viii significantly reduced ca - promoted binding of syt i to t - snares ( fig . in contrast , syt xii , the inhibitory syt that failed to suppress syt i - stimulated fusion , significantly reduced syt it - snare interactions only when added to the assay in a 3-fold molar excess of syt i ( fig . thus , it appears that inhibitory isoforms of syt can impact fusion in two ways : first , they directly inhibit snare - mediated fusion , and second , they displace syt i from the fusion complex . these properties are clearly apparent for syt iv and viii ; syt xii differs to some extent as this isoform exhibits less inhibitory activity in terms of both shutting down snare function and displacing syt i. moreover , these experiments provide further support for the idea that syts act , at least in part , via direct interactions with t - snares to regulate fusion . specificity of functional pairing between syts and t - snares during membrane fusion at present , approximately thirty - six snare proteins have been identified in humans ( 2 ) . it was proposed that pairing between specific v- and t - snares encodes the specificity of intracellular membrane fusion reactions ( 41 ) . 3c ) was plotted against the average amount of syt bound to ps - harboring vesicles in 1 mm ca from panel e. syts differ in their ability to bind ps and t - snares . 3c ) was plotted against the average amount of syt bound to ps - harboring vesicles in 1 mm ca from panel e. utilizing a subset of the plasma membrane snares ( snap-25b , snap-23 , syntaxin 1a , and syntaxin 4 ) and a ubiquitously expressed snare ( snap-29 ) , we asked whether sytt - snare pairing might also contribute to the specificity of membrane fusion reactions . we then tested the ability of syt i to stimulate fusion mediated by these different snare pairs . we found that syt i stimulated fusion whenever syntaxin 1a or snap-25b were present on the t - snare vesicle , but failed to efficiently stimulate fusion when both syntaxin 1a and snap-25b had been replaced with syntaxin 4 and snap-23 ( see also ref . in addition , casyt was able to activate otherwise weak snare - mediated fusion reactions ( e.g. moreover , syt i had only a slight effect on fusion between syb 2 vesicles and syntaxin 4/snap-23 vesicles even though this particular snare pair catalyzed fusion in a manner that was similar to reactions mediated by syntaxin 1a / snap-25b and syntaxin 1a / snap-23 . thus , syt i does not stimulate all snare - mediated fusion reactions equally , but rather , discriminates between t - snares , potentially adding a level of specificity to snare - mediated fusion reactions . we note that all isoforms of syt tested bound t - snares to some extent in egta . without exception , stimulatory syts bound to ps and t - snares in response to ca whereas inhibitory syts did not ( fig . moreover , a subset of inhibitory syts reduced the extent of syt i - stimulated fusion . below , we relate these in vitro findings with studies aimed at understanding the function of syt isoforms in cells . figure 5.syts iv and viii , but not xii , inhibit syt i - mediated stimulation of membrane fusion . syt i ( 10 m ) stimulates fusion between v- and t - snare vesicles in response to ca ( panels a , c , and e , red trace ) . syt i ( 10 m ) stimulates fusion between v- and t - snare vesicles in response to ca ( panels a , c , and e , red trace ) . in all plots , a , left , binding of syt i and iv to t - snare ( t ) vesicles was carried out in the absence ( - ) or presence ( + ) of ca . right , the amount of syt i bound to t - snares was quantified by densitometry , normalized to the snap-25 band in each lane , and plotted as a percentage of snap-25 . a , left , binding of syt i and iv to t - snare ( t ) vesicles was carried out in the absence ( - ) or presence ( + ) of ca . right , the amount of syt i bound to t - snares was quantified by densitometry , normalized to the snap-25 band in each lane , and plotted as a percentage of snap-25 . syt ii binds ps in response to ca and here we show that this isoform also interacts with the t - snares syntaxin 1a and snap-25b in a ca - promoted manner , and stimulates snare - catalyzed fusion . it was previously reported that syt ix does not bind to neuronal t - snares ( 22 ) but is able to rescue fast neurotransmitter release in syt i knock - out neurons ( 23 ) . however , another study employing three different kinds of binding assays demonstrated that syt ix does in fact bind neuronal t - snares in a ca - promoted manner ( 25 ) . here , we confirm these observations , as well as the fact that syt ix can directly stimulate snare - catalyzed fusion in response to ca . so , analogous to syts i and ii , syt ix is likely to regulate membrane fusion via ca - promoted interactions with both ps and t - snares . syt iv , a seizure - induced isoform with a single asp to ser substitution of one of the ca ligands in the c2a domain ( 29 ) is unable to bind ps and t - snares in a ca - dependent manner ( fig . up - regulation of syt iv might act homeostatically to dampen overactive circuits.6 here , we demonstrate that syt iv itself inhibits snare - catalyzed membrane fusion ( figs . we also show that syt iv down - regulates casyt i - stimulated membrane fusion in our reduced assay system ( fig . 5 , a and b ) , in part by competing with syt i for interactions with t - snares ( fig . interestingly , these two isoforms had opposite effects in the in vitro fusion assay ; syt iv inhibited snare - catalyzed fusion , whereas syt x stimulated fusion ( figs . if syt i and xii are indeed expressed on the same synaptic vesicles at similar levels , our results indicate that syt xii might not be able to modulate the function of syt i during synaptic transmission . in conclusion , we have shown that the ability of syts to mediate ca - triggered membrane fusion is strictly related to their abilities to engage ps and t - snares in a ca - dependent manner . we also identified a subset of inhibitory syts that antagonized syt i function during fusion by competing for sytsnare interactions , further establishing t - snares as critical syt effectors . finally , we further confirm that syt i is selective for certain snare pairs over others ( fig . 16 , 43 ) , supporting the hypothesis that during intracellular membrane transport and exocytosis , the action of syts on specific t - snares might contribute to the specificity of snare - mediated membrane fusion events . elucidation of the expression pattern and subcellular localization of each isoform of syt is needed to determine which t - snares a given syt isoform will encounter in vivo .

the impression of open surgery is characteristically that of prolonged painful recuperative periods with long scars . this has been transformed to more acceptable painless , cosmetically satisfying surgery with a short hospital stay due to the advent of operative laparoscopy . work - related musculoskeletal disorders ( wrmsds ) are being considered as an important health issue among surgeons performing minimally invasive surgeries ( miss ) , especially laparoscopic procedures . inadequate understanding of physical ergonomics , ergonomically deficient design of laparoscopic instruments and increased technological complexity have been cited as possible causes . hence laboratory research is prevalent in this field , frequently conducted in simulated environment using box trainers or other models . suturing and tying of knots have been the most commonly tested laparoscopic manipulation tasks utilised for assessing muscle activity , fatigue , task accuracy , pattern of joint movements , etc . the above - mentioned tasks , although very important for the performance of a successful surgery , occupy only a small percentage of the total surgical time . there is a limitation to generalise the applicability of the results of aforementioned tasks in surgical ergonomics . moreover , there is a dearth of ergonomic assessment studies analysing the important components such as dissection and exposure retraction . hence , in this study , we selected a basic , simulated , surgical protocol which incorporated tasks such as tissue dissection , exposing , retraction , clipping and suction with a surgical flow mimicking a real case scenario . the main purpose of this study was to conduct an ergonomic analysis of experienced and novice surgeons while performing a simulated laparoscopic cholecystectomy . we aimed to assess and report the cumulative ergonomic risk to the spine and the wrist during a procedure incorporating surgical flow as this may incorporate more natural movement patterns utilised by the surgeon . furthermore , the movements that occurred during task transition would be included in the final risk analysis . other purpose of this analysis was to analyse whether any difference exists in ergonomic between the experienced and novice surgeons . thirty - six right - handed laparoscopic surgeons were included in the study after obtaining informed consent . participants were divided into two groups according to the level of laparoscopic surgical experience : experts ( n = 16 ) and novices ( n = 16 ) . the expert group included the surgeons who were practising laparoscopic procedures for at least a minimum of 3 years and the novice group consisted of surgeons and residents who had no previous laparoscopic surgical experience else than observation of laparoscopic surgeries . demographic characteristics of the participating surgeons the simulated cholecystectomy was performed on lapvr simulator ( cae care manufacturer ) . in this virtual reality surgical simulator , there are three input portals , i.e. two trocars and one camera and a screen to visualise the procedure [ figure 1 ] . the procedure was selected at a basic level for aiding the completion by the novice surgeon . training session for familiarising with the surgical simulator controls was provided for the surgeons participating in the study . the height of the monitor and the trocars was fixed during all procedures in this study . the working height and dimension of the simulator monitor and trocar were as follows : lapvr surgical simulator monitor screen height from the ground - 168.5 cmmonitor screen width - 56 cmheight of the trocars from the ground - 108 cm . monitor screen height from the ground - 168.5 cm monitor screen width - 56 cm height of the trocars from the ground - 108 cm . a common surgical procedure was performed by the surgeons in both groups which comprised the three main tasks mentioned below . task 1 : exposure of the gallbladder and dissection of adhesionstask 2 : clipping and division of cystic structurestask 3 : mobilisation and removal of the gallbladder . task 1 : exposure of the gallbladder and dissection of adhesions task 2 : clipping and division of cystic structures task 3 : mobilisation and removal of the gallbladder . at the end of the simulation task , the performance measures and dexterity were displayed on the simulator screen as a compiled report . the motion analysis data and rapid upper limb assessment ( rula ) were used to estimate the surgeons ergonomics . ( a ) time required to complete the procedure and whether the designated time was exceeded . ( b ) proficiency was tested on analysis of items such as the total blood loss during the procedure , a number of clips dropped and used . there were 25 components on performance measures for which each surgeon was given a positive marking once the criteria were met . ( c ) dexterity was analysed on the basis of total path length of the left and right hand during the procedure . noraxon 's three - dimensional ( 3d ) myomotion analysis system consists of a set of sensors using inertial sensor technology [ figure 2 ] . based on fusion algorithms , the information from a 3d accelerometer , gyroscope and magnetometer is used to measure the 3d rotation angles of each sensor in absolute space . noraxon 's system software converts the quaternion data into anatomical angles using a rigid body model . noraxon myomotion inertial measurement unit sensors and display screen the motion sensors were attached to the following sites : foreheadthoracic spine - t1 ( first thoracic ) vertebralumbar spine - s2 ( second sacral ) levelmid - forearm - strapped on both left and right forearmshand - strapped around the dorsal aspect of the hand ( both left and right side ) . thoracic spine - t1 ( first thoracic ) vertebra lumbar spine - s2 ( second sacral ) level mid - forearm - strapped on both left and right forearms hand - strapped around the dorsal aspect of the hand ( both left and right side ) . the data from the software , the movement of the cervical and thoracolumbar ( tl ) spine , are computed in all three dimensions , namely , sagittal , coronal and transverse planes and movement of the wrist in the sagittal and coronal planes . at the cervical and tl spine flexion , the right side rotation and lateral flexion are displayed as positive , and at extension , the left side rotation and lateral flexion are designated negative signs . similarly , the wrist extension and radial deviation ( rd ) are designated positive and wrist flexion and ulnar deviation ( ud ) with negative signs . for example , 30 in the sagittal plane means 30 of extension and + 30 means 30 of flexion . accordingly , the joint movement values are assigned positive or negative signs [ tables 2 and 3 ] . the maximum angles in each direction were computed and used for comparison between the groups . maximum excursion of the cervical and thoracolumbar spine maximum excursion of the wrist joint the rula scale is a tool which helps draw conclusion on ergonomic risks related to an occupational task . the rula is divided into components such as upper arm , lower arm , wrist , neck , trunk and leg . the positions of the upper arm , lower arm and wrist are grouped together which give out a score , that is , clubbed alongside the similar score formed by the neck , trunk and leg . based on the outcomes of the score , further investigations and ergonomic changes can be decided . 1 or 2 = acceptable posture3 or 4 = further investigation , change may be needed5 or 6 = further investigation , change soon7 = investigate and implement change . 1 or 2 = acceptable posture 3 or 4 = further investigation , change may be needed 5 or 6 = further investigation , change soon 7 = investigate and implement change . ( a ) time required to complete the procedure and whether the designated time was exceeded . ( b ) proficiency was tested on analysis of items such as the total blood loss during the procedure , a number of clips dropped and used . there were 25 components on performance measures for which each surgeon was given a positive marking once the criteria were met . ( c ) dexterity was analysed on the basis of total path length of the left and right hand during the procedure . noraxon 's three - dimensional ( 3d ) myomotion analysis system consists of a set of sensors using inertial sensor technology [ figure 2 ] . based on fusion algorithms , the information from a 3d accelerometer , gyroscope and magnetometer is used to measure the 3d rotation angles of each sensor in absolute space . noraxon 's system software converts the quaternion data into anatomical angles using a rigid body model . noraxon myomotion inertial measurement unit sensors and display screen the motion sensors were attached to the following sites : foreheadthoracic spine - t1 ( first thoracic ) vertebralumbar spine - s2 ( second sacral ) levelmid - forearm - strapped on both left and right forearmshand - strapped around the dorsal aspect of the hand ( both left and right side ) . thoracic spine - t1 ( first thoracic ) vertebra lumbar spine - s2 ( second sacral ) level mid - forearm - strapped on both left and right forearms hand - strapped around the dorsal aspect of the hand ( both left and right side ) . the data from the software , the movement of the cervical and thoracolumbar ( tl ) spine , are computed in all three dimensions , namely , sagittal , coronal and transverse planes and movement of the wrist in the sagittal and coronal planes . at the cervical and tl spine flexion , the right side rotation and lateral flexion are displayed as positive , and at extension , the left side rotation and lateral flexion are designated negative signs . similarly , the wrist extension and radial deviation ( rd ) are designated positive and wrist flexion and ulnar deviation ( ud ) with negative signs . for example , 30 in the sagittal plane means 30 of extension and + 30 means 30 of flexion . accordingly , the joint movement values are assigned positive or negative signs [ tables 2 and 3 ] . the maximum angles in each direction were computed and used for comparison between the groups . the rula scale is a tool which helps draw conclusion on ergonomic risks related to an occupational task . the rula is divided into components such as upper arm , lower arm , wrist , neck , trunk and leg . the positions of the upper arm , lower arm and wrist are grouped together which give out a score , that is , clubbed alongside the similar score formed by the neck , trunk and leg . based on the outcomes of the score , further investigations and ergonomic changes can be decided . 1 or 2 = acceptable posture3 or 4 = further investigation , change may be needed5 or 6 = further investigation , change soon7 = investigate and implement change . 1 or 2 = acceptable posture 3 or 4 = further investigation , change may be needed 5 or 6 = further investigation , change soon 7 = investigate and implement change . the data of the maximum cervical and tl movement excursion [ table 2 ] , wrist movement excursion [ table 3 ] , rula scores [ table 4 ] and performance scores [ table 5 ] obtained during the simulated cholecystectomy was statistically compared between the novice and experienced surgeons . comparison of rapid upper limb assessment scores comparison of performance and dexterity scores during the simulated surgery , myomotion system - generated data of the cervical spine movements indicate that the maximum cervical flexion and the right side rotation were higher in novice whereas the right lateral flexion was higher in experienced surgeons . the results of the tl spine indicate that the novice showed more right tl rotation and the experienced surgeons showed more left tl rotation when maximum angles were compared . the results of motion analysis indicate the high left wrist flexion angles adopted by the surgeons , i.e. , maximum angles > 45 with concomitant rd . at the right wrist joint ( wj ) too , maximum angles of > 50 flexion and concomitant rd or ud were recorded in the sagittal and coronal plane . on comparison , there were no differences in the maximum angles of both left and right wjs between the experienced and novice surgeons . the result of the rula score indicates that there is a high postural risk during this surgical procedure . although the rula scores were higher in the novice surgeons , the difference was not statistically significant . for comparing path length , the performance score was higher in the experienced group ; however , the difference was not statistically significant . during the simulated surgery , myomotion system - generated data of the cervical spine movements indicate that the maximum cervical flexion and the right side rotation were higher in novice whereas the right lateral flexion was higher in experienced surgeons . the results of the tl spine indicate that the novice showed more right tl rotation and the experienced surgeons showed more left tl rotation when maximum angles were compared . the results of motion analysis indicate the high left wrist flexion angles adopted by the surgeons , i.e. , maximum angles > 45 with concomitant rd . at the right wrist joint ( wj ) too , maximum angles of > 50 flexion and concomitant rd or ud were recorded in the sagittal and coronal plane . on comparison , there were no differences in the maximum angles of both left and right wjs between the experienced and novice surgeons . the result of the rula score indicates that there is a high postural risk during this surgical procedure . although the rula scores were higher in the novice surgeons , the difference was not statistically significant . the performance score was higher in the experienced group ; however , the difference was not statistically significant . the primary objective of this study was to assess the ergonomics in the novice and experienced surgeons during a simulated laparoscopic cholecystectomy , and the secondary objective was to compare them for any differences in ergonomics . we studied the spinal and wrist postures during a simulated cholecystectomy using objective motion analysis . our study findings confirm that the surgeons head posture depends on the orientation of the monitor at the operation theatre as has been reported in other studies . in our study , most of the surgeons adopted neck extension posture during the simulated surgery , which they attributed to either screen height or habitual neck postures . the novice had higher maximum excursions into flexion and rotation which may be both due to limited visual cueing ( loss of 3d visual perspective and limited peripheral vision due to limited viewing spectrum ) and insufficient laparoscopic surgical experience . however , the reason for the higher maximum lateral flexion in the experienced surgeons remains inexplicable . overall , there was ergonomically acceptable upright tl posture adopted by the surgeons during the simulated surgery . our study results support the findings of berguer et al . which state that although the posture of the surgeon is more upright during laparoscopy , it seems to be accompanied by substantially less body movement and weight shifting than during open surgery . this situation could account for increased static postural fatigue as the operation lasts for minimum period of 60 min and can extend to a couple of hours . have revealed that even relatively low levels ( 5% of maximum voluntary contraction [ mvc ] ) of muscle contraction , if sustained for an hour , can lead to muscle fatigue . jonsson et al . have suggested that the static muscle contraction should not exceed 5% of mvc for tasks of prolonged duration . the probable reason for this as elucidated by sjogaard could be that during low - level contractions , the muscle can maintain homoeostasis with respect to energy turnover but not with respect to intracellular / extracellular potassium concentration . the static trunk posture could be one of the reasons that increase the risk of musculoskeletal problems and pain in minimal access surgeons . when compared , there were no statistically significant differences in the maximum ranges between the experienced and novice surgeons except in tl rotation . the reason for higher right side tl rotation in novice could be due to their incorrect starting position . their entire body was rotated to the left due to the inappropriate feet placement , due to which there was a compensatory right side neck and trunk rotation . in our study , no surgeon scored in an acceptable range of 12 on the neck and trunk rula . although the tl spine was in near neutral postures , the positions adopted by the cervical spine scores increased the rula scores during the simulated surgery . the surgeons who scored high on the rula ( > 5 ) had adopted cervical extension along with rotation and lateral flexion . the surgeons who scored lower on the rula ( 3 or 4 ) had adopted either near neutral postures of the cervical spine or lesser degrees of flexion during the surgery . inappropriate monitor position , limited ergonomic awareness and habitual extension and lateral flexion postures adopted during the simulation surgery , culminated to the high neck and trunk rula scores in both groups . hence , there was no statistically significant difference between the groups on neck and trunk rula . these findings further strengthen the existing body of evidence which indicates that the monitor position can greatly influence the adoption of wrong postures of the neck . the static tl posture adds to the musculoskeletal risk at the spine in both novice and experienced surgeons . the role of the left upper extremity is predominantly to hold and manoeuvre the tissue which had to be operated on . the typical trend observed in both experienced and novice surgeons at the left wrist was flexion > 45 with radial angular deviation . however , at higher wrist flexion angles , the wrist was positioned in either neutral or ud [ figure 3 ] . the right and left wrist awkward position while using ring - handled instrument during the simulated surgery the right upper extremity had to function to manoeuvre the different instruments ( maryland dissector , clip applicator , endoscopic shear , l - hook electrocautery and suction ) for various tasks of the simulated surgery . in our study , the maximum right wrist flexion was above 50 in both experienced and novice surgeons . the frequently adopted wrist position in the frontal plane was of ud [ figure 3 ] , with intermittent rd . there were periodic changes in position of the right wrist in both sagittal and frontal plane while performing tasks such as dissection , clipping and cauterisation . no surgeon in both experienced and novice group adopted a neutral wrist posture . when we compared maximum right and left wrist excursions , there was no significant difference between the experienced and novice surgeons . there is a reduction in grip strength as well as an increase in the carpal tunnel pressure with wrist in flexed posture . keir et al . had revealed that the hydrostatic pressure in the carpal tunnel was found to be affected by both wrist posture and tendon load . muscular loading elevated carpal tunnel pressure , particularly the loading of palmaris longus with the wrist in extension and the digital flexors with the wrist flexed . correspondingly , the local contact forces by the digital flexors indicated the highest loads on the median nerve with the wrist flexed . anteriorly positioned nerves were flattened in the anteroposterior plane between the tendon and flexor retinaculum ; this was greatest with flexion and least with extension . furthermore , maximum grip efficiency has been reported in 2035 of extension and relatively low angles ( 57 ) of ud by other researchers . hence , in light of the aforementioned reasons , the high wrist flexion angles occurring during laparoscopic surgeries should be reduced by novel ergonomic handle designs . matern et al . have discussed earlier that a drawback of mis handles is their deviation from the longitudinal axis of the forearm , which causes uncomfortable ud and rd of the wrist and requires large - scale arm movements for simple instrument rotation along the longitudinal axis . many previous studies have highlighted on sagittal plane angles ( flexion and extension ) at the wrist . however , a limited number of studies have emphasised on frontal plane movements ( wrist rd / ud ) during laparoscopic procedures . although ud has been more commonly reported , occurrence of rd during laparoscopic procedures has also been mentioned in previous research . nguyen et al . have reported that at the upper extremity , there are more wrist supination and wrist ud and rd during a laparoscopic procedure as compared to open surgery . furthermore , the method utilised in previous research more commonly was video analysis . during video analysis , if the camera is not aligned well or the participant under study changes his / her position with respect to the camera , some movements and the precise degree to which they occur can be overlooked . since the accelerometer sensor used in this study is attached to the forearm and hand , even with a change in upper limb position , the angular movements are recorded with sufficient precision at the wrist . although higher flexion angles and ud predominate , the presence of rd can not be ignored . furthermore , rd can cause a reduction in the grip force and add to the mechanical inefficiency . in our study , the wrist rula score was high , i.e. , 4 , for majority of the surgeons ( 25 on the left and 29 on the right side ) , with few surgeons ( 7 surgeons on the left and 3 on the right side ) scoring 3 . the results of our study support the findings of snchez - margallo et al . , which indicate that the previous laparoscopic experience has no favourable effect while using ring - handled instrument . the rula score results of their study suggest that all study surgeons adopt an inadequate position of the wrist associated with a marked hyperflexion of this joint . they computed the rula scores for the risk based on sagittal plane deviations ( i.e. , flexion - extension ) . in our study , we included the wj deflections in the both sagittal and frontal planes to compute wrist rula . the surgeons in our study performed the simulated surgical procedure with ring - handled instrument handles in both hands . in biomechanical terms , these elongated instruments increase the leverage of the load arm leading to greater angles of wrist deviations in the sagittal and coronal plane . this leads to an increased postural stress at the wj and may contribute to wrist and hand pain . previous research indicates that ring handled instruments are less preferred due to the awkward wrist position imposed by them , when compared to axial handled instruments . compared the muscle tension ( surface electromyography ) caused by two different grasping modalities during the use of ring - handled laparoscopic scissors , showing that it is beneficial to use larger palm support instead of introducing the thumb through the rings of the handle . in our study , we observed that all the surgeons placed their fingers through the rings in the scissors handle contrary to berguer 's recommendations , with the exception of one surgeon who used palmar grasp on the left hand to hold the ring - handled instrument . this reflects the fact that reported ergonomic guidelines are usually ignored by operating laparoscopic surgeons , similarly to evidence reported by wauben et al . in their study . in comparison to previous research , we did not find any statistically significant difference in performance measures between the experienced and novice surgeons [ table 5 ] . the probable reason for this could be because basic level surgery was chosen for comparison ; as well , to the goal - oriented task , nature of the surgical task could have led to greater motivation for task completion in the novice surgeons . our study findings confirm that the surgeons head posture depends on the orientation of the monitor at the operation theatre as has been reported in other studies . in our study , most of the surgeons adopted neck extension posture during the simulated surgery , which they attributed to either screen height or habitual neck postures . the novice had higher maximum excursions into flexion and rotation which may be both due to limited visual cueing ( loss of 3d visual perspective and limited peripheral vision due to limited viewing spectrum ) and insufficient laparoscopic surgical experience . however , the reason for the higher maximum lateral flexion in the experienced surgeons remains inexplicable . overall , there was ergonomically acceptable upright tl posture adopted by the surgeons during the simulated surgery . our study results support the findings of berguer et al . which state that although the posture of the surgeon is more upright during laparoscopy , it seems to be accompanied by substantially less body movement and weight shifting than during open surgery . this situation could account for increased static postural fatigue as the operation lasts for minimum period of 60 min and can extend to a couple of hours . have revealed that even relatively low levels ( 5% of maximum voluntary contraction [ mvc ] ) of muscle contraction , if sustained for an hour , can lead to muscle fatigue . jonsson et al . have suggested that the static muscle contraction should not exceed 5% of mvc for tasks of prolonged duration . the probable reason for this as elucidated by sjogaard could be that during low - level contractions , the muscle can maintain homoeostasis with respect to energy turnover but not with respect to intracellular / extracellular potassium concentration . . the static trunk posture could be one of the reasons that increase the risk of musculoskeletal problems and pain in minimal access surgeons . when compared , there were no statistically significant differences in the maximum ranges between the experienced and novice surgeons except in tl rotation . the reason for higher right side tl rotation in novice could be due to their incorrect starting position . their entire body was rotated to the left due to the inappropriate feet placement , due to which there was a compensatory right side neck and trunk rotation . in our study , no surgeon scored in an acceptable range of 12 on the neck and trunk rula . although the tl spine was in near neutral postures , the positions adopted by the cervical spine scores increased the rula scores during the simulated surgery . the surgeons who scored high on the rula ( > 5 ) had adopted cervical extension along with rotation and lateral flexion . the surgeons who scored lower on the rula ( 3 or 4 ) had adopted either near neutral postures of the cervical spine or lesser degrees of flexion during the surgery . inappropriate monitor position , limited ergonomic awareness and habitual extension and lateral flexion postures adopted during the simulation surgery , culminated to the high neck and trunk rula scores in both groups . hence , there was no statistically significant difference between the groups on neck and trunk rula . these findings further strengthen the existing body of evidence which indicates that the monitor position can greatly influence the adoption of wrong postures of the neck . the static tl posture adds to the musculoskeletal risk at the spine in both novice and experienced surgeons . the role of the left upper extremity is predominantly to hold and manoeuvre the tissue which had to be operated on . the typical trend observed in both experienced and novice surgeons at the left wrist was flexion > 45 with radial angular deviation . however , at higher wrist flexion angles , the wrist was positioned in either neutral or ud [ figure 3 ] . the right and left wrist awkward position while using ring - handled instrument during the simulated surgery the right upper extremity had to function to manoeuvre the different instruments ( maryland dissector , clip applicator , endoscopic shear , l - hook electrocautery and suction ) for various tasks of the simulated surgery . in our study , the maximum right wrist flexion was above 50 in both experienced and novice surgeons . the frequently adopted wrist position in the frontal plane was of ud [ figure 3 ] , with intermittent rd . there were periodic changes in position of the right wrist in both sagittal and frontal plane while performing tasks such as dissection , clipping and cauterisation . no surgeon in both experienced and novice group adopted a neutral wrist posture . when we compared maximum right and left wrist excursions , there was no significant difference between the experienced and novice surgeons . there is a reduction in grip strength as well as an increase in the carpal tunnel pressure with wrist in flexed posture . keir et al . had revealed that the hydrostatic pressure in the carpal tunnel was found to be affected by both wrist posture and tendon load . muscular loading elevated carpal tunnel pressure , particularly the loading of palmaris longus with the wrist in extension and the digital flexors with the wrist flexed . correspondingly , the local contact forces by the digital flexors indicated the highest loads on the median nerve with the wrist flexed . anteriorly positioned nerves were flattened in the anteroposterior plane between the tendon and flexor retinaculum ; this was greatest with flexion and least with extension . furthermore , maximum grip efficiency has been reported in 2035 of extension and relatively low angles ( 57 ) of ud by other researchers . hence , in light of the aforementioned reasons , the high wrist flexion angles occurring during laparoscopic surgeries should be reduced by novel ergonomic handle designs . matern et al . have discussed earlier that a drawback of mis handles is their deviation from the longitudinal axis of the forearm , which causes uncomfortable ud and rd of the wrist and requires large - scale arm movements for simple instrument rotation along the longitudinal axis . many previous studies have highlighted on sagittal plane angles ( flexion and extension ) at the wrist . however , a limited number of studies have emphasised on frontal plane movements ( wrist rd / ud ) during laparoscopic procedures . although ud has been more commonly reported , occurrence of rd during laparoscopic procedures has also been mentioned in previous research . nguyen et al . have reported that at the upper extremity , there are more wrist supination and wrist ud and rd during a laparoscopic procedure as compared to open surgery . furthermore , the method utilised in previous research more commonly was video analysis . during video analysis , if the camera is not aligned well or the participant under study changes his / her position with respect to the camera , some movements and the precise degree to which they occur can be overlooked . since the accelerometer sensor used in this study is attached to the forearm and hand , even with a change in upper limb position , the angular movements are recorded with sufficient precision at the wrist . although higher flexion angles and ud predominate , the presence of rd can not be ignored . furthermore , rd can cause a reduction in the grip force and add to the mechanical inefficiency . in our study , the wrist rula score was high , i.e. , 4 , for majority of the surgeons ( 25 on the left and 29 on the right side ) , with few surgeons ( 7 surgeons on the left and 3 on the right side ) scoring 3 . the results of our study support the findings of snchez - margallo et al . , which indicate that the previous laparoscopic experience has no favourable effect while using ring - handled instrument . the rula score results of their study suggest that all study surgeons adopt an inadequate position of the wrist associated with a marked hyperflexion of this joint . they computed the rula scores for the risk based on sagittal plane deviations ( i.e. , flexion - extension ) . in our study , we included the wj deflections in the both sagittal and frontal planes to compute wrist rula . the surgeons in our study performed the simulated surgical procedure with ring - handled instrument handles in both hands . in biomechanical terms , these elongated instruments increase the leverage of the load arm leading to greater angles of wrist deviations in the sagittal and coronal plane . this leads to an increased postural stress at the wj and may contribute to wrist and hand pain . previous research indicates that ring handled instruments are less preferred due to the awkward wrist position imposed by them , when compared to axial handled instruments . compared the muscle tension ( surface electromyography ) caused by two different grasping modalities during the use of ring - handled laparoscopic scissors , showing that it is beneficial to use larger palm support instead of introducing the thumb through the rings of the handle . in our study , we observed that all the surgeons placed their fingers through the rings in the scissors handle contrary to berguer 's recommendations , with the exception of one surgeon who used palmar grasp on the left hand to hold the ring - handled instrument . this reflects the fact that reported ergonomic guidelines are usually ignored by operating laparoscopic surgeons , similarly to evidence reported by wauben et al . in their study . in comparison to previous research , we did not find any statistically significant difference in performance measures between the experienced and novice surgeons [ table 5 ] . the probable reason for this could be because basic level surgery was chosen for comparison ; as well , to the goal - oriented task , nature of the surgical task could have led to greater motivation for task completion in the novice surgeons . an objective analysis of the wrist shows that both expert and novice surgeons adopt disadvantageous postures of both right and left wrists while using axial - handled instruments on the simulator . the spinal analysis indicates that the monitor position greatly influences the neck posture , and predominantly static postures are adopted at the tl spine . we conclude that ergonomic inadequacies can lead to the physical postural risk , during the minimally invasive surgical procedure in both experienced and novice surgeons .

introduction : there is a rise in prevalence of work - related musculoskeletal disorders in surgeons performing laparoscopic surgeries due to lack of ergonomic considerations to the minimal access surgical environment . the objective of this study was to assess the physical ergonomics in experienced and novice surgeons during a simulated laparoscopic cholecystectomy.methodology:thirty-two surgeons participated in this study and were distributed in two groups ( experienced and novices ) based on the inclusion criteria . both groups were screened for the spinal and wrist movements on the orientation sensor - based , motion analysis device while performing a simulated laparoscopic cholecystectomy . simultaneous video recording was used to estimate the other joint positions . the rula ( rapid upper limb assessment ) ergonomic risk scores were estimated with the acquired data.results:we found that surgeons in both novice and experienced groups scored a high on the rula . limited awareness of the influence of monitor position on the postural risk caused surgeons to adopt non - neutral range cervical postures . the thoracolumbar spine is subjected to static postural demand . awkward wrist postures were adopted during the surgery by both groups . there was no statistically significant difference in the rula scores between the novice and experienced , but some differences in maximum joint excursions between them as detected on the motion analysis system.conclusion:both experienced and novice surgeons adopted poor spinal and wrist ergonomics during simulated cholecystectomy . we concluded that the physical ergonomic risk is medium as estimated by the rula scoring method , during this minimally invasive surgical procedure , demanding implementation of change in the ergonomic practices .

INTRODUCTION METHODOLOGY Metrics evaluation system available on the simulation equipment Objective record of wrist joint and spinal movements with the help of movement analysis system and software Observational record of upper limb posture and risk assessment on rapid upper limb assessment scale RESULTS Objective movement analysis data Rapid upper limb analysis scores Performance and dexterity measures DISCUSSION Spinal ergonomic analysis Neck and trunk rapid upper limb assessment Wrist and hand ergonomic analysis CONCLUSION Financial support and sponsorship Conflicts of interest

this has been transformed to more acceptable painless , cosmetically satisfying surgery with a short hospital stay due to the advent of operative laparoscopy . work - related musculoskeletal disorders ( wrmsds ) are being considered as an important health issue among surgeons performing minimally invasive surgeries ( miss ) , especially laparoscopic procedures . the above - mentioned tasks , although very important for the performance of a successful surgery , occupy only a small percentage of the total surgical time . there is a limitation to generalise the applicability of the results of aforementioned tasks in surgical ergonomics . moreover , there is a dearth of ergonomic assessment studies analysing the important components such as dissection and exposure retraction . hence , in this study , we selected a basic , simulated , surgical protocol which incorporated tasks such as tissue dissection , exposing , retraction , clipping and suction with a surgical flow mimicking a real case scenario . the main purpose of this study was to conduct an ergonomic analysis of experienced and novice surgeons while performing a simulated laparoscopic cholecystectomy . we aimed to assess and report the cumulative ergonomic risk to the spine and the wrist during a procedure incorporating surgical flow as this may incorporate more natural movement patterns utilised by the surgeon . other purpose of this analysis was to analyse whether any difference exists in ergonomic between the experienced and novice surgeons . participants were divided into two groups according to the level of laparoscopic surgical experience : experts ( n = 16 ) and novices ( n = 16 ) . the expert group included the surgeons who were practising laparoscopic procedures for at least a minimum of 3 years and the novice group consisted of surgeons and residents who had no previous laparoscopic surgical experience else than observation of laparoscopic surgeries . demographic characteristics of the participating surgeons the simulated cholecystectomy was performed on lapvr simulator ( cae care manufacturer ) . the procedure was selected at a basic level for aiding the completion by the novice surgeon . training session for familiarising with the surgical simulator controls was provided for the surgeons participating in the study . the height of the monitor and the trocars was fixed during all procedures in this study . a common surgical procedure was performed by the surgeons in both groups which comprised the three main tasks mentioned below . at the end of the simulation task , the performance measures and dexterity were displayed on the simulator screen as a compiled report . the motion analysis data and rapid upper limb assessment ( rula ) were used to estimate the surgeons ergonomics . ( b ) proficiency was tested on analysis of items such as the total blood loss during the procedure , a number of clips dropped and used . ( c ) dexterity was analysed on the basis of total path length of the left and right hand during the procedure . based on fusion algorithms , the information from a 3d accelerometer , gyroscope and magnetometer is used to measure the 3d rotation angles of each sensor in absolute space . noraxon myomotion inertial measurement unit sensors and display screen the motion sensors were attached to the following sites : foreheadthoracic spine - t1 ( first thoracic ) vertebralumbar spine - s2 ( second sacral ) levelmid - forearm - strapped on both left and right forearmshand - strapped around the dorsal aspect of the hand ( both left and right side ) . the data from the software , the movement of the cervical and thoracolumbar ( tl ) spine , are computed in all three dimensions , namely , sagittal , coronal and transverse planes and movement of the wrist in the sagittal and coronal planes . maximum excursion of the cervical and thoracolumbar spine maximum excursion of the wrist joint the rula scale is a tool which helps draw conclusion on ergonomic risks related to an occupational task . the positions of the upper arm , lower arm and wrist are grouped together which give out a score , that is , clubbed alongside the similar score formed by the neck , trunk and leg . based on the outcomes of the score , further investigations and ergonomic changes can be decided . ( b ) proficiency was tested on analysis of items such as the total blood loss during the procedure , a number of clips dropped and used . ( c ) dexterity was analysed on the basis of total path length of the left and right hand during the procedure . based on fusion algorithms , the information from a 3d accelerometer , gyroscope and magnetometer is used to measure the 3d rotation angles of each sensor in absolute space . noraxon myomotion inertial measurement unit sensors and display screen the motion sensors were attached to the following sites : foreheadthoracic spine - t1 ( first thoracic ) vertebralumbar spine - s2 ( second sacral ) levelmid - forearm - strapped on both left and right forearmshand - strapped around the dorsal aspect of the hand ( both left and right side ) . the data from the software , the movement of the cervical and thoracolumbar ( tl ) spine , are computed in all three dimensions , namely , sagittal , coronal and transverse planes and movement of the wrist in the sagittal and coronal planes . the rula scale is a tool which helps draw conclusion on ergonomic risks related to an occupational task . the positions of the upper arm , lower arm and wrist are grouped together which give out a score , that is , clubbed alongside the similar score formed by the neck , trunk and leg . based on the outcomes of the score , further investigations and ergonomic changes can be decided . the data of the maximum cervical and tl movement excursion [ table 2 ] , wrist movement excursion [ table 3 ] , rula scores [ table 4 ] and performance scores [ table 5 ] obtained during the simulated cholecystectomy was statistically compared between the novice and experienced surgeons . comparison of rapid upper limb assessment scores comparison of performance and dexterity scores during the simulated surgery , myomotion system - generated data of the cervical spine movements indicate that the maximum cervical flexion and the right side rotation were higher in novice whereas the right lateral flexion was higher in experienced surgeons . the results of the tl spine indicate that the novice showed more right tl rotation and the experienced surgeons showed more left tl rotation when maximum angles were compared . the results of motion analysis indicate the high left wrist flexion angles adopted by the surgeons , i.e. on comparison , there were no differences in the maximum angles of both left and right wjs between the experienced and novice surgeons . the result of the rula score indicates that there is a high postural risk during this surgical procedure . although the rula scores were higher in the novice surgeons , the difference was not statistically significant . for comparing path length , the performance score was higher in the experienced group ; however , the difference was not statistically significant . during the simulated surgery , myomotion system - generated data of the cervical spine movements indicate that the maximum cervical flexion and the right side rotation were higher in novice whereas the right lateral flexion was higher in experienced surgeons . the results of the tl spine indicate that the novice showed more right tl rotation and the experienced surgeons showed more left tl rotation when maximum angles were compared . the results of motion analysis indicate the high left wrist flexion angles adopted by the surgeons , i.e. on comparison , there were no differences in the maximum angles of both left and right wjs between the experienced and novice surgeons . the result of the rula score indicates that there is a high postural risk during this surgical procedure . although the rula scores were higher in the novice surgeons , the difference was not statistically significant . the performance score was higher in the experienced group ; however , the difference was not statistically significant . the primary objective of this study was to assess the ergonomics in the novice and experienced surgeons during a simulated laparoscopic cholecystectomy , and the secondary objective was to compare them for any differences in ergonomics . we studied the spinal and wrist postures during a simulated cholecystectomy using objective motion analysis . our study findings confirm that the surgeons head posture depends on the orientation of the monitor at the operation theatre as has been reported in other studies . in our study , most of the surgeons adopted neck extension posture during the simulated surgery , which they attributed to either screen height or habitual neck postures . the novice had higher maximum excursions into flexion and rotation which may be both due to limited visual cueing ( loss of 3d visual perspective and limited peripheral vision due to limited viewing spectrum ) and insufficient laparoscopic surgical experience . however , the reason for the higher maximum lateral flexion in the experienced surgeons remains inexplicable . overall , there was ergonomically acceptable upright tl posture adopted by the surgeons during the simulated surgery . the static trunk posture could be one of the reasons that increase the risk of musculoskeletal problems and pain in minimal access surgeons . when compared , there were no statistically significant differences in the maximum ranges between the experienced and novice surgeons except in tl rotation . their entire body was rotated to the left due to the inappropriate feet placement , due to which there was a compensatory right side neck and trunk rotation . although the tl spine was in near neutral postures , the positions adopted by the cervical spine scores increased the rula scores during the simulated surgery . the surgeons who scored high on the rula ( > 5 ) had adopted cervical extension along with rotation and lateral flexion . the surgeons who scored lower on the rula ( 3 or 4 ) had adopted either near neutral postures of the cervical spine or lesser degrees of flexion during the surgery . inappropriate monitor position , limited ergonomic awareness and habitual extension and lateral flexion postures adopted during the simulation surgery , culminated to the high neck and trunk rula scores in both groups . hence , there was no statistically significant difference between the groups on neck and trunk rula . these findings further strengthen the existing body of evidence which indicates that the monitor position can greatly influence the adoption of wrong postures of the neck . the static tl posture adds to the musculoskeletal risk at the spine in both novice and experienced surgeons . the typical trend observed in both experienced and novice surgeons at the left wrist was flexion > 45 with radial angular deviation . the right and left wrist awkward position while using ring - handled instrument during the simulated surgery the right upper extremity had to function to manoeuvre the different instruments ( maryland dissector , clip applicator , endoscopic shear , l - hook electrocautery and suction ) for various tasks of the simulated surgery . in our study , the maximum right wrist flexion was above 50 in both experienced and novice surgeons . there were periodic changes in position of the right wrist in both sagittal and frontal plane while performing tasks such as dissection , clipping and cauterisation . no surgeon in both experienced and novice group adopted a neutral wrist posture . when we compared maximum right and left wrist excursions , there was no significant difference between the experienced and novice surgeons . there is a reduction in grip strength as well as an increase in the carpal tunnel pressure with wrist in flexed posture . had revealed that the hydrostatic pressure in the carpal tunnel was found to be affected by both wrist posture and tendon load . correspondingly , the local contact forces by the digital flexors indicated the highest loads on the median nerve with the wrist flexed . anteriorly positioned nerves were flattened in the anteroposterior plane between the tendon and flexor retinaculum ; this was greatest with flexion and least with extension . hence , in light of the aforementioned reasons , the high wrist flexion angles occurring during laparoscopic surgeries should be reduced by novel ergonomic handle designs . have reported that at the upper extremity , there are more wrist supination and wrist ud and rd during a laparoscopic procedure as compared to open surgery . since the accelerometer sensor used in this study is attached to the forearm and hand , even with a change in upper limb position , the angular movements are recorded with sufficient precision at the wrist . furthermore , rd can cause a reduction in the grip force and add to the mechanical inefficiency . , 4 , for majority of the surgeons ( 25 on the left and 29 on the right side ) , with few surgeons ( 7 surgeons on the left and 3 on the right side ) scoring 3 . the rula score results of their study suggest that all study surgeons adopt an inadequate position of the wrist associated with a marked hyperflexion of this joint . they computed the rula scores for the risk based on sagittal plane deviations ( i.e. the surgeons in our study performed the simulated surgical procedure with ring - handled instrument handles in both hands . in biomechanical terms , these elongated instruments increase the leverage of the load arm leading to greater angles of wrist deviations in the sagittal and coronal plane . previous research indicates that ring handled instruments are less preferred due to the awkward wrist position imposed by them , when compared to axial handled instruments . compared the muscle tension ( surface electromyography ) caused by two different grasping modalities during the use of ring - handled laparoscopic scissors , showing that it is beneficial to use larger palm support instead of introducing the thumb through the rings of the handle . in our study , we observed that all the surgeons placed their fingers through the rings in the scissors handle contrary to berguer 's recommendations , with the exception of one surgeon who used palmar grasp on the left hand to hold the ring - handled instrument . in comparison to previous research , we did not find any statistically significant difference in performance measures between the experienced and novice surgeons [ table 5 ] . the probable reason for this could be because basic level surgery was chosen for comparison ; as well , to the goal - oriented task , nature of the surgical task could have led to greater motivation for task completion in the novice surgeons . our study findings confirm that the surgeons head posture depends on the orientation of the monitor at the operation theatre as has been reported in other studies . in our study , most of the surgeons adopted neck extension posture during the simulated surgery , which they attributed to either screen height or habitual neck postures . the novice had higher maximum excursions into flexion and rotation which may be both due to limited visual cueing ( loss of 3d visual perspective and limited peripheral vision due to limited viewing spectrum ) and insufficient laparoscopic surgical experience . however , the reason for the higher maximum lateral flexion in the experienced surgeons remains inexplicable . overall , there was ergonomically acceptable upright tl posture adopted by the surgeons during the simulated surgery . the static trunk posture could be one of the reasons that increase the risk of musculoskeletal problems and pain in minimal access surgeons . when compared , there were no statistically significant differences in the maximum ranges between the experienced and novice surgeons except in tl rotation . their entire body was rotated to the left due to the inappropriate feet placement , due to which there was a compensatory right side neck and trunk rotation . although the tl spine was in near neutral postures , the positions adopted by the cervical spine scores increased the rula scores during the simulated surgery . the surgeons who scored high on the rula ( > 5 ) had adopted cervical extension along with rotation and lateral flexion . the surgeons who scored lower on the rula ( 3 or 4 ) had adopted either near neutral postures of the cervical spine or lesser degrees of flexion during the surgery . inappropriate monitor position , limited ergonomic awareness and habitual extension and lateral flexion postures adopted during the simulation surgery , culminated to the high neck and trunk rula scores in both groups . hence , there was no statistically significant difference between the groups on neck and trunk rula . these findings further strengthen the existing body of evidence which indicates that the monitor position can greatly influence the adoption of wrong postures of the neck . the static tl posture adds to the musculoskeletal risk at the spine in both novice and experienced surgeons . the typical trend observed in both experienced and novice surgeons at the left wrist was flexion > 45 with radial angular deviation . the right and left wrist awkward position while using ring - handled instrument during the simulated surgery the right upper extremity had to function to manoeuvre the different instruments ( maryland dissector , clip applicator , endoscopic shear , l - hook electrocautery and suction ) for various tasks of the simulated surgery . in our study , the maximum right wrist flexion was above 50 in both experienced and novice surgeons . there were periodic changes in position of the right wrist in both sagittal and frontal plane while performing tasks such as dissection , clipping and cauterisation . no surgeon in both experienced and novice group adopted a neutral wrist posture . when we compared maximum right and left wrist excursions , there was no significant difference between the experienced and novice surgeons . there is a reduction in grip strength as well as an increase in the carpal tunnel pressure with wrist in flexed posture . had revealed that the hydrostatic pressure in the carpal tunnel was found to be affected by both wrist posture and tendon load . correspondingly , the local contact forces by the digital flexors indicated the highest loads on the median nerve with the wrist flexed . anteriorly positioned nerves were flattened in the anteroposterior plane between the tendon and flexor retinaculum ; this was greatest with flexion and least with extension . hence , in light of the aforementioned reasons , the high wrist flexion angles occurring during laparoscopic surgeries should be reduced by novel ergonomic handle designs . since the accelerometer sensor used in this study is attached to the forearm and hand , even with a change in upper limb position , the angular movements are recorded with sufficient precision at the wrist . furthermore , rd can cause a reduction in the grip force and add to the mechanical inefficiency . , 4 , for majority of the surgeons ( 25 on the left and 29 on the right side ) , with few surgeons ( 7 surgeons on the left and 3 on the right side ) scoring 3 . the rula score results of their study suggest that all study surgeons adopt an inadequate position of the wrist associated with a marked hyperflexion of this joint . they computed the rula scores for the risk based on sagittal plane deviations ( i.e. the surgeons in our study performed the simulated surgical procedure with ring - handled instrument handles in both hands . in biomechanical terms , these elongated instruments increase the leverage of the load arm leading to greater angles of wrist deviations in the sagittal and coronal plane . previous research indicates that ring handled instruments are less preferred due to the awkward wrist position imposed by them , when compared to axial handled instruments . compared the muscle tension ( surface electromyography ) caused by two different grasping modalities during the use of ring - handled laparoscopic scissors , showing that it is beneficial to use larger palm support instead of introducing the thumb through the rings of the handle . in our study , we observed that all the surgeons placed their fingers through the rings in the scissors handle contrary to berguer 's recommendations , with the exception of one surgeon who used palmar grasp on the left hand to hold the ring - handled instrument . in comparison to previous research , we did not find any statistically significant difference in performance measures between the experienced and novice surgeons [ table 5 ] . the probable reason for this could be because basic level surgery was chosen for comparison ; as well , to the goal - oriented task , nature of the surgical task could have led to greater motivation for task completion in the novice surgeons . an objective analysis of the wrist shows that both expert and novice surgeons adopt disadvantageous postures of both right and left wrists while using axial - handled instruments on the simulator . the spinal analysis indicates that the monitor position greatly influences the neck posture , and predominantly static postures are adopted at the tl spine . we conclude that ergonomic inadequacies can lead to the physical postural risk , during the minimally invasive surgical procedure in both experienced and novice surgeons .

there are four major groups of autoimmune blistering diseases , including pemphigus ( pemphigus vulgaris pemphigus foliaceus [ pf ] , pemphigus erythematosus , paraneoplastic pemphigus , immunoglobulin a [ iga ] pemphigus ) , pemphigoids ( bullous pemphigoid [ bp ] , pemphigoid gestationis , mucous membrane pemphigoid [ mmp ] , linear iga disease ) , epidermolysis bullosa acquisita ( eba ) , and dermatitis herpetiformis . within the autoimmune blistering diseases , autoantibodies play a critical role in destruction of skin in different ways , including loss of cell cell adhesion , dermo - epithelial dysadhesion , or mixed form of them . autoantibody against desmoglein 1 ( dsg1 ) , dsg3 , bp180 , and bp230 is the most important player in the majority of autoimmune blistering diseases . in the mmp , bp180 , laminin-332 , and 64 integrin mmp , which also known as cicatricial pemphigoid ( cp ) , is characterized by subepithelial bullae , less commonly on the skin , and more associated with mucous membranes . this disease is predominant in females , and it usually occurs in individuals with older age , between 60 and 80 years old . however , various studies reported childhood mmp . different subclasses of igg and iga autoantibodies , especially igg1 and igg4 subclasses , are mainly responsible for mmp development . , different parts of mucosal membranes including oral , nasal , ocular , laryngeal , esophageal , and anogenital could be damaged . between patients with mmp , scarring of the mmp is also common among patients , which may result in severe life - threatening sequelae . when the cornea of the eye is affected , repeated scarring may result in blindness . similar to other autoimmune diseases , mmp severity is mainly controlled with corticosteroids and different immunosuppressant agents . recently , using of biological agents has been discussed by different authors . in this study , it was tried to analyze the efficiency and the safety of intravenous ig ( ivig ) in patients with mmp . a systematic literature searching for all the published articles associated with the use of ivig and mmp which was conducted by databases of pubmed and google scholar was performed . all the associated studies until september 2015 were considered , using the keywords such as cicatricial pemphigoid or ocular pemphigoid or mucous membrane pemphigoid or mmp and intravenous immunoglobulin or ivig to find all the relevant studies . among the searched items , it is worthy of note that although the combination therapy rituximab with ivig was included in the study , it was not considered as the study associated with the role of ivig in the treatment of mmp . all the extracted data including year of publishing , number of patients , their age and sex , the dose of administrated ivig , response time , outcome , and ivig - related side effects were categorized and then entered into a database . in addition , all the data were rechecked after preparing the database . various therapeutic strategies are available for treatment of patients with mmp , but not all the patients respond to those treatments . choice of appropriate treatment depends on several factors including site involved , severity of disease , and its progression . topical and systemic treatments are usually used for mild and severe mmp , respectively . in patients with more severe and progressive diseases , a combination of both topical and systemic treatment may be required to control disease progression . overall , systemic corticosteroids , adjuvant immunosuppressive therapy , antibiotics , biologic agents such as rituximab and tumor necrosis factor- ( tnf- ) inhibitors , and immunomodulatory procedures such as ivig are the main therapeutic agents used in treating of mmp . excellent oral care has been emphasized as being an important part of the treatment of mmp . systemic corticosteroids which are the first - line treatment in severe mmp ( ocular mmp , esophageal , laryngeal , severe gingival , and/or severe anogenital ) usually response rapidly once treatment is initiated . however , several adverse effects are associated to this type of treatment . in more severe cases , using of immunosuppressive adjuvant therapies including azathioprine ( aza ) , mycophenolate mofetil ( mmf ) , cyclophosphamide ( ctx ) , and methotrexate ( mtx ) could help to better control of disease . in addition , a combination of these therapies may be effective for induction of remission the disease . in rare cases , surgical treatment is necessary if scarring narrows the airway and breathing becomes difficult or in some cases with ocular cp ( ocp ) . in patients with ocp , eye surgery must be undertaken with care because it may lead to the disease reactivation . mild mmp that is limited to oral cavity cloud be controlled by topical treatments alone . however , in those with severe mmf , the systemic therapies may be essential . prednisone is the most common first - line treatment which usually is given at a dose of 11.5 mg / kg / day while this treatment is the major source of side effects . ctx is another treatment in mmp , which could be used in more severe cases or those with rapid progression . it is an alkylating agent that suppresses b - lymphocytes ( b - cells ) function greater than t - lymphocytes ( t - cells ) function , which could result in several side effects . in cases with mild mmp and without rapid progression , dosage ranges from 1 to 4 mg / kg / day , whereas it is recommended to be used at the dose of 12 mg / kg / day that could be increased to 5 mg / kg / day . mtx is another used therapy for patients with mmp , which was reported in few studies . in addition in addition to conventional treatments , some biologic agents including rituximab , etanercept , infliximab , and immunomodulatory agents , such as ivig , have been reported to be effective in the treatment mmp . this caused the emergence the idea to inhibit this cytokine with different agents ( etanercept , infliximab ) . anti - tnf therapy is a common treatment in rheumatoid arthritis ( ra ) which is considered an autoimmune disorder . rituximab is a chimeric , monoclonal antibody that binds to the molecule cd20 expressed on the cell surface of b - cells . first , it was approved by the us food and drug administration ( fda ) for the treatment of non - hodgkin 's lymphoma in 1997 . subsequently , its usage was extended to treatment of various off - label diseases . during recent years , rituximab is increasingly used in autoimmune blister diseases . ivig therapy considered as another option for management of mmp patients with rapidly disease progression or those who did not respond to previous treatments . it is not the first - line therapy but could be used as the adjuvant therapy in patients with mentioned conditions . over the last decade ivig is a biologic immune modulatory agent , composed of polyclonal antibodies , derived from the plasma of a large pool of healthy donors . it was approved by the fda for use in immune thrombocytopenic purpura , primary immunodeficiency , secondary immunodeficiency , pediatric human immunodeficiency virus ( hiv ) infection , kawasaki disease , prevention of graft versus host disease , and infection in bone marrow transplant recipients . in addition to these approved conditions , it is increasingly used for various off - label autoimmune disorders including pemphigus , pemphigoid , systemic lupus erythematosus ( sle ) , ra , and multiple sclerosis ( ms ) . in autoimmune diseases , however , it is generally accepted that ivig therapy should be limited to patients who fail conventional therapy , demonstrate serious side effects , contraindications to conventional therapy , or patients with rapidly progressive disease . despite the several case reports and case series in efficiency of ivig therapy for treatment of autoimmune diseases , it remained a controversial topic . this type of therapy may increase the risk of infection due to suppression of immune system . for instance , in hepatitis b virus or hepatitis c virus carriers who are at risk of reactivation of infection during or after immunosuppressant , adding ivig to treatment protocol may be effective in both controlling autoimmune disease and reduction of reactivation risk . considering that the ivig is not immunosuppressive and has a favorable side effect profile in compression to immunosuppressive therapy , it could be an appropriate treatment choice for patients who are at risk of infections or viral reactivation . sometimes , ivig is used at a replacement dose of 200400 mg / kg body weight . however , high - dose ivig ( hdivig ) is most frequently at 2 g / kg , which is given monthly for major autoimmune and inflammatory disorders . the precise mechanism by which ivig functions as an anti - inflammatory agent remains unclear . it provides the large amounts of immunoregulatory substances which have the capacity to regulate the immune system in different ways . ivig can act by potential actions including , anti - idiotype antibody production , competitive inhibition of binding to activating fc - receptors ( fcrs ) , upregulation of inhibitory fcrs , increase clearance of autoantibodies by reticuloendothelial system , decreased half - life of autoantibodies due to competitive binding to fcrs , interference with the activation of complement and the cytokine network , and t - cell modulation . in overall , however , there are several reports of ivig adverse effects which vary in a wide range . different factors including age , type of disease , dose of ivig , and rate of the infusion play role in severity of adverse effects due to ivig therapy . the first group of adverse effects includes headache , flushing of the face , malaise , chest tightness , fever , chills , myalgia , fatigue , dyspnea , back pain , nausea , vomiting , diarrhea , change in blood pressure , and tachycardia . the most common adverse reaction related to ivig therapy is an infusion reaction , and symptoms may include flushing , headache , chills , nausea , tachycardia , hypotension , and wheezing . ivig is used in various patients with mmp for different reasons . in 1997 , urcelay et al . reported two cases who had successful treatment with ivig therapy . in the first case , a 50-year - old woman was diagnosed at mmp in 1992 ; betamethasone and cyclosporine did not control the disease completely . in addition , dapsone and aza were not tolerated . moreover , tetracycline and nicotinamide were unhelpful . thus , ivig was initiated at a dose of 2 g / kg / cycle in 1995 . it caused an improvement , and the patient was lesion - free after six cycles . as the second patient on the same study , a 40-year - old man was diagnosed at mmp in 1995 . after treatment with dapsone and betamethasone , ivig was started with dosage of 2 g / kg / cycle . however , it was stopped due to development of urticarial in the seventh infusion . in 1999 , foster and ahmed reported ten cases that all of them were more than 50 years old . duration of systemic therapy before initiation of ivig varied from 3 to 14 years , with a mean of 8.3 years . various treatments were used , but all of them were gradually stopped due to different reasons including side effects , ineffectiveness , and lack of response . thereby , ivig was initiated at the dosage of 23 g / kg / cycle , which led to fully control of the disease , during a minimum of 4 months . the next successful ivig therapy in mmp patients was reported by ahmed and coln , who have analyzed the clinical outcomes as well as the disease progression in those with oral pemphigoid in a retrospective study in 2001 . on that study , eight patients ( containing six females and two males aged 4367 years ) who compared to 12 patients treated without using ivig therapy were contributed on ivig therapy . the dose of ivig for patients in the first group was varied between 1 and 2 g / kg per cycle , which was divided into three doses for infusion during three consecutive days . pemphigoid disease in all the patients , in both groups , was limited to oral cavity at the time of enrolling in the study . average duration of treatment in the first group was considerably shorter than second ( 32.9 vs. 41.8 months ) . in addition , it resulted in lower average number of side effects ( 0.4 vs. 3.2 ) , significantly lower number of relapse rate ( 0.1 vs. 2.1 ) , higher remission rate ( 1 vs. 0.4 ) , and considerable higher quality of live ( lower than the poor index vs. higher than tolerant of reasonable index ) . interestingly , among the patients in the first group , no one developed oral pemphigoid in any other mucous membrane . in contrast , more than half of patients in the second group were developed oral pemphigoid at extraoral sites . in issue of adverse effect however , in the conventional therapy group , all the 12 patients experienced various side effects . reported a successful disease control in a 74-year - old female patient who was newly diagnosed with pemphigoid . moreover , she did not respond to mmf and high - doses of corticosteroids reasonably . indeed , despite the reduction in disease severity , lesions persisted . in addition , she experienced widespread erosions on the gingiva , marked conjunctivitis , and rapid scarring of the left eye after 6 months . this led to initiation of ivig ( 1 g / kg / cycle ) to control disease activity . a total of six cycles each 4 weeks were administrated which resulted in remission of disease for more than 12 months . sami et al . presented the ivig therapy in 15 patients with severe mmp who were nonresponsive to the conventional treatments or developed multiple side effects . all other conventional agents were also discontinued , and ivig was eventually used as the monotherapy . in the absence of any treatments , all patients experienced remission for a period ranging from 12 to 72 months ( mean , 23.9 ) . average month with side effects decreased considerably ( 5.8 vs. 0.6 ) ; relapse rate average decreased ( 7.33 vs. 1.47 ) , and remission rate was increased ( 0 vs. 1 ) after initiation of ivig . furthermore , quality of life increased markedly ( near unsatisfactory vs. almost high quality ) . with reports that confirmed effectiveness of ivig therapy in mmp patients , more patients were treated with this immunomodulatory agent . in 2004 , letko et al . compared the clinical outcomes of ivig therapy to conventional immunosuppressive therapy in patients with mmp , whose disease progressed to involve the eye . ivig and conventional therapy groups were clinically remitted within 4 and 8.5 months , respectively . however , the recurrence of the disease was observed in five patients in the other group . thus , it led to a decreasing in the rate of infusions which resulted in the resolution of those side effects . however , all the patients in the conventional therapy group demonstrated side effects . as the final outcome , all the patients who were treated with ivig had been controlled completely . conversely , in the other group , only three patients were completely controlled ; three were partially controlled , and two remained uncontrolled . in the same year , a case series included ten patients with a diagnosis of ocp , reported successfully using of ivig in patients with mmp . in eight patients who completed treatment , a sustained clinical remission was observed . in contrast , other two patients were excluded from the treatment protocol , which resulted in progression of ocp . on that study , the total number of cycles and duration of ivig therapy reported ranged from 20 to 42 ( mean , 32 ) and from 25 to 43 months ( mean , 35 ) , respectively . the next report was published by segura et al . , which contains four cases with mmp ( three females and one male ) . one of them did not respond to ivig therapy while another one had a complete response . the ivig therapy was stopped due to toxicity on patients with partial remission . however , other two patients did not experience any adverse effect . in 2008 , mignogna et al . reported a total of six patients with severe mmp , containing three males and three females aged 5880 years ( mean , 69.5 ) . in all of them , gingival , buccal , and palate lesions were the initial signs of mmp , followed by chronic conjunctivitis and later extended to conjunctival shrinkage . within the average of 9.1 months , an actual clinical remission ( healing of previous oral lesions and resolution of ocular scarring without development new lesion ) was achieved . patients remained on systemic steroids and immunosuppressants that were gradually reduced by 50% in the first 3 months of ivig therapy and further 20% during the last cycles of maintenance therapy . a case report of treatment refractory ocp with ivig was published in 2008 . on that study , a 65-year - old man clinically diagnosed as having cryptogenic organizing pneumonia . initial treatment included methylprednisolone , prednisone , and aza that were unable to control disease progression . thus , immunosuppressive treatment with ctx was initiated while inflammation could not be brought under control . to control disease progression , after initiation ivig , a rapid improvement was reported , which led to controlling of inflammation in both eyes after seven cycles . despite the hdivig and relatively short time of repetition , no side effect was observed . according to those results , it was suggested that ivig therapy could be more effective than conventional immunosuppression treatment for controlling inflammation and avoiding disease progression . in 2009 , a study of ten patients with autoimmune mucocutaneous blistering diseases , including seven pemphigus vulgaris ( pv ) and three mmp patients ( two females and one male aged 4276 [ mean , 66 ] ) , confirmed the efficiency of ivig therapy at the dose of 2 g / kg in controlling of mmp patients . subsequently , foster et al . analyzed the efficiency of combination of rituximab with ivig compared to conventional therapy all the patients in the first group who were treated using conventional therapy were blind in one eye . four in the second group who were treated with the combination of rituximab and ivig therapy were also blind in one of their eyes . completion of the treatment protocol in the second group ranged from 3 to 19 months ( mean , 11 ) . as the result of that study , in contrast , no one in the second group experienced new blindness , in addition to stopping disease progression . barbosa ldo et al . reported a 75-year - old female with mmp who went into remission following ivig infusion at the dose of 1.5 g / kg . recently , a case with oral mmp was reported . despite the starting oral prednisolone and keeping for 6 months , no improvement was observed . after revealing that dapsone can not help , ivig was administrated at a dose of 2 g / kg / cycle which caused complete remission . with maintaining ivig therapy for 6 months the effectivness , safety , and associated factors of intravenous immunoglobulin treatment in mucous membrane pemphigoid , based on reviewed studies various therapeutic strategies are available for treatment of patients with mmp , but not all the patients respond to those treatments . choice of appropriate treatment depends on several factors including site involved , severity of disease , and its progression . topical and systemic treatments are usually used for mild and severe mmp , respectively . in patients with more severe and progressive diseases , a combination of both topical and systemic treatment may be required to control disease progression . overall , systemic corticosteroids , adjuvant immunosuppressive therapy , antibiotics , biologic agents such as rituximab and tumor necrosis factor- ( tnf- ) inhibitors , and immunomodulatory procedures such as ivig are the main therapeutic agents used in treating of mmp . excellent oral care has been emphasized as being an important part of the treatment of mmp . systemic corticosteroids which are the first - line treatment in severe mmp ( ocular mmp , esophageal , laryngeal , severe gingival , and/or severe anogenital ) usually response rapidly once treatment is initiated . however , several adverse effects are associated to this type of treatment . in more severe cases , using of immunosuppressive adjuvant therapies including azathioprine ( aza ) , mycophenolate mofetil ( mmf ) , cyclophosphamide ( ctx ) , and methotrexate ( mtx ) could help to better control of disease . in addition , a combination of these therapies may be effective for induction of remission the disease . in rare cases , surgical treatment is necessary if scarring narrows the airway and breathing becomes difficult or in some cases with ocular cp ( ocp ) . in patients with ocp , eye surgery must be undertaken with care because it may lead to the disease reactivation . mild mmp that is limited to oral cavity cloud be controlled by topical treatments alone . however , in those with severe mmf , the systemic therapies may be essential . prednisone is the most common first - line treatment which usually is given at a dose of 11.5 mg / kg / day while this treatment is the major source of side effects . ctx is another treatment in mmp , which could be used in more severe cases or those with rapid progression . it is an alkylating agent that suppresses b - lymphocytes ( b - cells ) function greater than t - lymphocytes ( t - cells ) function , which could result in several side effects . in cases with mild mmp and without rapid progression dosage ranges from 1 to 4 mg / kg / day , whereas it is recommended to be used at the dose of 12 mg / kg / day that could be increased to 5 mg / kg / day . mtx is another used therapy for patients with mmp , which was reported in few studies . in addition , there is much evidence that a combination of these treatments may be helpful . in addition to conventional treatments , some biologic agents including rituximab , etanercept , infliximab , and immunomodulatory agents , such as ivig , have been reported to be effective in the treatment mmp . this caused the emergence the idea to inhibit this cytokine with different agents ( etanercept , infliximab ) . anti - tnf therapy is a common treatment in rheumatoid arthritis ( ra ) which is considered an autoimmune disorder . rituximab is a chimeric , monoclonal antibody that binds to the molecule cd20 expressed on the cell surface of b - cells . first , it was approved by the us food and drug administration ( fda ) for the treatment of non - hodgkin 's lymphoma in 1997 . subsequently , its usage was extended to treatment of various off - label diseases . during recent years , rituximab is increasingly used in autoimmune blister diseases . ivig therapy considered as another option for management of mmp patients with rapidly disease progression or those who did not respond to previous treatments . it is not the first - line therapy but could be used as the adjuvant therapy in patients with mentioned conditions . over the last decade ivig is a biologic immune modulatory agent , composed of polyclonal antibodies , derived from the plasma of a large pool of healthy donors . it was approved by the fda for use in immune thrombocytopenic purpura , primary immunodeficiency , secondary immunodeficiency , pediatric human immunodeficiency virus ( hiv ) infection , kawasaki disease , prevention of graft versus host disease , and infection in bone marrow transplant recipients . in addition to these approved conditions , it is increasingly used for various off - label autoimmune disorders including pemphigus , pemphigoid , systemic lupus erythematosus ( sle ) , ra , and multiple sclerosis ( ms ) . in autoimmune diseases , however , it is generally accepted that ivig therapy should be limited to patients who fail conventional therapy , demonstrate serious side effects , contraindications to conventional therapy , or patients with rapidly progressive disease . despite the several case reports and case series in efficiency of ivig therapy for treatment of autoimmune diseases , it remained a controversial topic . this type of therapy may increase the risk of infection due to suppression of immune system . for instance , in hepatitis b virus or hepatitis c virus carriers who are at risk of reactivation of infection during or after immunosuppressant , adding ivig to treatment protocol may be effective in both controlling autoimmune disease and reduction of reactivation risk . considering that the ivig is not immunosuppressive and has a favorable side effect profile in compression to immunosuppressive therapy , it could be an appropriate treatment choice for patients who are at risk of infections or viral reactivation . sometimes , ivig is used at a replacement dose of 200400 mg / kg body weight . however , high - dose ivig ( hdivig ) is most frequently at 2 g / kg , which is given monthly for major autoimmune and inflammatory disorders . the precise mechanism by which ivig functions as an anti - inflammatory agent remains unclear . it provides the large amounts of immunoregulatory substances which have the capacity to regulate the immune system in different ways . ivig can act by potential actions including , anti - idiotype antibody production , competitive inhibition of binding to activating fc - receptors ( fcrs ) , upregulation of inhibitory fcrs , increase clearance of autoantibodies by reticuloendothelial system , decreased half - life of autoantibodies due to competitive binding to fcrs , interference with the activation of complement and the cytokine network , and t - cell modulation . in overall , however , there are several reports of ivig adverse effects which vary in a wide range . different factors including age , type of disease , dose of ivig , and rate of the infusion play role in severity of adverse effects due to ivig therapy . some of them could be appeared during or immediately after infusion . in contrast , some others arise with a delay . the first group of adverse effects includes headache , flushing of the face , malaise , chest tightness , fever , chills , myalgia , fatigue , dyspnea , back pain , nausea , vomiting , diarrhea , change in blood pressure , and tachycardia . the most common adverse reaction related to ivig therapy is an infusion reaction , and symptoms may include flushing , headache , chills , nausea , tachycardia , hypotension , and wheezing . ivig is used in various patients with mmp for different reasons . in 1997 , urcelay et al . reported two cases who had successful treatment with ivig therapy . in the first case , a 50-year - old woman was diagnosed at mmp in 1992 ; betamethasone and cyclosporine did not control the disease completely . in addition , dapsone and aza were not tolerated . moreover , tetracycline and nicotinamide were unhelpful . thus , ivig was initiated at a dose of 2 g / kg / cycle in 1995 . it caused an improvement , and the patient was lesion - free after six cycles . as the second patient on the same study , a 40-year - old man was diagnosed at mmp in 1995 . after treatment with dapsone and betamethasone , ivig was started with dosage of 2 g / kg / cycle . however , it was stopped due to development of urticarial in the seventh infusion . in 1999 , foster and ahmed reported ten cases that all of them were more than 50 years old . duration of systemic therapy before initiation of ivig varied from 3 to 14 years , with a mean of 8.3 years . various treatments were used , but all of them were gradually stopped due to different reasons including side effects , ineffectiveness , and lack of response . thereby , ivig was initiated at the dosage of 23 g / kg / cycle , which led to fully control of the disease , during a minimum of 4 months . the next successful ivig therapy in mmp patients was reported by ahmed and coln , who have analyzed the clinical outcomes as well as the disease progression in those with oral pemphigoid in a retrospective study in 2001 . on that study , eight patients ( containing six females and two males aged 4367 years ) who compared to 12 patients treated without using ivig therapy were contributed on ivig therapy . the dose of ivig for patients in the first group was varied between 1 and 2 g / kg per cycle , which was divided into three doses for infusion during three consecutive days . pemphigoid disease in all the patients , in both groups , was limited to oral cavity at the time of enrolling in the study . average duration of treatment in the first group was considerably shorter than second ( 32.9 vs. 41.8 months ) . in addition , it resulted in lower average number of side effects ( 0.4 vs. 3.2 ) , significantly lower number of relapse rate ( 0.1 vs. 2.1 ) , higher remission rate ( 1 vs. 0.4 ) , and considerable higher quality of live ( lower than the poor index vs. higher than tolerant of reasonable index ) . interestingly , among the patients in the first group , no one developed oral pemphigoid in any other mucous membrane . in contrast , more than half of patients in the second group were developed oral pemphigoid at extraoral sites . in issue of adverse effect however , in the conventional therapy group , all the 12 patients experienced various side effects . reported a successful disease control in a 74-year - old female patient who was newly diagnosed with pemphigoid . on that patient , moreover , she did not respond to mmf and high - doses of corticosteroids reasonably . indeed , despite the reduction in disease severity , lesions persisted . in addition , she experienced widespread erosions on the gingiva , marked conjunctivitis , and rapid scarring of the left eye after 6 months . this led to initiation of ivig ( 1 g / kg / cycle ) to control disease activity . a total of six cycles each 4 weeks were administrated which resulted in remission of disease for more than 12 months . sami et al . presented the ivig therapy in 15 patients with severe mmp who were nonresponsive to the conventional treatments or developed multiple side effects . all 15 study patients were treated with an ivig dose of 12 g / kg / cycle which was divided into three equal doses . all other conventional agents were also discontinued , and ivig was eventually used as the monotherapy . in the absence of any treatments , all patients experienced remission for a period ranging from 12 to 72 months ( mean , 23.9 ) . average month with side effects decreased considerably ( 5.8 vs. 0.6 ) ; relapse rate average decreased ( 7.33 vs. 1.47 ) , and remission rate was increased ( 0 vs. 1 ) after initiation of ivig . furthermore , quality of life increased markedly ( near unsatisfactory vs. almost high quality ) . with reports that confirmed effectiveness of ivig therapy in mmp patients , more patients were treated with this immunomodulatory agent . in 2004 , letko et al . compared the clinical outcomes of ivig therapy to conventional immunosuppressive therapy in patients with mmp , whose disease progressed to involve the eye . ivig and conventional therapy groups were clinically remitted within 4 and 8.5 months , respectively . however , the recurrence of the disease was observed in five patients in the other group . thus , it led to a decreasing in the rate of infusions which resulted in the resolution of those side effects . however , all the patients in the conventional therapy group demonstrated side effects . as the final outcome , all the patients who were treated with ivig had been controlled completely . conversely , in the other group , only three patients were completely controlled ; three were partially controlled , and two remained uncontrolled . in the same year , a case series included ten patients with a diagnosis of ocp , reported successfully using of ivig in patients with mmp . in eight patients who completed treatment , a sustained clinical remission was observed . in contrast , other two patients were excluded from the treatment protocol , which resulted in progression of ocp . on that study , the total number of cycles and duration of ivig therapy reported ranged from 20 to 42 ( mean , 32 ) and from 25 to 43 months ( mean , 35 ) , respectively . one of them did not respond to ivig therapy while another one had a complete response . the ivig therapy was stopped due to toxicity on patients with partial remission . however , other two patients did not experience any adverse effect . in 2008 , mignogna et al . reported a total of six patients with severe mmp , containing three males and three females aged 5880 years ( mean , 69.5 ) . in all of them , gingival , buccal , and palate lesions were the initial signs of mmp , followed by chronic conjunctivitis and later extended to conjunctival shrinkage . within the average of 9.1 months , an actual clinical remission ( healing of previous oral lesions and resolution of ocular scarring without development new lesion ) patients remained on systemic steroids and immunosuppressants that were gradually reduced by 50% in the first 3 months of ivig therapy and further 20% during the last cycles of maintenance therapy . a case report of treatment refractory ocp with ivig was published in 2008 . on that study , a 65-year - old man clinically diagnosed as having cryptogenic organizing pneumonia . initial treatment included methylprednisolone , prednisone , and aza that were unable to control disease progression . thus , immunosuppressive treatment with ctx was initiated while inflammation could not be brought under control . to control disease progression , ivig was planned to be administrated at a total dose of 3 g / kg / cycle which was repeated every 2 weeks . after initiation ivig , a rapid improvement was reported , which led to controlling of inflammation in both eyes after seven cycles . despite the hdivig and relatively short time of repetition , no side effect was observed . according to those results , it was suggested that ivig therapy could be more effective than conventional immunosuppression treatment for controlling inflammation and avoiding disease progression . in 2009 , a study of ten patients with autoimmune mucocutaneous blistering diseases , including seven pemphigus vulgaris ( pv ) and three mmp patients ( two females and one male aged 4276 [ mean , 66 ] ) , confirmed the efficiency of ivig therapy at the dose of 2 g / kg in controlling of mmp patients . subsequently , foster et al . analyzed the efficiency of combination of rituximab with ivig compared to conventional therapy all the patients in the first group who were treated using conventional therapy were blind in one eye . four in the second group who were treated with the combination of rituximab and ivig therapy were also blind in one of their eyes . completion of the treatment protocol in the second group ranged from 3 to 19 months ( mean , 11 ) . as the result of that study , in contrast , no one in the second group experienced new blindness , in addition to stopping disease progression . barbosa ldo et al . reported a 75-year - old female with mmp who went into remission following ivig infusion at the dose of 1.5 g / kg . recently , a case with oral mmp was reported . despite the starting oral prednisolone and keeping for 6 months , no improvement was observed . after revealing that dapsone can not help , ivig was administrated at a dose of 2 g / kg / cycle which caused complete remission . with maintaining ivig therapy for 6 months the effectivness , safety , and associated factors of intravenous immunoglobulin treatment in mucous membrane pemphigoid , based on reviewed studies in addition to conventional therapies , some other treatments are used to treat various autoimmune diseases . some of them act through the relatively clear signaling pathways , such as several biological agents . for example , targeting a certain cytokine , molecule , or cell could be categorized in the first group . recently , tavakolpour suggested that targeting the interleukin ( il)-4 could be a possible treatment in pemphigus . thereby , dupilumab , an anti - il-4 receptor alpha monoclonal antibody , was introduced as an effective drug to treat those with pemphigus . in contrast to these types of treatments , ivig does not block or induce a certain signaling pathway . ivig is used at replacement dose to treat patients with primary antibody deficiencies while hdivig is used as an immunomodulatory agent in a wide range of autoimmune diseases . it also used for inflammatory disorders and bacterial and viral infections that could not be controlled by conventional therapy . there are several reports of using successfully ivig therapy in different autoimmune diseases or diseases that caused by vigorous and uncontrolled immune responses , including ms , sle , ra , autoimmune hemolytic anemia , asthma , pv , pf , eba , bp , and mmp . in contrast , some other reports are available that did not confirm effectiveness of ivig therapy in various autoimmune diseases . table 2 summarizes the reported outcomes of ivig therapy in some inflammatory and autoimmune diseases . the effectiveness of intravenous immunoglobulin treatment in selected inflammatory and autoimmune diseases ivig therapy can cause prevention or even treating of infection . the use of ivig in children with hiv infection and low peripheral cd4 t - cell count was approved by the fda early in the 1990s . in addition , a study demonstrated the possible role of hdivig therapy in adults with hiv infection . ivig reduced the incidence of cytomegalovirus infection and interstitial pneumonia in allogeneic bone marrow transplant recipients in the era before ganciclovir . furthermore , there are several proofs that it can be helpful in patients with some other infections such as respiratory syncytial virus . thus , it may be suitable treatment in a patient with those infections who need to immunosuppressant for discussed autoimmune diseases . ivig may help to suppress high viral replication due to immunosuppressant , in addition to controlling the autoimmune disease in several pathways . in addition , there is some evidence of passive transfer of hepatitis b antibodies from ivig . in patients with mmp , ivig can be considered an effective treatment with minimum adverse effect compared to the conventional treatments . in this review , 13 studies with a total of seventy patients who were diagnosed at mmp and were considered for ivig therapy were analyzed . sixty - five patients who continued therapy were treated successfully with ivig while two patients in the study of sami et al . did not completely treated and experienced ocp progression . segura et al . reported four cases with mmp under ivig therapy , in which only one of them demonstrated a complete response . one unsuccessful treatment , who completed therapy , and two patients who showed a partial response by ivig therapy were also reported . in both cases with partial response , ivig therapy was discontinued because of the ivig - related adverse effects . with analyzing published studies , it can be concluded that ivig is a relatively safe and fast response treatment compared to conventional treatments of mmp . majority of patients included in this review did not demonstrate serious adverse effects . in those studies that compared two groups of conventional and ivig therapy , a faster clinical response this review revealed that ivig therapy is a promising treatment for patients with mmp , who did not respond to conventional therapy or experienced adverse effects due to conventional therapy . another lesson that can be learned is related to cessation of ivig therapy before complete remission . it was shown that decreasing ivig dose or cessation of that may result in relapse of the disease . all the included studies were used moderate ivig and hdivig , varied between 1 and 3 g / kg / cycle . response time was strongly variable in analyzed studies , but it seems that it is considerably lower than conventional therapy in mmp . considering the relatively low number of patients who were treated with ivig , finding a reasonable association between dose of ivig and other involved factors in response to ivig , including age , sex , and prior treatments among those with mmp is not possible . however , further studies are needed to clarify these factors and even the optimal dose of ivig .

background : mucous membrane pemphigoid ( mmp ) is considered an autoimmune blistering disease that predominantly affects mucous membranes . various treatments are available for controlling the diseases , but not all of them may respond.materials and methods : pubmed and google scholar were searched for all the associated studies until 2015 , using the keywords such as cicatricial pemphigoid or ocular pemphigoid or mucous membrane pemphigoid or mmp and intravenous immunoglobulin or ivig to find all the relevant studies . the last search update was for september 2 , 2015 . among the searched items , only english studies were included in the review.results:after excluding nonrelevant studies , 13 studies with a total number of seventy patients with mmp who were under treatment with ivig were analyzed . the 65 patients responded completely , one did not respond , two had partially responded , and the remaining two patients stopped ivig therapy , which resulted in ocular cicatricial pemphigoid progression . majority of the studies reported mild adverse effects while two of them did not report any unwanted side effect . the most common side effect was headache , followed by nausea . most of the patients who had a cessation of ivig therapy before achieving clinical remission experienced the disease progression.conclusion:overall , it can be concluded that ivig therapy was very helpful in treatment of mmp patients who did not respond to conventional therapy or stopped using them for various side effects . adverse effects associated with ivig therapy were considerably lower than conventional therapy that can lead toward treatment with this agent in patients who suffer from severe side effects .

INTRODUCTION MATERIALS AND METHODS Conventional therapy for mucous membrane pemphigoid Emerging treatments Intravenous immunoglobulin treatment Intravenous immunoglobulin as a treatment in mucous membrane pemphigoid DISCUSSION CONCLUSION Financial support and sponsorship Conflicts of interest AUTHOR'S CONTRIBUTION

there are four major groups of autoimmune blistering diseases , including pemphigus ( pemphigus vulgaris pemphigus foliaceus [ pf ] , pemphigus erythematosus , paraneoplastic pemphigus , immunoglobulin a [ iga ] pemphigus ) , pemphigoids ( bullous pemphigoid [ bp ] , pemphigoid gestationis , mucous membrane pemphigoid [ mmp ] , linear iga disease ) , epidermolysis bullosa acquisita ( eba ) , and dermatitis herpetiformis . within the autoimmune blistering diseases , autoantibodies play a critical role in destruction of skin in different ways , including loss of cell cell adhesion , dermo - epithelial dysadhesion , or mixed form of them . autoantibody against desmoglein 1 ( dsg1 ) , dsg3 , bp180 , and bp230 is the most important player in the majority of autoimmune blistering diseases . in the mmp , bp180 , laminin-332 , and 64 integrin mmp , which also known as cicatricial pemphigoid ( cp ) , is characterized by subepithelial bullae , less commonly on the skin , and more associated with mucous membranes . between patients with mmp , scarring of the mmp is also common among patients , which may result in severe life - threatening sequelae . in this study , it was tried to analyze the efficiency and the safety of intravenous ig ( ivig ) in patients with mmp . a systematic literature searching for all the published articles associated with the use of ivig and mmp which was conducted by databases of pubmed and google scholar was performed . all the associated studies until september 2015 were considered , using the keywords such as cicatricial pemphigoid or ocular pemphigoid or mucous membrane pemphigoid or mmp and intravenous immunoglobulin or ivig to find all the relevant studies . among the searched items , it is worthy of note that although the combination therapy rituximab with ivig was included in the study , it was not considered as the study associated with the role of ivig in the treatment of mmp . all the extracted data including year of publishing , number of patients , their age and sex , the dose of administrated ivig , response time , outcome , and ivig - related side effects were categorized and then entered into a database . various therapeutic strategies are available for treatment of patients with mmp , but not all the patients respond to those treatments . in patients with more severe and progressive diseases , a combination of both topical and systemic treatment may be required to control disease progression . overall , systemic corticosteroids , adjuvant immunosuppressive therapy , antibiotics , biologic agents such as rituximab and tumor necrosis factor- ( tnf- ) inhibitors , and immunomodulatory procedures such as ivig are the main therapeutic agents used in treating of mmp . excellent oral care has been emphasized as being an important part of the treatment of mmp . in patients with ocp , eye surgery must be undertaken with care because it may lead to the disease reactivation . in addition in addition to conventional treatments , some biologic agents including rituximab , etanercept , infliximab , and immunomodulatory agents , such as ivig , have been reported to be effective in the treatment mmp . ivig therapy considered as another option for management of mmp patients with rapidly disease progression or those who did not respond to previous treatments . in autoimmune diseases , however , it is generally accepted that ivig therapy should be limited to patients who fail conventional therapy , demonstrate serious side effects , contraindications to conventional therapy , or patients with rapidly progressive disease . despite the several case reports and case series in efficiency of ivig therapy for treatment of autoimmune diseases , it remained a controversial topic . considering that the ivig is not immunosuppressive and has a favorable side effect profile in compression to immunosuppressive therapy , it could be an appropriate treatment choice for patients who are at risk of infections or viral reactivation . different factors including age , type of disease , dose of ivig , and rate of the infusion play role in severity of adverse effects due to ivig therapy . the first group of adverse effects includes headache , flushing of the face , malaise , chest tightness , fever , chills , myalgia , fatigue , dyspnea , back pain , nausea , vomiting , diarrhea , change in blood pressure , and tachycardia . the most common adverse reaction related to ivig therapy is an infusion reaction , and symptoms may include flushing , headache , chills , nausea , tachycardia , hypotension , and wheezing . reported two cases who had successful treatment with ivig therapy . in the first case , a 50-year - old woman was diagnosed at mmp in 1992 ; betamethasone and cyclosporine did not control the disease completely . duration of systemic therapy before initiation of ivig varied from 3 to 14 years , with a mean of 8.3 years . various treatments were used , but all of them were gradually stopped due to different reasons including side effects , ineffectiveness , and lack of response . thereby , ivig was initiated at the dosage of 23 g / kg / cycle , which led to fully control of the disease , during a minimum of 4 months . the next successful ivig therapy in mmp patients was reported by ahmed and coln , who have analyzed the clinical outcomes as well as the disease progression in those with oral pemphigoid in a retrospective study in 2001 . the dose of ivig for patients in the first group was varied between 1 and 2 g / kg per cycle , which was divided into three doses for infusion during three consecutive days . in addition , it resulted in lower average number of side effects ( 0.4 vs. 3.2 ) , significantly lower number of relapse rate ( 0.1 vs. 2.1 ) , higher remission rate ( 1 vs. 0.4 ) , and considerable higher quality of live ( lower than the poor index vs. higher than tolerant of reasonable index ) . interestingly , among the patients in the first group , no one developed oral pemphigoid in any other mucous membrane . in issue of adverse effect however , in the conventional therapy group , all the 12 patients experienced various side effects . a total of six cycles each 4 weeks were administrated which resulted in remission of disease for more than 12 months . presented the ivig therapy in 15 patients with severe mmp who were nonresponsive to the conventional treatments or developed multiple side effects . average month with side effects decreased considerably ( 5.8 vs. 0.6 ) ; relapse rate average decreased ( 7.33 vs. 1.47 ) , and remission rate was increased ( 0 vs. 1 ) after initiation of ivig . with reports that confirmed effectiveness of ivig therapy in mmp patients , more patients were treated with this immunomodulatory agent . compared the clinical outcomes of ivig therapy to conventional immunosuppressive therapy in patients with mmp , whose disease progressed to involve the eye . thus , it led to a decreasing in the rate of infusions which resulted in the resolution of those side effects . however , all the patients in the conventional therapy group demonstrated side effects . as the final outcome , all the patients who were treated with ivig had been controlled completely . in the same year , a case series included ten patients with a diagnosis of ocp , reported successfully using of ivig in patients with mmp . in contrast , other two patients were excluded from the treatment protocol , which resulted in progression of ocp . on that study , the total number of cycles and duration of ivig therapy reported ranged from 20 to 42 ( mean , 32 ) and from 25 to 43 months ( mean , 35 ) , respectively . one of them did not respond to ivig therapy while another one had a complete response . in all of them , gingival , buccal , and palate lesions were the initial signs of mmp , followed by chronic conjunctivitis and later extended to conjunctival shrinkage . patients remained on systemic steroids and immunosuppressants that were gradually reduced by 50% in the first 3 months of ivig therapy and further 20% during the last cycles of maintenance therapy . according to those results , it was suggested that ivig therapy could be more effective than conventional immunosuppression treatment for controlling inflammation and avoiding disease progression . in 2009 , a study of ten patients with autoimmune mucocutaneous blistering diseases , including seven pemphigus vulgaris ( pv ) and three mmp patients ( two females and one male aged 4276 [ mean , 66 ] ) , confirmed the efficiency of ivig therapy at the dose of 2 g / kg in controlling of mmp patients . analyzed the efficiency of combination of rituximab with ivig compared to conventional therapy all the patients in the first group who were treated using conventional therapy were blind in one eye . four in the second group who were treated with the combination of rituximab and ivig therapy were also blind in one of their eyes . with maintaining ivig therapy for 6 months the effectivness , safety , and associated factors of intravenous immunoglobulin treatment in mucous membrane pemphigoid , based on reviewed studies various therapeutic strategies are available for treatment of patients with mmp , but not all the patients respond to those treatments . in patients with more severe and progressive diseases , a combination of both topical and systemic treatment may be required to control disease progression . overall , systemic corticosteroids , adjuvant immunosuppressive therapy , antibiotics , biologic agents such as rituximab and tumor necrosis factor- ( tnf- ) inhibitors , and immunomodulatory procedures such as ivig are the main therapeutic agents used in treating of mmp . excellent oral care has been emphasized as being an important part of the treatment of mmp . in patients with ocp , eye surgery must be undertaken with care because it may lead to the disease reactivation . prednisone is the most common first - line treatment which usually is given at a dose of 11.5 mg / kg / day while this treatment is the major source of side effects . mtx is another used therapy for patients with mmp , which was reported in few studies . in addition to conventional treatments , some biologic agents including rituximab , etanercept , infliximab , and immunomodulatory agents , such as ivig , have been reported to be effective in the treatment mmp . anti - tnf therapy is a common treatment in rheumatoid arthritis ( ra ) which is considered an autoimmune disorder . ivig therapy considered as another option for management of mmp patients with rapidly disease progression or those who did not respond to previous treatments . in addition to these approved conditions , it is increasingly used for various off - label autoimmune disorders including pemphigus , pemphigoid , systemic lupus erythematosus ( sle ) , ra , and multiple sclerosis ( ms ) . in autoimmune diseases , however , it is generally accepted that ivig therapy should be limited to patients who fail conventional therapy , demonstrate serious side effects , contraindications to conventional therapy , or patients with rapidly progressive disease . despite the several case reports and case series in efficiency of ivig therapy for treatment of autoimmune diseases , it remained a controversial topic . considering that the ivig is not immunosuppressive and has a favorable side effect profile in compression to immunosuppressive therapy , it could be an appropriate treatment choice for patients who are at risk of infections or viral reactivation . different factors including age , type of disease , dose of ivig , and rate of the infusion play role in severity of adverse effects due to ivig therapy . the first group of adverse effects includes headache , flushing of the face , malaise , chest tightness , fever , chills , myalgia , fatigue , dyspnea , back pain , nausea , vomiting , diarrhea , change in blood pressure , and tachycardia . the most common adverse reaction related to ivig therapy is an infusion reaction , and symptoms may include flushing , headache , chills , nausea , tachycardia , hypotension , and wheezing . reported two cases who had successful treatment with ivig therapy . duration of systemic therapy before initiation of ivig varied from 3 to 14 years , with a mean of 8.3 years . various treatments were used , but all of them were gradually stopped due to different reasons including side effects , ineffectiveness , and lack of response . thereby , ivig was initiated at the dosage of 23 g / kg / cycle , which led to fully control of the disease , during a minimum of 4 months . the next successful ivig therapy in mmp patients was reported by ahmed and coln , who have analyzed the clinical outcomes as well as the disease progression in those with oral pemphigoid in a retrospective study in 2001 . the dose of ivig for patients in the first group was varied between 1 and 2 g / kg per cycle , which was divided into three doses for infusion during three consecutive days . pemphigoid disease in all the patients , in both groups , was limited to oral cavity at the time of enrolling in the study . in addition , it resulted in lower average number of side effects ( 0.4 vs. 3.2 ) , significantly lower number of relapse rate ( 0.1 vs. 2.1 ) , higher remission rate ( 1 vs. 0.4 ) , and considerable higher quality of live ( lower than the poor index vs. higher than tolerant of reasonable index ) . interestingly , among the patients in the first group , no one developed oral pemphigoid in any other mucous membrane . in issue of adverse effect however , in the conventional therapy group , all the 12 patients experienced various side effects . a total of six cycles each 4 weeks were administrated which resulted in remission of disease for more than 12 months . presented the ivig therapy in 15 patients with severe mmp who were nonresponsive to the conventional treatments or developed multiple side effects . average month with side effects decreased considerably ( 5.8 vs. 0.6 ) ; relapse rate average decreased ( 7.33 vs. 1.47 ) , and remission rate was increased ( 0 vs. 1 ) after initiation of ivig . with reports that confirmed effectiveness of ivig therapy in mmp patients , more patients were treated with this immunomodulatory agent . compared the clinical outcomes of ivig therapy to conventional immunosuppressive therapy in patients with mmp , whose disease progressed to involve the eye . however , the recurrence of the disease was observed in five patients in the other group . thus , it led to a decreasing in the rate of infusions which resulted in the resolution of those side effects . however , all the patients in the conventional therapy group demonstrated side effects . as the final outcome , all the patients who were treated with ivig had been controlled completely . conversely , in the other group , only three patients were completely controlled ; three were partially controlled , and two remained uncontrolled . in the same year , a case series included ten patients with a diagnosis of ocp , reported successfully using of ivig in patients with mmp . in contrast , other two patients were excluded from the treatment protocol , which resulted in progression of ocp . on that study , the total number of cycles and duration of ivig therapy reported ranged from 20 to 42 ( mean , 32 ) and from 25 to 43 months ( mean , 35 ) , respectively . one of them did not respond to ivig therapy while another one had a complete response . the ivig therapy was stopped due to toxicity on patients with partial remission . in all of them , gingival , buccal , and palate lesions were the initial signs of mmp , followed by chronic conjunctivitis and later extended to conjunctival shrinkage . within the average of 9.1 months , an actual clinical remission ( healing of previous oral lesions and resolution of ocular scarring without development new lesion ) patients remained on systemic steroids and immunosuppressants that were gradually reduced by 50% in the first 3 months of ivig therapy and further 20% during the last cycles of maintenance therapy . according to those results , it was suggested that ivig therapy could be more effective than conventional immunosuppression treatment for controlling inflammation and avoiding disease progression . in 2009 , a study of ten patients with autoimmune mucocutaneous blistering diseases , including seven pemphigus vulgaris ( pv ) and three mmp patients ( two females and one male aged 4276 [ mean , 66 ] ) , confirmed the efficiency of ivig therapy at the dose of 2 g / kg in controlling of mmp patients . analyzed the efficiency of combination of rituximab with ivig compared to conventional therapy all the patients in the first group who were treated using conventional therapy were blind in one eye . four in the second group who were treated with the combination of rituximab and ivig therapy were also blind in one of their eyes . with maintaining ivig therapy for 6 months the effectivness , safety , and associated factors of intravenous immunoglobulin treatment in mucous membrane pemphigoid , based on reviewed studies in addition to conventional therapies , some other treatments are used to treat various autoimmune diseases . in contrast , some other reports are available that did not confirm effectiveness of ivig therapy in various autoimmune diseases . furthermore , there are several proofs that it can be helpful in patients with some other infections such as respiratory syncytial virus . in patients with mmp , ivig can be considered an effective treatment with minimum adverse effect compared to the conventional treatments . in this review , 13 studies with a total of seventy patients who were diagnosed at mmp and were considered for ivig therapy were analyzed . sixty - five patients who continued therapy were treated successfully with ivig while two patients in the study of sami et al . reported four cases with mmp under ivig therapy , in which only one of them demonstrated a complete response . one unsuccessful treatment , who completed therapy , and two patients who showed a partial response by ivig therapy were also reported . in both cases with partial response , ivig therapy was discontinued because of the ivig - related adverse effects . with analyzing published studies , it can be concluded that ivig is a relatively safe and fast response treatment compared to conventional treatments of mmp . majority of patients included in this review did not demonstrate serious adverse effects . in those studies that compared two groups of conventional and ivig therapy , a faster clinical response this review revealed that ivig therapy is a promising treatment for patients with mmp , who did not respond to conventional therapy or experienced adverse effects due to conventional therapy . another lesson that can be learned is related to cessation of ivig therapy before complete remission . response time was strongly variable in analyzed studies , but it seems that it is considerably lower than conventional therapy in mmp . considering the relatively low number of patients who were treated with ivig , finding a reasonable association between dose of ivig and other involved factors in response to ivig , including age , sex , and prior treatments among those with mmp is not possible .

progressive supranuclear palsy ( psp ) is the second most common cause of parkinsonism [ 1 , 2 ] . common symptoms of psp include parkinsonism with progressive vertical gaze palsy , postural instability with falls , akinesia , and cognitive impairment [ 3 , 4 ] . further , psp causes neuronal degeneration in the brainstem , including in the substantia nigra , tegmentum , and superior colliculus ; tau pathology that accompanies tuft - shaped astrocytes has also been observed in the superior and middle frontal gyri in psp . earlier magnetic resonance imaging ( mri ) studies have primarily used regions of interest analyses to examine brain atrophy [ 7 , 8 , 9 , 10 , 11 ] . however , the results of such analyses depend largely on the arbitrary locus and size that examiners select retrospectively . also , this method is time - consuming and only measures relatively large areas , potentially overlooking finer differences . voxel - based analysis using statistical parametric mapping ( spm ) was developed to examine focal differences in brain anatomy . this method can eliminate the effects of differences in size , and voxel - by - voxel comparison allows one to perform a prospective statistical analysis of differences in gray matter loss and changes in metabolism . using voxel - based morphometry ( vbm ) with mri [ 12 , 13 ] and voxel - based positron emission tomography ( pet ) analysis [ 14 , 15 , 16 , 17 , 18 ] of psp cases , reductions in gray matter and metabolic activity have been observed primarily in the frontal lobe and basal midbrain . however , patients in previous pet and mri studies have differed with regard to the clinical stage of the disease . thus , a comparison between the metabolic changes and decreases in gray matter in patients is difficult to make . the strength of the present study is a direct comparison of fluorodeoxyglucose ( fdg ) and vbm in the same psp subjects . based on the success of large multicenter alzheimer disease neuroimaging studies ( e.g. adni and aibl ) , there is increasing interest in neuroimaging measures as secondary or surrogate markers of outcome for therapy trials on neurodegenerative diseases . the modality that can diagnose psp in its early stages more adequately remains to be identified . the primary aim of this study was to compare the distribution of glucose hypometabolism and atrophy simultaneously in the same group of patients . the cognitive disturbances that occur in psp are referred to as subcortical dementia [ 19 , 20 ] . psp patients demonstrate significant deficits in recall and moderate forgetfulness , despite intact short - term and implicit perceptual memory . psp is also accompanied by memory loss and more common deficits , including executive dysfunction , decreased working memory , psychomotor slowing , non - fluent aphasia , and , at times , profound behavioral changes that mimic frontotemporal dementia [ 21 , 22 ] . despite the clinical significance of dementia , no study has examined the brain structures that are affected during cognitive impairment in patients with psp . in this study , we selected psp patients with relatively mild cognitive impairment and examined the changes in the associated brain regions . in this study , we included 16 probable psp patients [ 5 females and 11 males ; age : 64.9 ( 6.4 ) years ; mini - mental state examination ( mmse ) score : 21.0 ( 4.4 ) ] and 20 cognitively normal controls [ 8 females and 12 males ; age : 64.8 ( 6.4 ) years ; mmse score : 29.8 ( 0.6 ) ] . these patients had been admitted to the infirmary at our institution between april 1996 and march 2002 to undergo an evaluation for dementia and had agreed to be registered in the database ; they were followed up for > 2 years . in order to ensure that the imaging protocols and the types of pet and mri scanners were the same throughout the observation period the participants informed consents were obtained when they registered in the dementia registry database , but due to the retrospective design of this study informed consent was not required . all patients were examined by neurologists and psychiatrists and underwent standard neurological and neuropsychological examinations , laboratory tests , electroencephalography , cranial mri , and f - fdg - pet . the criteria for psp of the national institute of neurological disorders and stroke society were used to select patients for inclusion : a gradually progressive disorder ; onset at 40 years of age ; vertical ( upward and downward gaze ) supranuclear palsy and prominent postural instability with falls in the 1st year of the disease , and no evidence of other diseases that could explain the features above . our patient group had mild cognitive impairment [ mmse score : 21.0 ( 4.4 ) ] . all patients fulfilled the criteria for probable psp , and all subjects who were included had been followed up for 3 years after study entry . the diagnosis of psp was confirmed in all cases at the follow - up evaluation . the patients characteristics , including extrapyramidal signs , parkinsonism , and cortical symptoms , are shown in table 1 . all control subjects had mmse scores 28 and exhibited no clinical evidence of cognitive deficits or neurological disease . we did not detect any abnormal findings with regard to age - related atrophy or changes in white matter , including white matter hyperintensities . a 1.5-tesla signa horizon mri system was used for this study ( ge medical systems , milwaukee , wisc . sagittal , coronal , and axial t1-weighted spin echo images ( tr / te / nex : 550/15/2 ; 5 mm thick , 2.5-mm gap ) and axial t2-weighted fast spin echo images ( 3,000/21,105/2 ) were obtained to detect abnormalities . vbm analyses were conducted using coronal 3d spoiled gradient echo ( spgr ) images [ 23 , 24](14/3/2 , 20 flip angle , 220-mm field of view , 256 256 matrix , 124 1.5 mm contiguous sections ) . the 3d - spgr images were constructed with a voxel size of 0.86 0.86 1.5 mm . then , the images were reconstructed into 2.0-mm isotropic voxel images by the spm / vbm methods . the detailed pet procedure has been described previously . in brief , fdg - pet images were obtained using a headtome iv scanner ( shimadzu , kyoto , japan ) . the subjects were examined in the resting condition with their eyes closed and ears unplugged . after a transmission scan , a 12-min emission scan was started 60 min after an intravenous injection of 185370 mbq fdg . the slice number was 14 , and the slice interval was 6.5 mm in the z - motion mode . anatomical normalization and statistical processing of the pet and mr images were performed using spm for windows , version 8 ( spm 8 ; wellcome department of cognitive neurology , london , uk ) . the calculations and image matrix manipulations were performed using matlab r2009b ( mathworks , natick , mass . all coronal spgr mri datasets were reconstructed into axial datasets and subsequently converted to analyze format and displayed with the right hemisphere on the right side of the image . next , we applied dartel ( an algorithm for accurate diffeomorphic image registration ) , implemented as a toolbox in spm 8 , to create a set of group - specific templates . using these templates finally , the gray matter probability values were smoothed using an 8-mm full - width at half maximum gaussian kernel to match the pet analysis . the dartel procedure , in addition to the previous spatial normalization methods that were developed by ashburner , improves anatomical precision . for fdg - pet , all individual pet images were co - registered with mri . the images were transformed into a standard stereotactic anatomical space using the dartel flow that was created in the mri normalization . further , all pet and mri images were smoothed using an isotropic 8-mm gaussian kernel to increase the signal - to - noise ratio and compensate for differences in gyral anatomy between individuals . the individual fdg images were adjusted by proportional scaling to a mean value of 5.0 mg 100 ml min . the metabolic activity , gray matter , and white matter were compared between patients and controls and analyzed by a 2-sample t test . the correlations between mmse scores and brain structures were then examined with regard to metabolic activity , gray matter , and white matter by voxel - wise regression analysis , considering total intracranial volume , gender , and age as nuisance parameters . each comparison between patients and controls was analyzed by a statistical design in the 2-sample t test . in this study , we applied an uncorrected threshold of p < 0.001 on the basis of the results obtained in the previous studies , in which the frontal lobes and the midbrain were found as the regions of hypometabolism [ 15 , 16 , 17 , 18 ] or decreased gray matter [ 12 , 13 ] in patients with psp . in this study , we included 16 probable psp patients [ 5 females and 11 males ; age : 64.9 ( 6.4 ) years ; mini - mental state examination ( mmse ) score : 21.0 ( 4.4 ) ] and 20 cognitively normal controls [ 8 females and 12 males ; age : 64.8 ( 6.4 ) years ; mmse score : 29.8 ( 0.6 ) ] . these patients had been admitted to the infirmary at our institution between april 1996 and march 2002 to undergo an evaluation for dementia and had agreed to be registered in the database ; they were followed up for > 2 years . in order to ensure that the imaging protocols and the types of pet and mri scanners were the same throughout the observation period the participants informed consents were obtained when they registered in the dementia registry database , but due to the retrospective design of this study informed consent was not required . all patients were examined by neurologists and psychiatrists and underwent standard neurological and neuropsychological examinations , laboratory tests , electroencephalography , cranial mri , and f - fdg - pet . the criteria for psp of the national institute of neurological disorders and stroke society were used to select patients for inclusion : a gradually progressive disorder ; onset at 40 years of age ; vertical ( upward and downward gaze ) supranuclear palsy and prominent postural instability with falls in the 1st year of the disease , and no evidence of other diseases that could explain the features above . our patient group had mild cognitive impairment [ mmse score : 21.0 ( 4.4 ) ] . all patients fulfilled the criteria for probable psp , and all subjects who were included had been followed up for 3 years after study entry . the diagnosis of psp was confirmed in all cases at the follow - up evaluation . the patients characteristics , including extrapyramidal signs , parkinsonism , and cortical symptoms , are shown in table 1 . all control subjects had mmse scores 28 and exhibited no clinical evidence of cognitive deficits or neurological disease . we did not detect any abnormal findings with regard to age - related atrophy or changes in white matter , including white matter hyperintensities . a 1.5-tesla signa horizon mri system was used for this study ( ge medical systems , milwaukee , wisc . sagittal , coronal , and axial t1-weighted spin echo images ( tr / te / nex : 550/15/2 ; 5 mm thick , 2.5-mm gap ) and axial t2-weighted fast spin echo images ( 3,000/21,105/2 ) were obtained to detect abnormalities . vbm analyses were conducted using coronal 3d spoiled gradient echo ( spgr ) images [ 23 , 24](14/3/2 , 20 flip angle , 220-mm field of view , 256 256 matrix , 124 1.5 mm contiguous sections ) . the 3d - spgr images were constructed with a voxel size of 0.86 0.86 1.5 mm . then , the images were reconstructed into 2.0-mm isotropic voxel images by the spm / vbm methods . the detailed pet procedure has been described previously . in brief , fdg - pet images were obtained using a headtome iv scanner ( shimadzu , kyoto , japan ) . the subjects were examined in the resting condition with their eyes closed and ears unplugged . after a transmission scan , a 12-min emission scan was started 60 min after an intravenous injection of 185370 mbq fdg . the slice number was 14 , and the slice interval was 6.5 mm in the z - motion mode . anatomical normalization and statistical processing of the pet and mr images were performed using spm for windows , version 8 ( spm 8 ; wellcome department of cognitive neurology , london , uk ) . the calculations and image matrix manipulations were performed using matlab r2009b ( mathworks , natick , mass . all coronal spgr mri datasets were reconstructed into axial datasets and subsequently converted to analyze format and displayed with the right hemisphere on the right side of the image . next , we applied dartel ( an algorithm for accurate diffeomorphic image registration ) , implemented as a toolbox in spm 8 , to create a set of group - specific templates . using these templates finally , the gray matter probability values were smoothed using an 8-mm full - width at half maximum gaussian kernel to match the pet analysis . the dartel procedure , in addition to the previous spatial normalization methods that were developed by ashburner , improves anatomical precision . for fdg - pet , all individual pet images were co - registered with mri . the images were transformed into a standard stereotactic anatomical space using the dartel flow that was created in the mri normalization . further , all pet and mri images were smoothed using an isotropic 8-mm gaussian kernel to increase the signal - to - noise ratio and compensate for differences in gyral anatomy between individuals . the individual fdg images were adjusted by proportional scaling to a mean value of 5.0 mg 100 ml min . the metabolic activity , gray matter , and white matter were compared between patients and controls and analyzed by a 2-sample t test . the correlations between mmse scores and brain structures were then examined with regard to metabolic activity , gray matter , and white matter by voxel - wise regression analysis , considering total intracranial volume , gender , and age as nuisance parameters . each comparison between patients and controls was analyzed by a statistical design in the 2-sample t test . in this study , we applied an uncorrected threshold of p < 0.001 on the basis of the results obtained in the previous studies , in which the frontal lobes and the midbrain were found as the regions of hypometabolism [ 15 , 16 , 17 , 18 ] or decreased gray matter [ 12 , 13 ] in patients with psp . using fdg - pet , we observed a reduction in metabolic activity in the bilateral frontal lobes and midbrain tegmentum in the psp group compared with normal controls ( p < 0.001 , uncorrected ; fig . , we compared gray matter volume in the bilateral frontal lobes , anterior cingulate gyrus , right middle temporal gyrus , and right midbrain between patients and normal controls ( p < 0.001 , uncorrected ; fig . 1b ; table 2 ) . white matter volume decreased significantly in the bilateral thalami , midbrain tegmentum , and right frontal lobes ( p < 0.001 , uncorrected ; fig . 1c ; table 2 ) . the decrease in white matter in the midbrain was more marked than the reduction in gray matter in the visual analysis . the extent to which the white matter decreased was less than the decrease in hypometabolism in psp patients . a positive metabolic and structural correlation with mmse was observed in the left frontal lobe in regression analysis ( p < 0.001 , uncorrected ; fig . 2 ; table 3 ) . additional reductions in gray matter were noted in the right frontal lobe and bilateral rectal gyri ( fig . 2 ; table 3 ) . 3b ) images of a male patient with psp ( mmse score : 23 ) and a normal female subject ( mmse score : 30 ) of the same age ( 68 years ) . slight midbrain and bilateral frontal lobe atrophy was observed on visual inspection of the conventional t1-weighted image of the psp patient compared with the healthy control ( fig . 3a ) ; however , the psp patient experienced more severe hypometabolism in the midbrain and bilateral frontal lobes , including the medial and lateral frontal lobes . 4b ) images of a demented male patient with psp ( mmse score : 17 ) and a normal female subject ( mmse score : 30 ) of the same age ( 70 years ) . abnormalities in the midbrain were not observed on visual inspection of the conventional t1-weighted image and fdg - pet images of the psp patient compared with the healthy control ; however , slight atrophy and more severe hypometabolism were noted in the psp patient in the bilateral frontal lobes ( fig . using fdg - pet , we observed a reduction in metabolic activity in the bilateral frontal lobes and midbrain tegmentum in the psp group compared with normal controls ( p < 0.001 , uncorrected ; fig . , we compared gray matter volume in the bilateral frontal lobes , anterior cingulate gyrus , right middle temporal gyrus , and right midbrain between patients and normal controls ( p < 0.001 , uncorrected ; fig . 1b ; table 2 ) . white matter volume decreased significantly in the bilateral thalami , midbrain tegmentum , and right frontal lobes ( p < 0.001 , uncorrected ; fig . 1c ; table 2 ) . the decrease in white matter in the midbrain was more marked than the reduction in gray matter in the visual analysis . the extent to which the white matter decreased was less than the decrease in hypometabolism in psp patients . a positive metabolic and structural correlation with mmse was observed in the left frontal lobe in regression analysis ( p < 0.001 , uncorrected ; fig . 2 ; table 3 ) . additional reductions in gray matter were noted in the right frontal lobe and bilateral rectal gyri ( fig . 2 ; table 3 ) . figure 3 shows mr ( fig . 3a ) and fdg - pet ( fig . 3b ) images of a male patient with psp ( mmse score : 23 ) and a normal female subject ( mmse score : 30 ) of the same age ( 68 years ) . slight midbrain and bilateral frontal lobe atrophy was observed on visual inspection of the conventional t1-weighted image of the psp patient compared with the healthy control ( fig . 3a ) ; however , the psp patient experienced more severe hypometabolism in the midbrain and bilateral frontal lobes , including the medial and lateral frontal lobes . 4b ) images of a demented male patient with psp ( mmse score : 17 ) and a normal female subject ( mmse score : 30 ) of the same age ( 70 years ) . abnormalities in the midbrain were not observed on visual inspection of the conventional t1-weighted image and fdg - pet images of the psp patient compared with the healthy control ; however , slight atrophy and more severe hypometabolism were noted in the psp patient in the bilateral frontal lobes ( fig . this study is the first head - to - head comparison of fdg and vbm in the same group of psp subjects . the fdg - pet and mri scans showed that relatively similar regions experienced significant reductions in metabolic activity , gray matter , and white matter primarily the frontal lobe and midbrain tegmentum . first , the visual extents of the regions in which there were significant changes were much greater on fdg - pet than on mri . further , the correlation between the frontal lobe and mmse scores in psp patients was similar in all evaluations . compared with atrophy , the cluster volume of gray matter hypometabolism was greater , which implies that fdg - pet is more important in the differential diagnosis of psp . fdg - pet revealed hypometabolism in the bilateral frontal lobes and midbrain tegmentum , in agreement with previous pet studies [ 14 , 15 , 16 , 17 , 18 ] . vbm analysis disclosed significant reductions in gray matter in the lateral frontal lobe , including the middle and inferior frontal gyri , midbrain , and middle frontal gyrus . additional gray matter reductions in the hippocampus and parahippocampal gyrus were noted in another study . the cluster volume in our study appeared to be smaller than in previous vbm studies . such misregistrations could be observed in the periventricular area and the deep brain structures , such as the striatal nucleus and brainstem . the greater reduction in midbrain gray matter ( compared with white matter ) that was observed in one study might be explained by the misregistration of structures . however , there would be some space to discuss the structural changes in the midbrain . in this study , t1-weighted imaging was used for vbm analysis . t1-weighted imaging is less sensitive to changes in iron deposits in the brainstem , which complicates the discrimination of mr signals between gray matter and white matter in the brainstem . another study that used surface rendering without segmenting the gray and white matter observed brainstem atrophy in psp patients ; however , due to limitations in measuring the volumes of regional structures , a new method for measuring brainstem atrophy in psp should be developed our procedure vbm using dartel revealed greater reductions in the white than in the gray matter in the midbrain tegmentum . further , the cluster volume of gray matter hypometabolism was much higher compared with gray matter atrophy in psp patients . our results show that atrophy and hypometabolism in the frontal lobe are as important as in the basal midbrain in the diagnosis of psp patients exhibiting cognitive dysfunction . in fact , as shown in figure 4 , we identified a demented psp patient who only experienced hypometabolism and atrophy in the frontal lobe but not in the basal midbrain . if we consider psp with regard to dementia , disease progression in the frontal lobe versus the midbrain is not synchronized . the cognitive changes that occur in psp are referred to as subcortical dementia , which is associated with cognitive impairment , including prominent deficits in recall and moderate forgetfulness , despite intact short - term and implicit perceptual memory . neuropsychological studies employing the frontal behavioral inventory suggest the presence of frontal lobe dysfunction in psp patients . moreover , neuropathological studies have demonstrated that the middle frontal gyrus is a representative region in which abnormal glial cells , accompanied by abnormal tau protein , appear , further supporting our data . the relationship between the unified parkinson 's disease rating scale score and structural changes has to be elucidated in future studies . our results suggest that in addition to the basal midbrain , the frontal lobe is a part not to be missed in the diagnosis of demented psp patients who do not experience hypometabolism or atrophy in the midbrain . a limitation of this study is that the diagnosis of psp in our patients was not confirmed by autopsy . second , we did not compare the diagnostic performance between mri and pet in diagnosing psp . third , this study used an old type of pet scanner and the slice interval of 6.5 mm may be relatively large to evaluate midbrain metabolism precisely . further , we compared the pet and mri findings only with raw data and did not perform the atrophy correction . atrophy may have contributed to the extent of the reduced signal shown in the pet scan . however , the methods for atrophy correction are still in the developing stages , and future studies have to develop an appropriate technique for atrophy correction . finally , the use of vbm with the dartel algorithm continues to be refined ; advances in its precision and accuracy might pave the way for a more detailed study of psp pathology . compared with atrophy , the cluster volume of gray matter hypometabolism was greater , which implies that fdg - pet is more important in the differential diagnosis of psp . fdg - pet revealed hypometabolism in the bilateral frontal lobes and midbrain tegmentum , in agreement with previous pet studies [ 14 , 15 , 16 , 17 , 18 ] . vbm analysis disclosed significant reductions in gray matter in the lateral frontal lobe , including the middle and inferior frontal gyri , midbrain , and middle frontal gyrus . additional gray matter reductions in the hippocampus and parahippocampal gyrus were noted in another study . the cluster volume in our study appeared to be smaller than in previous vbm studies . such misregistrations could be observed in the periventricular area and the deep brain structures , such as the striatal nucleus and brainstem . the greater reduction in midbrain gray matter ( compared with white matter ) that was observed in one study might be explained by the misregistration of structures . however , there would be some space to discuss the structural changes in the midbrain . in this study , t1-weighted imaging was used for vbm analysis . t1-weighted imaging is less sensitive to changes in iron deposits in the brainstem , which complicates the discrimination of mr signals between gray matter and white matter in the brainstem . another study that used surface rendering without segmenting the gray and white matter observed brainstem atrophy in psp patients ; however , due to limitations in measuring the volumes of regional structures , a new method for measuring brainstem atrophy in psp should be developed our procedure vbm using dartel revealed greater reductions in the white than in the gray matter in the midbrain tegmentum . further , the cluster volume of gray matter hypometabolism was much higher compared with gray matter atrophy in psp patients . our results show that atrophy and hypometabolism in the frontal lobe are as important as in the basal midbrain in the diagnosis of psp patients exhibiting cognitive dysfunction . in fact , as shown in figure 4 , we identified a demented psp patient who only experienced hypometabolism and atrophy in the frontal lobe but not in the basal midbrain . if we consider psp with regard to dementia , disease progression in the frontal lobe versus the midbrain is not synchronized . the cognitive changes that occur in psp are referred to as subcortical dementia , which is associated with cognitive impairment , including prominent deficits in recall and moderate forgetfulness , despite intact short - term and implicit perceptual memory . neuropsychological studies employing the frontal behavioral inventory suggest the presence of frontal lobe dysfunction in psp patients . moreover , neuropathological studies have demonstrated that the middle frontal gyrus is a representative region in which abnormal glial cells , accompanied by abnormal tau protein , appear , further supporting our data . the relationship between the unified parkinson 's disease rating scale score and structural changes has to be elucidated in future studies . our results suggest that in addition to the basal midbrain , the frontal lobe is a part not to be missed in the diagnosis of demented psp patients who do not experience hypometabolism or atrophy in the midbrain . a limitation of this study is that the diagnosis of psp in our patients was not confirmed by autopsy . second , we did not compare the diagnostic performance between mri and pet in diagnosing psp . third , this study used an old type of pet scanner and the slice interval of 6.5 mm may be relatively large to evaluate midbrain metabolism precisely . further , we compared the pet and mri findings only with raw data and did not perform the atrophy correction . atrophy may have contributed to the extent of the reduced signal shown in the pet scan . however , the methods for atrophy correction are still in the developing stages , and future studies have to develop an appropriate technique for atrophy correction . finally , the use of vbm with the dartel algorithm continues to be refined ; advances in its precision and accuracy might pave the way for a more detailed study of psp pathology . this head - to - head study of fdg - pet and mri demonstrates that in psp patients the extent of metabolic changes is greater than that of changes in gray and white matter . moreover , mmse scores were associated with changes in the frontal lobe in psp patients , which will be beneficial in diagnosing psp patients with cognitive impairment who do not develop hypometabolism or atrophy in the midbrain and will contribute to future neuropathological and neurophysiological studies of psp .

background / aimsthe aim of this study was to compare differences in morphological and functional changes in brain regions in individual patients with progressive supranuclear palsy ( psp ) and correlate their mini - mental state examination ( mmse ) score with anatomy and function using magnetic resonance imaging ( mri ) and 18f - fluorodeoxyglucose positron emission tomography ( fdg - pet).methodssixteen psp patients and 20 age - matched healthy volunteers underwent fdg - pet and 3-dimensional mri . gray matter , white matter and metabolic activity were compared between patients and normal controls . in addition , possible correlations between the mmse score and brain function / anatomy were examined.resultsthe psp group had reduced cerebral glucose metabolism , and lower gray and white matter volumes in the frontal lobes and midbrain compared with normal controls . in psp subjects , the metabolic changes observed in the pet scans were greater than the loss in gray and white matter observed in the mri scans . the mmse scores were positively correlated with volume and fdg uptake in the frontal lobe.conclusionfdg-pet is a more effective tool in the diagnosis of psp than mri . atrophy and hypometabolism in the frontal lobe are as important as in the basal midbrain for differentiating psp patients who primarily exhibit cognitive dysfunction from normal controls .

Introduction Patients and Methods Patient Selection Magnetic Resonance Imaging Positron Emission Tomography Data Analysis Results Comparison with Normal Controls Metabolic and Structural Correlations with MMSE Representative Cases Discussion Comparison between Cortical Atrophy and Hypometabolism in PSP Patients Brain Structures Correlate with Cognitive Impairment in PSP Patients Conclusions Disclosure Statement

progressive supranuclear palsy ( psp ) is the second most common cause of parkinsonism [ 1 , 2 ] . common symptoms of psp include parkinsonism with progressive vertical gaze palsy , postural instability with falls , akinesia , and cognitive impairment [ 3 , 4 ] . further , psp causes neuronal degeneration in the brainstem , including in the substantia nigra , tegmentum , and superior colliculus ; tau pathology that accompanies tuft - shaped astrocytes has also been observed in the superior and middle frontal gyri in psp . earlier magnetic resonance imaging ( mri ) studies have primarily used regions of interest analyses to examine brain atrophy [ 7 , 8 , 9 , 10 , 11 ] . voxel - based analysis using statistical parametric mapping ( spm ) was developed to examine focal differences in brain anatomy . this method can eliminate the effects of differences in size , and voxel - by - voxel comparison allows one to perform a prospective statistical analysis of differences in gray matter loss and changes in metabolism . using voxel - based morphometry ( vbm ) with mri [ 12 , 13 ] and voxel - based positron emission tomography ( pet ) analysis [ 14 , 15 , 16 , 17 , 18 ] of psp cases , reductions in gray matter and metabolic activity have been observed primarily in the frontal lobe and basal midbrain . thus , a comparison between the metabolic changes and decreases in gray matter in patients is difficult to make . the strength of the present study is a direct comparison of fluorodeoxyglucose ( fdg ) and vbm in the same psp subjects . the primary aim of this study was to compare the distribution of glucose hypometabolism and atrophy simultaneously in the same group of patients . in this study , we selected psp patients with relatively mild cognitive impairment and examined the changes in the associated brain regions . in this study , we included 16 probable psp patients [ 5 females and 11 males ; age : 64.9 ( 6.4 ) years ; mini - mental state examination ( mmse ) score : 21.0 ( 4.4 ) ] and 20 cognitively normal controls [ 8 females and 12 males ; age : 64.8 ( 6.4 ) years ; mmse score : 29.8 ( 0.6 ) ] . in order to ensure that the imaging protocols and the types of pet and mri scanners were the same throughout the observation period the participants informed consents were obtained when they registered in the dementia registry database , but due to the retrospective design of this study informed consent was not required . all patients were examined by neurologists and psychiatrists and underwent standard neurological and neuropsychological examinations , laboratory tests , electroencephalography , cranial mri , and f - fdg - pet . the criteria for psp of the national institute of neurological disorders and stroke society were used to select patients for inclusion : a gradually progressive disorder ; onset at 40 years of age ; vertical ( upward and downward gaze ) supranuclear palsy and prominent postural instability with falls in the 1st year of the disease , and no evidence of other diseases that could explain the features above . the diagnosis of psp was confirmed in all cases at the follow - up evaluation . we did not detect any abnormal findings with regard to age - related atrophy or changes in white matter , including white matter hyperintensities . the subjects were examined in the resting condition with their eyes closed and ears unplugged . using these templates finally , the gray matter probability values were smoothed using an 8-mm full - width at half maximum gaussian kernel to match the pet analysis . for fdg - pet , all individual pet images were co - registered with mri . the metabolic activity , gray matter , and white matter were compared between patients and controls and analyzed by a 2-sample t test . the correlations between mmse scores and brain structures were then examined with regard to metabolic activity , gray matter , and white matter by voxel - wise regression analysis , considering total intracranial volume , gender , and age as nuisance parameters . each comparison between patients and controls was analyzed by a statistical design in the 2-sample t test . in this study , we applied an uncorrected threshold of p < 0.001 on the basis of the results obtained in the previous studies , in which the frontal lobes and the midbrain were found as the regions of hypometabolism [ 15 , 16 , 17 , 18 ] or decreased gray matter [ 12 , 13 ] in patients with psp . in this study , we included 16 probable psp patients [ 5 females and 11 males ; age : 64.9 ( 6.4 ) years ; mini - mental state examination ( mmse ) score : 21.0 ( 4.4 ) ] and 20 cognitively normal controls [ 8 females and 12 males ; age : 64.8 ( 6.4 ) years ; mmse score : 29.8 ( 0.6 ) ] . in order to ensure that the imaging protocols and the types of pet and mri scanners were the same throughout the observation period the participants informed consents were obtained when they registered in the dementia registry database , but due to the retrospective design of this study informed consent was not required . all patients were examined by neurologists and psychiatrists and underwent standard neurological and neuropsychological examinations , laboratory tests , electroencephalography , cranial mri , and f - fdg - pet . the criteria for psp of the national institute of neurological disorders and stroke society were used to select patients for inclusion : a gradually progressive disorder ; onset at 40 years of age ; vertical ( upward and downward gaze ) supranuclear palsy and prominent postural instability with falls in the 1st year of the disease , and no evidence of other diseases that could explain the features above . the diagnosis of psp was confirmed in all cases at the follow - up evaluation . we did not detect any abnormal findings with regard to age - related atrophy or changes in white matter , including white matter hyperintensities . sagittal , coronal , and axial t1-weighted spin echo images ( tr / te / nex : 550/15/2 ; 5 mm thick , 2.5-mm gap ) and axial t2-weighted fast spin echo images ( 3,000/21,105/2 ) were obtained to detect abnormalities . the subjects were examined in the resting condition with their eyes closed and ears unplugged . the slice number was 14 , and the slice interval was 6.5 mm in the z - motion mode . using these templates finally , the gray matter probability values were smoothed using an 8-mm full - width at half maximum gaussian kernel to match the pet analysis . for fdg - pet , all individual pet images were co - registered with mri . the images were transformed into a standard stereotactic anatomical space using the dartel flow that was created in the mri normalization . further , all pet and mri images were smoothed using an isotropic 8-mm gaussian kernel to increase the signal - to - noise ratio and compensate for differences in gyral anatomy between individuals . the metabolic activity , gray matter , and white matter were compared between patients and controls and analyzed by a 2-sample t test . the correlations between mmse scores and brain structures were then examined with regard to metabolic activity , gray matter , and white matter by voxel - wise regression analysis , considering total intracranial volume , gender , and age as nuisance parameters . each comparison between patients and controls was analyzed by a statistical design in the 2-sample t test . in this study , we applied an uncorrected threshold of p < 0.001 on the basis of the results obtained in the previous studies , in which the frontal lobes and the midbrain were found as the regions of hypometabolism [ 15 , 16 , 17 , 18 ] or decreased gray matter [ 12 , 13 ] in patients with psp . using fdg - pet , we observed a reduction in metabolic activity in the bilateral frontal lobes and midbrain tegmentum in the psp group compared with normal controls ( p < 0.001 , uncorrected ; fig . , we compared gray matter volume in the bilateral frontal lobes , anterior cingulate gyrus , right middle temporal gyrus , and right midbrain between patients and normal controls ( p < 0.001 , uncorrected ; fig . white matter volume decreased significantly in the bilateral thalami , midbrain tegmentum , and right frontal lobes ( p < 0.001 , uncorrected ; fig . the decrease in white matter in the midbrain was more marked than the reduction in gray matter in the visual analysis . the extent to which the white matter decreased was less than the decrease in hypometabolism in psp patients . a positive metabolic and structural correlation with mmse was observed in the left frontal lobe in regression analysis ( p < 0.001 , uncorrected ; fig . additional reductions in gray matter were noted in the right frontal lobe and bilateral rectal gyri ( fig . 3b ) images of a male patient with psp ( mmse score : 23 ) and a normal female subject ( mmse score : 30 ) of the same age ( 68 years ) . slight midbrain and bilateral frontal lobe atrophy was observed on visual inspection of the conventional t1-weighted image of the psp patient compared with the healthy control ( fig . 3a ) ; however , the psp patient experienced more severe hypometabolism in the midbrain and bilateral frontal lobes , including the medial and lateral frontal lobes . 4b ) images of a demented male patient with psp ( mmse score : 17 ) and a normal female subject ( mmse score : 30 ) of the same age ( 70 years ) . abnormalities in the midbrain were not observed on visual inspection of the conventional t1-weighted image and fdg - pet images of the psp patient compared with the healthy control ; however , slight atrophy and more severe hypometabolism were noted in the psp patient in the bilateral frontal lobes ( fig . using fdg - pet , we observed a reduction in metabolic activity in the bilateral frontal lobes and midbrain tegmentum in the psp group compared with normal controls ( p < 0.001 , uncorrected ; fig . , we compared gray matter volume in the bilateral frontal lobes , anterior cingulate gyrus , right middle temporal gyrus , and right midbrain between patients and normal controls ( p < 0.001 , uncorrected ; fig . white matter volume decreased significantly in the bilateral thalami , midbrain tegmentum , and right frontal lobes ( p < 0.001 , uncorrected ; fig . the decrease in white matter in the midbrain was more marked than the reduction in gray matter in the visual analysis . the extent to which the white matter decreased was less than the decrease in hypometabolism in psp patients . a positive metabolic and structural correlation with mmse was observed in the left frontal lobe in regression analysis ( p < 0.001 , uncorrected ; fig . additional reductions in gray matter were noted in the right frontal lobe and bilateral rectal gyri ( fig . 3a ) and fdg - pet ( fig . 3b ) images of a male patient with psp ( mmse score : 23 ) and a normal female subject ( mmse score : 30 ) of the same age ( 68 years ) . 3a ) ; however , the psp patient experienced more severe hypometabolism in the midbrain and bilateral frontal lobes , including the medial and lateral frontal lobes . 4b ) images of a demented male patient with psp ( mmse score : 17 ) and a normal female subject ( mmse score : 30 ) of the same age ( 70 years ) . abnormalities in the midbrain were not observed on visual inspection of the conventional t1-weighted image and fdg - pet images of the psp patient compared with the healthy control ; however , slight atrophy and more severe hypometabolism were noted in the psp patient in the bilateral frontal lobes ( fig . this study is the first head - to - head comparison of fdg and vbm in the same group of psp subjects . the fdg - pet and mri scans showed that relatively similar regions experienced significant reductions in metabolic activity , gray matter , and white matter primarily the frontal lobe and midbrain tegmentum . first , the visual extents of the regions in which there were significant changes were much greater on fdg - pet than on mri . further , the correlation between the frontal lobe and mmse scores in psp patients was similar in all evaluations . compared with atrophy , the cluster volume of gray matter hypometabolism was greater , which implies that fdg - pet is more important in the differential diagnosis of psp . fdg - pet revealed hypometabolism in the bilateral frontal lobes and midbrain tegmentum , in agreement with previous pet studies [ 14 , 15 , 16 , 17 , 18 ] . vbm analysis disclosed significant reductions in gray matter in the lateral frontal lobe , including the middle and inferior frontal gyri , midbrain , and middle frontal gyrus . additional gray matter reductions in the hippocampus and parahippocampal gyrus were noted in another study . such misregistrations could be observed in the periventricular area and the deep brain structures , such as the striatal nucleus and brainstem . the greater reduction in midbrain gray matter ( compared with white matter ) that was observed in one study might be explained by the misregistration of structures . however , there would be some space to discuss the structural changes in the midbrain . t1-weighted imaging is less sensitive to changes in iron deposits in the brainstem , which complicates the discrimination of mr signals between gray matter and white matter in the brainstem . another study that used surface rendering without segmenting the gray and white matter observed brainstem atrophy in psp patients ; however , due to limitations in measuring the volumes of regional structures , a new method for measuring brainstem atrophy in psp should be developed our procedure vbm using dartel revealed greater reductions in the white than in the gray matter in the midbrain tegmentum . further , the cluster volume of gray matter hypometabolism was much higher compared with gray matter atrophy in psp patients . our results show that atrophy and hypometabolism in the frontal lobe are as important as in the basal midbrain in the diagnosis of psp patients exhibiting cognitive dysfunction . in fact , as shown in figure 4 , we identified a demented psp patient who only experienced hypometabolism and atrophy in the frontal lobe but not in the basal midbrain . if we consider psp with regard to dementia , disease progression in the frontal lobe versus the midbrain is not synchronized . neuropsychological studies employing the frontal behavioral inventory suggest the presence of frontal lobe dysfunction in psp patients . the relationship between the unified parkinson 's disease rating scale score and structural changes has to be elucidated in future studies . our results suggest that in addition to the basal midbrain , the frontal lobe is a part not to be missed in the diagnosis of demented psp patients who do not experience hypometabolism or atrophy in the midbrain . a limitation of this study is that the diagnosis of psp in our patients was not confirmed by autopsy . further , we compared the pet and mri findings only with raw data and did not perform the atrophy correction . however , the methods for atrophy correction are still in the developing stages , and future studies have to develop an appropriate technique for atrophy correction . finally , the use of vbm with the dartel algorithm continues to be refined ; advances in its precision and accuracy might pave the way for a more detailed study of psp pathology . compared with atrophy , the cluster volume of gray matter hypometabolism was greater , which implies that fdg - pet is more important in the differential diagnosis of psp . fdg - pet revealed hypometabolism in the bilateral frontal lobes and midbrain tegmentum , in agreement with previous pet studies [ 14 , 15 , 16 , 17 , 18 ] . vbm analysis disclosed significant reductions in gray matter in the lateral frontal lobe , including the middle and inferior frontal gyri , midbrain , and middle frontal gyrus . additional gray matter reductions in the hippocampus and parahippocampal gyrus were noted in another study . such misregistrations could be observed in the periventricular area and the deep brain structures , such as the striatal nucleus and brainstem . the greater reduction in midbrain gray matter ( compared with white matter ) that was observed in one study might be explained by the misregistration of structures . t1-weighted imaging is less sensitive to changes in iron deposits in the brainstem , which complicates the discrimination of mr signals between gray matter and white matter in the brainstem . another study that used surface rendering without segmenting the gray and white matter observed brainstem atrophy in psp patients ; however , due to limitations in measuring the volumes of regional structures , a new method for measuring brainstem atrophy in psp should be developed our procedure vbm using dartel revealed greater reductions in the white than in the gray matter in the midbrain tegmentum . further , the cluster volume of gray matter hypometabolism was much higher compared with gray matter atrophy in psp patients . our results show that atrophy and hypometabolism in the frontal lobe are as important as in the basal midbrain in the diagnosis of psp patients exhibiting cognitive dysfunction . in fact , as shown in figure 4 , we identified a demented psp patient who only experienced hypometabolism and atrophy in the frontal lobe but not in the basal midbrain . if we consider psp with regard to dementia , disease progression in the frontal lobe versus the midbrain is not synchronized . neuropsychological studies employing the frontal behavioral inventory suggest the presence of frontal lobe dysfunction in psp patients . the relationship between the unified parkinson 's disease rating scale score and structural changes has to be elucidated in future studies . our results suggest that in addition to the basal midbrain , the frontal lobe is a part not to be missed in the diagnosis of demented psp patients who do not experience hypometabolism or atrophy in the midbrain . a limitation of this study is that the diagnosis of psp in our patients was not confirmed by autopsy . further , we compared the pet and mri findings only with raw data and did not perform the atrophy correction . atrophy may have contributed to the extent of the reduced signal shown in the pet scan . however , the methods for atrophy correction are still in the developing stages , and future studies have to develop an appropriate technique for atrophy correction . finally , the use of vbm with the dartel algorithm continues to be refined ; advances in its precision and accuracy might pave the way for a more detailed study of psp pathology . this head - to - head study of fdg - pet and mri demonstrates that in psp patients the extent of metabolic changes is greater than that of changes in gray and white matter . moreover , mmse scores were associated with changes in the frontal lobe in psp patients , which will be beneficial in diagnosing psp patients with cognitive impairment who do not develop hypometabolism or atrophy in the midbrain and will contribute to future neuropathological and neurophysiological studies of psp .

one of the goals of periodontal therapy is the predictable regeneration of the periodontium in areas previously affected by periodontal disease . histological and ultrastructural studies have demonstrated that dental roots that have been exposed to the oral cavity or to the periodontal pocket present reduced collagen fiber insertion , changes in their mineral density and root contamination by bacteria and its products . scaling and root planing alone are not able to fully eliminate the etiological contaminants and produce a compact smear layer covering the instrumented surface which inhibits periodontal tissue reattachment . these alterations have become the rationale for the use of demineralizing agents as adjunct to periodontal therapy due to their potential for removing smear layer and exposing the underlying radicular collagen fibrils , funneling dentin tubules and modifying dentin permeability , restoring the biocompatibility of the roots . it has been shown that demineralization of the root surface can exert neutralizing effects on endotoxins from periodontal pathogens in vitro , e.g. , inhibition of fibroblast proliferation , synthesis and attachment . other studies have shown that , when compared to non - conditioned ones , acid conditioned dental roots are more effective in maintaining fibrin clot and exposing collagen fibrils and associated proteoglycans . in vivo animal and human histological studies have shown improved biological response when decalcifying agents are used to condition the root surface . the most used demineralizing agents for these purposes are citric acid , phosphoric acid , ethylenediaminetetraacetic acid ( edta ) and tetracycline hydrochloride . nevertheless , the great variability of protocols employed by clinicians and researchers has prevented consistent comparisons among them . clinical trials have also provided insufficient evidence that acid conditioning of diseased dental roots present any additional new attachment when compared to non - conditioned ones . the only systematic review found on this subject was published by mariotti ( 2006 ) who concluded that the use of citric acid , tetracycline or edta to modify the root surface provides no benefit of clinical significance to regeneration in patients with chronic periodontitis . on the other hand , the author identified that several factors as lack of controls , non - calibrated examiners , masked reference standards and small sample sizes , among others , reduced the observational quality of relevant studies . as a consequence , mariotti ( 2006 ) stated that the overall conclusion of his review must be carefully considered . some authors have measured the number and diameter of dentin tubules exposed after root conditioning in order to relate these parameters with potential for cell adhesion and for intertubular collagen exposure . ( 1992 ) have found a time - dependent increase in the mean dentin tubule orifice diameter after treating dentin surfaces with citric acid or tetracycline hcl and ruggeri , et al . ( 2007 ) stressed that the exposure of the dentin matrix of root surface allows the formation of a proper fibrin clot , which is a determinant factor for the positive outcome of the early wound healing events . this would facilitate the integration between the root surface and the connective tissue favoring migration and attachment of gingival fibroblasts . there still is a remarkable controversy concerning to the type of chemical conditioner , time of its application and even the need of its use , which justifies the search for parameters that can support the option for this procedure in periodontal treatments . to the best of our knowledge , there is no standardized study comparing several chemical root conditioners for their ability of smear layer removing and dentin tubule widening . therefore , the aim of this study was to contribute with reliable data to analyze and compare diseased dental root surfaces treated by manual scaling followed by conditioning with four commonly used demineralizing agents . twenty - five human single - rooted teeth scheduled for extraction due to advanced periodontal disease at the bauru school of dentistry , university of so paulo , brazil , were selected for this study after signing an informed consent form . the selected teeth had to meet the following inclusion criteria : 1 ) no history of scaling and root planing in the previous 6 months ; 2 ) proximal attachment loss of 5 mm or more ; 3 ) absence of decay lesions or restorations near the cementoenamel junction ( cej ) . the freshly extracted teeth were cleaned from blood and other debris in saline solution and visible calculus was gently removed using manual scalers ( figure 1a ) . the teeth were stored in formalin at 10% for no more than 7 days . the crowns were removed by transversal sectioning at the cej with a water cooled high speed bur ( figure 1b ) . the diseased parts of the roots were visually identified with the aid of a magnifying glass ( lactona - 4 of magnification ; lactona , so paulo , sp , brazil ) as the area showing absence of remnants of the periodontal ligament ( figure 1a ) . then , each root received a second section made 2 mm away from the first in the apical direction , resulting in radicular dishes ( figure 1c ) . on the mesial and distal surfaces of the dishes , 2 grooves separated by a distance of 4 mm were made with the same previously used bur , determining an area measuring 2 mm x 4 mm ( figures 1d and 1e ) approximately . this area was scaled by the same operator with 20 strokes of gracey curettes ( hu - friedy ; hu - friedy do brasil , rio de janeiro , rj , brazil ) ( figure 1f ) and then , the mesial and distal halves of the dishes were sectioned , producing 50 radicular fragments with an scaled area delimited by the grooves in which demineralizing agents were tested ( figure 1 g ) . the radicular fragments were randomly divided into 5 groups of 10 fragments each according to the treatment given to the scaled surface as follows : 1 ) ca : demineralization with a liquid solution of citric acid at 50% and ph1 ( pharmcia specfica ; farmcias e drogarias , bauru , sp , brazil ) for 3 min ; 2 ) tc - hcl : demineralization with a liquid solution of tetracycline hydrochloride at 50 mg / ml ( laboratrio teuto , anpolis , go , brazil ) for 3 min ; 3 ) edta : demineralization by a gel of edta at 24% ( prefgel ; biora ab , malm , sweden ) for 3 min ; 4 ) pa : demineralization with a liquid solution of 37% phosphoric acid ( pharmcia specfica ; farmcias e drogarias ) for 3 min ; 5 ) control : treatment with saline solution for 3 min . all the agents were applied to the roots surfaces by burnishing them with a sterile cotton pellet changed every 30 s ( figure 1h ) ; the surfaces were then flushed profusely with distilled water . after the treatments , the dental fragments were immediately processed for analysis by conventional scanning electron microscopy ( sem ) . specimen preparation . a : extracted teeth in which the diseased part of the root was identified by the absence of periodontal ligament remnants ( * ) ; b : tooth crown being transversely cut at the cementoenamel with a water - cooled high - speed bur ; c : radicular dish obtained by sectioning the root 2 mm away from the first cut in the apical direction ; d : pencil marks made 4 mm apart from each other on the mesial and distal surfaces of the dishes where grooves were made ( e ) , delimiting an area of 2 mm x 4 mm approximately ; f : scaling of the area determined in e ; g : mesial and distal halves of the dishes separated before receiving the burnishing of the demineralizing agents with a sterile cotton pellet ( h ) the dental fragments were prepared for sem analysis as described by braidotti , et al . ( 2000 ) and observed at a jsm-5600 lv scanning electron microscope ( joel , tokyo , japan ) . digital images were taken at 1,000 and 2,000 magnification and at zero tilt angle . the sem micrographs were transferred to a computer and analyzed by image j software ( available from http://rbs.info.nih.gov/ij/ ) . the core of the groove - delimited area on each specimen was chosen for analysis . the roots surfaces were examined for general morphologic characteristics and for the presence of smear layer at magnification of 1,000. the field shown at magnification of 1,000 was also taken as reference for the area in which the number of exposed dentin tubules was counted . the field shown at magnification of 2,000 was taken as reference for the total area ( 266 , 240 m ) in which the percentage of area occupied by the enlarged dentin tubules was calculated . all the morphometric measurements were performed by a single investigator who was unaware of the origin of the specimens . this investigator was calibrated with repeated measurements until a 90% level of reproducibility was attained . the data were analyzed statistically by one - way anova , kruskal - wallis and dunn post - tests at significance level of 0.05 . twenty - five human single - rooted teeth scheduled for extraction due to advanced periodontal disease at the bauru school of dentistry , university of so paulo , brazil , were selected for this study after signing an informed consent form . the selected teeth had to meet the following inclusion criteria : 1 ) no history of scaling and root planing in the previous 6 months ; 2 ) proximal attachment loss of 5 mm or more ; 3 ) absence of decay lesions or restorations near the cementoenamel junction ( cej ) . the freshly extracted teeth were cleaned from blood and other debris in saline solution and visible calculus was gently removed using manual scalers ( figure 1a ) . the teeth were stored in formalin at 10% for no more than 7 days . the crowns were removed by transversal sectioning at the cej with a water cooled high speed bur ( figure 1b ) . the diseased parts of the roots were visually identified with the aid of a magnifying glass ( lactona - 4 of magnification ; lactona , so paulo , sp , brazil ) as the area showing absence of remnants of the periodontal ligament ( figure 1a ) . then , each root received a second section made 2 mm away from the first in the apical direction , resulting in radicular dishes ( figure 1c ) . on the mesial and distal surfaces of the dishes , 2 grooves separated by a distance of 4 mm were made with the same previously used bur , determining an area measuring 2 mm x 4 mm ( figures 1d and 1e ) approximately . this area was scaled by the same operator with 20 strokes of gracey curettes ( hu - friedy ; hu - friedy do brasil , rio de janeiro , rj , brazil ) ( figure 1f ) and then , the mesial and distal halves of the dishes were sectioned , producing 50 radicular fragments with an scaled area delimited by the grooves in which demineralizing agents were tested ( figure 1 g ) . the radicular fragments were randomly divided into 5 groups of 10 fragments each according to the treatment given to the scaled surface as follows : 1 ) ca : demineralization with a liquid solution of citric acid at 50% and ph1 ( pharmcia specfica ; farmcias e drogarias , bauru , sp , brazil ) for 3 min ; 2 ) tc - hcl : demineralization with a liquid solution of tetracycline hydrochloride at 50 mg / ml ( laboratrio teuto , anpolis , go , brazil ) for 3 min ; 3 ) edta : demineralization by a gel of edta at 24% ( prefgel ; biora ab , malm , sweden ) for 3 min ; 4 ) pa : demineralization with a liquid solution of 37% phosphoric acid ( pharmcia specfica ; farmcias e drogarias ) for 3 min ; 5 ) control : treatment with saline solution for 3 min . all the agents were applied to the roots surfaces by burnishing them with a sterile cotton pellet changed every 30 s ( figure 1h ) ; the surfaces were then flushed profusely with distilled water . after the treatments , the dental fragments were immediately processed for analysis by conventional scanning electron microscopy ( sem ) . specimen preparation . a : extracted teeth in which the diseased part of the root was identified by the absence of periodontal ligament remnants ( * ) ; b : tooth crown being transversely cut at the cementoenamel with a water - cooled high - speed bur ; c : radicular dish obtained by sectioning the root 2 mm away from the first cut in the apical direction ; d : pencil marks made 4 mm apart from each other on the mesial and distal surfaces of the dishes where grooves were made ( e ) , delimiting an area of 2 mm x 4 mm approximately ; f : scaling of the area determined in e ; g : mesial and distal halves of the dishes separated before receiving the burnishing of the demineralizing agents with a sterile cotton pellet ( h ) the dental fragments were prepared for sem analysis as described by braidotti , et al . ( 2000 ) and observed at a jsm-5600 lv scanning electron microscope ( joel , tokyo , japan ) . digital images were taken at 1,000 and 2,000 magnification and at zero tilt angle . the sem micrographs were transferred to a computer and analyzed by image j software ( available from http://rbs.info.nih.gov/ij/ ) . the core of the groove - delimited area on each specimen was chosen for analysis . the roots surfaces were examined for general morphologic characteristics and for the presence of smear layer at magnification of 1,000. the field shown at magnification of 1,000 was also taken as reference for the area in which the number of exposed dentin tubules was counted . the field shown at magnification of 2,000 was taken as reference for the total area ( 266 , 240 m ) in which the percentage of area occupied by the enlarged dentin tubules was calculated . all the morphometric measurements were performed by a single investigator who was unaware of the origin of the specimens . this investigator was calibrated with repeated measurements until a 90% level of reproducibility was attained . the data were analyzed statistically by one - way anova , kruskal - wallis and dunn post - tests at significance level of 0.05 . debris or other particles were not observed on the examined surfaces ( figure 2a ) . dentin tubules were totally exposed at the 1,000 field and the average percentage of the area occupied by tubules was 0.12% ( ranging from 8.67 m to 159 m at the magnification of 2,000 ( figure 2b ) . . a : representative specimen of the ca group in which no smear layer can be noted and dentin tubules are totally exposed ( 1,000 ) ; b : 2,000 magnification of the central part of a showing dentin tubules widened ; c : representative specimen of the tc - hcl group at 1,000 showing absence of smear layer and exposed dentin tubules ; d : 2,000 magnification of the central part of c showing widened dentin tubules ; e : representative specimen of edta group presenting smear layer and few exposed tubules ( 1,000 ) ; f : 2,000 magnification of the central part of e ; g : representative specimen of pa group at 1,000 of magnification covered by smear layer and some exposed tubules ; h : 2,000 magnification of the central part of g showing discrete widening of the tubules ; i : representative specimen of the control group at 1,000 showing smear layer without evident tubule exposure ; j : 2,000 magnification of the central part of i in which the tubules are not enlarged enough to measure their corresponding area even though all the tubules were exposed in 8 specimens of the tc - hcl group at the 1,000 field ( figure 2c ) , residual smear layer and some debris were present in 33.3% of them . the mean percentage of area occupied by exposed tubules was 0.08% ( ranging from 0 m to 48.18 m ) at the magnification of 2,000 ( figure 2d ) . the majority of the specimens of edta group presented with smear layer ( 80% ) and there were no exposed tubules 5 of them . the mean number of exposed tubules in this group was 4.4 at the 1,000 field ( figure 2e ) , occupying an area equivalent to 0.01% of the total ( ranging from 0 m to 5.82 m ) at the 2,000 of magnification ( figure 2f ) . all of them also showed a foamy surface ( figure 2 g ) probably due to the precipitation of an insoluble calcium phosphate layer . the mean number of exposed tubules at 1,000 of magnification was 12.1 ( figure 2 g ) and these tubules accounted for a mean area of 0.03% ( ranging from 0 m to 46.25 m ) at 2,000 of magnification ( figure 2h ) . although all specimens of this group showed smear layer , open tubules could be seen in 2 of them which were responsible for the mean number of 2.3 tubules for this group at the 1,000 magnification field ( figure 2i ) . nevertheless , most of them were not enlarged enough to measure their corresponding area and the mean value for this group was 0% of the total area at 2,000 of magnification ( figure 2j ) . the mean number of exposed dentin tubules by the different treatments decreased according to the following order : tc - hcl > ca > pa > edta nevertheless , statistically significant differences were only found when compared the groups tc - hcl , and c ; tc - hcl and edta ; ca and c and ca and edta ( table 1 ) . mean number of exposed dentin tubules and corresponding area after conditioning with the four different demineralizing agents same uppercase or lowercase letter in the same line means no statistically significant difference ( p>0.05 ) . when comparing the area occupied by dentin tubule openings , ca produced the greatest tubule widening followed by tc - hcl , pa , edta and saline ( table 1 ) . there was no statistically significant difference between ca and tc - hcl groups , and ca was different from all other groups . tc - hcl group differed only from group c. the exposure of dentin tubules in group c did not result in widening . debris or other particles were not observed on the examined surfaces ( figure 2a ) . dentin tubules were totally exposed at the 1,000 field and the average percentage of the area occupied by tubules was 0.12% ( ranging from 8.67 m to 159 m at the magnification of 2,000 ( figure 2b ) . . a : representative specimen of the ca group in which no smear layer can be noted and dentin tubules are totally exposed ( 1,000 ) ; b : 2,000 magnification of the central part of a showing dentin tubules widened ; c : representative specimen of the tc - hcl group at 1,000 showing absence of smear layer and exposed dentin tubules ; d : 2,000 magnification of the central part of c showing widened dentin tubules ; e : representative specimen of edta group presenting smear layer and few exposed tubules ( 1,000 ) ; f : 2,000 magnification of the central part of e ; g : representative specimen of pa group at 1,000 of magnification covered by smear layer and some exposed tubules ; h : 2,000 magnification of the central part of g showing discrete widening of the tubules ; i : representative specimen of the control group at 1,000 showing smear layer without evident tubule exposure ; j : 2,000 magnification of the central part of i in which the tubules are not enlarged enough to measure their corresponding area even though all the tubules were exposed in 8 specimens of the tc - hcl group at the 1,000 field ( figure 2c ) , residual smear layer and some debris were present in 33.3% of them . the mean percentage of area occupied by exposed tubules was 0.08% ( ranging from 0 m to 48.18 m ) at the magnification of 2,000 ( figure 2d ) . the majority of the specimens of edta group presented with smear layer ( 80% ) and there were no exposed tubules 5 of them . the mean number of exposed tubules in this group was 4.4 at the 1,000 field ( figure 2e ) , occupying an area equivalent to 0.01% of the total ( ranging from 0 m to 5.82 m ) at the 2,000 of magnification ( figure 2f ) . all of them also showed a foamy surface ( figure 2 g ) probably due to the precipitation of an insoluble calcium phosphate layer . the mean number of exposed tubules at 1,000 of magnification was 12.1 ( figure 2 g ) and these tubules accounted for a mean area of 0.03% ( ranging from 0 m to 46.25 m ) at 2,000 of magnification ( figure 2h ) . although all specimens of this group showed smear layer , open tubules could be seen in 2 of them which were responsible for the mean number of 2.3 tubules for this group at the 1,000 magnification field ( figure 2i ) . nevertheless , most of them were not enlarged enough to measure their corresponding area and the mean value for this group was 0% of the total area at 2,000 of magnification ( figure 2j ) . the mean number of exposed dentin tubules by the different treatments decreased according to the following order : tc - hcl > ca > nevertheless , statistically significant differences were only found when compared the groups tc - hcl , and c ; tc - hcl and edta ; ca and c and ca and edta ( table 1 ) . mean number of exposed dentin tubules and corresponding area after conditioning with the four different demineralizing agents same uppercase or lowercase letter in the same line means no statistically significant difference ( p>0.05 ) . when comparing the area occupied by dentin tubule openings , ca produced the greatest tubule widening followed by tc - hcl , pa , edta and saline ( table 1 ) . there was no statistically significant difference between ca and tc - hcl groups , and ca was different from all other groups . tc - hcl group differed only from group c. the exposure of dentin tubules in group c did not result in widening . this study compared the 4 most commonly used chemical agents for root conditioning as adjunctive therapy for teeth affected by periodontitis . the presented data suggest that citric acid and tetracycline - hcl , in this particular way of use , are more effective in removing smear layer and in exposing and widening dentin tubules than phosphoric acid and edta . since register and burdick ( 1975 ) compared root conditioning with citric acid ( ph 1 for 2 - 3 min ) and other chemical substances and found optimal cementogenesis and connective tissue new attachment , several investigators have devoted considerable time studying conditioning agents to improve periodontal regeneration . unfortunately , numerous and often uncontrolled histological and clinical studies have created controversy and confusion about the positive or negative effects of those agents . the inconsistency of these studies may be due to differences in experimental systems and techniques . nevertheless , there is a common acceptance that it is not possible to decontaminate periodontitis - affected root surfaces by mechanical means alone . it is also well documented that hand or ultrasonic scaling of root surface produces a nonbiocompatible smear layer that must be removed to expose the underlying collagen in order to favor fibroblast migration , attachment , and orientation . tetracycline became one of the most widely used and studied demineralizing agents since in vitro studies of terranova , et al . many concentrations and application times were tested ranging from 0.5% to 200% and from 0.5 to 10 min . most of the studies , however , found the best results with concentrations between 50 mg / ml and 125 mg / ml during 3 to 4 min of application by burnishing technique . ( 2000 ) concluded that concentrations between 50 mg / ml and 125 mg / ml might alter dentin surfaces by removing the smear layer and also maximize tubule openings in a short period if repeated applications are performed . based on these studies , we adopted the concentration of 50 mg / ml for tc - hcl . it has also been shown that burnishing the demineralizing agent on the root surface results in efficient removal of smear layer and exposure of the underlying tubules due to demineralization action of fresh acid solution . this was the rationale for the use of burnishing technique and for changing the cotton pellet at every 30 s in this study . the low ph of the saturated solutions of citric acid and tetracycline - hcl were suggested as one of the reasons for the reduced cellular insertion and for the unpredictability of the results , once it could denature the organic matrix of dentin . it was also suggested that acid etching would interfere on periodontal healing by its necrotizing effect on the surrounding progenitor cells . thus , edta at 12%-24% , neutral ph for 30 s to 3 min was introduced aiming at removing smear layer and widening dentin tubules without damaging biological structures . ( 2007 ) presented reliable findings using monoclonal antibodies and field emission in - lens scanning electron microscopy ( feisem ) showing that both citric acid and edta treatments are able to etch and expose collagen fibrils and proteoglycans without any degradation of dentin collagen matrix . our findings demonstrated that edta , in this way of use , failed to properly remove smear layer and expose dentin tubules . five specimens ( 50% ) from the edta group had none of their dentin tubules exposed and only 2 were totally clean from smear layer . it must be emphasized that even though ca and tc - hcl did not differ significantly on tubule number and widening evaluations , tc - hcl could not be considered different from the other groups . this result was the responsible for classifying tc - hcl in second place in our analysis . another interesting finding was that ca and pa exposed similar numbers of dentin tubules , but ca produced greater enlargement as reflected by the area measurements . edta and pa had the same behavior as saline solution on both evaluations , suggesting that these agents failed on efficiently conditioning the roots surfaces . all specimens from the pa group showed a foamy surface , probably due to a chemical acid / basic reaction between a strong acid and the hydroxyapatite , leading to calcium phosphate deposition on the roots surfaces . this occurrence can be attributed to the extended time of acid contact with the roots ( 3 min ) . it may produce an insoluble form of that salt , which precipitates , blocking tubule openings and annulling its demineralizing effect . as the superficial composition of these deposits was not characterized in this study , this interpretation is speculative . however , it is suggestive that phosphoric acid is not appropriate for root conditioning in this particular way of using . relevant findings from other researchers have reinforced that acid etching plays a decisive role in the establishment of new connective tissue attachment influencing the early healing events , i.e. , adsorption and adhesion of blood elements and fibrin to the root surface . in this aspect , baker , et al . ( 2005 ) have clearly demonstrated the superiority of citric acid in comparison to edta when applied for 5 min on planed dentin root surfaces . fibrin clot adhesion was better supported by the ca - treated than edta - treated dentin surfaces and forces produced by three 5-min rinses in pbs under agitation on a rotary shaker table partially removed the fibrin clot from edta - treated surfaces but not from ca - treated surfaces . the authors stressed that a fibrin network was firmly attached directly on dentin surface were the tubules were exposed and widened by the citric acid treated fragments . these findings led us to believe that the more exposed and widened the dentin tubules are , the more retained the fibrin clot will be on the root surface , perhaps within limits still to be determined . citric acid and tetracycline behaved very similarly in this particular aspect , suggesting that both can be equally effective as conditioning agents . the number and diameter of exposed dentin tubules in laser - conditioned roots were calculated in sem micrographs by herrero , et al . ( 2009 ) who related increased numbers and diameters to better root conditioning , once widening tubules openings causes higher exposure of the underlying dentin favoring connective tissue attachment . gamal and mailhot ( 2003 ) also observed that periodontal ligament fibroblasts adhere and differentiate on edta - conditioned roots surfaces that were free from smear layer and presented exposed round to oval dentin tubule orifices . nevertheless , there is no study comparing the number of exposed dentin tubules and their corresponding area after conditioning with several conditioners for comparison with our data . the findings of this study corroborate previous data stressing the advantages of using conditioning agents on diseased root surfaces and provide additional relevant information when choosing an agent for root conditioning . before extrapolation of data to the clinical conditions can be done , the correspondence between capacity for tubule exposure / widening and intensity of connective tissue attachment should be further investigated in in vivo surveys . the comparison among four of the most frequently used chemical root conditioners according to their efficiency on smear layer removal and dentin tubule widening showed that citric acid was the most effective followed by tetracycline - hcl , phosphoric acid and edta . this information can be of value as an extra parameter for choosing one of them for root conditioning .

dental roots that have been exposed to the oral cavity and periodontal pocket environment present superficial changes , which can prevent connective tissue reattachment . demineralizing agents have been used as an adjunct to the periodontal treatment aiming at restoring the biocompatibility of roots.objectivethis study compared four commonly used demineralizing agents for their capacity of removing smear layer and opening dentin tubules . methodsfifty fragments of human dental roots previously exposed to periodontal disease were scaled and randomly divided into the following groups of treatment : 1 ) ca : demineralization with citric acid for 3 min ; 2 ) tc - hcl : demineralization with tetracycline - hcl for 3 min ; 3 ) edta : demineralization with edta for 3 min ; 4 ) pa : demineralization with 37% phosphoric acid for 3 min ; 5)control : rubbing of saline solution for 3 min . scanning electron microscopy was used to check for the presence of residual smear layer and for measuring the number and area of exposed dentin tubules . resultssmear layer was present in 100% of the specimens from the groups pa and control ; in 80% from edta group ; in 33.3% from tc - hcl group and 0% from ca group . the mean numbers of exposed dentin tubules in a standardized area were : tc - hcl=43.825.2 ; ca=39.337 ; pa=12.116.3 ; edta=4.47.5 and control=2.35.7 . the comparison showed significant differences between the following pairs of groups : tc - hcl and control ; tc - hcl and edta ; ca and control ; and ca and edta . the mean percentages of area occupied by exposed dentin tubules were : ca=0.120.17% ; tc - hcl=0.080.06% ; pa=0.030.05% ; edta=0.010.01% and control=00% . the ca group differed significantly from the others except for the tc - hcl group . conclusionthere was a decreasing ability for smear layer removal and dentin tubule widening as follows : ac > tc - hcl > pa > edta . this information can be of value as an extra parameter for choosing one of them for root conditioning .

INTRODUCTION MATERIAL AND METHODS Specimen preparation SEM analysis Quantitative and Qualitative Measurements RESULTS Group CA Group TC-HCl Group EDTA Group PA Control group DISCUSSION CONCLUSION

one of the goals of periodontal therapy is the predictable regeneration of the periodontium in areas previously affected by periodontal disease . histological and ultrastructural studies have demonstrated that dental roots that have been exposed to the oral cavity or to the periodontal pocket present reduced collagen fiber insertion , changes in their mineral density and root contamination by bacteria and its products . these alterations have become the rationale for the use of demineralizing agents as adjunct to periodontal therapy due to their potential for removing smear layer and exposing the underlying radicular collagen fibrils , funneling dentin tubules and modifying dentin permeability , restoring the biocompatibility of the roots . the most used demineralizing agents for these purposes are citric acid , phosphoric acid , ethylenediaminetetraacetic acid ( edta ) and tetracycline hydrochloride . some authors have measured the number and diameter of dentin tubules exposed after root conditioning in order to relate these parameters with potential for cell adhesion and for intertubular collagen exposure . ( 1992 ) have found a time - dependent increase in the mean dentin tubule orifice diameter after treating dentin surfaces with citric acid or tetracycline hcl and ruggeri , et al . ( 2007 ) stressed that the exposure of the dentin matrix of root surface allows the formation of a proper fibrin clot , which is a determinant factor for the positive outcome of the early wound healing events . to the best of our knowledge , there is no standardized study comparing several chemical root conditioners for their ability of smear layer removing and dentin tubule widening . therefore , the aim of this study was to contribute with reliable data to analyze and compare diseased dental root surfaces treated by manual scaling followed by conditioning with four commonly used demineralizing agents . the selected teeth had to meet the following inclusion criteria : 1 ) no history of scaling and root planing in the previous 6 months ; 2 ) proximal attachment loss of 5 mm or more ; 3 ) absence of decay lesions or restorations near the cementoenamel junction ( cej ) . the radicular fragments were randomly divided into 5 groups of 10 fragments each according to the treatment given to the scaled surface as follows : 1 ) ca : demineralization with a liquid solution of citric acid at 50% and ph1 ( pharmcia specfica ; farmcias e drogarias , bauru , sp , brazil ) for 3 min ; 2 ) tc - hcl : demineralization with a liquid solution of tetracycline hydrochloride at 50 mg / ml ( laboratrio teuto , anpolis , go , brazil ) for 3 min ; 3 ) edta : demineralization by a gel of edta at 24% ( prefgel ; biora ab , malm , sweden ) for 3 min ; 4 ) pa : demineralization with a liquid solution of 37% phosphoric acid ( pharmcia specfica ; farmcias e drogarias ) for 3 min ; 5 ) control : treatment with saline solution for 3 min . a : extracted teeth in which the diseased part of the root was identified by the absence of periodontal ligament remnants ( * ) ; b : tooth crown being transversely cut at the cementoenamel with a water - cooled high - speed bur ; c : radicular dish obtained by sectioning the root 2 mm away from the first cut in the apical direction ; d : pencil marks made 4 mm apart from each other on the mesial and distal surfaces of the dishes where grooves were made ( e ) , delimiting an area of 2 mm x 4 mm approximately ; f : scaling of the area determined in e ; g : mesial and distal halves of the dishes separated before receiving the burnishing of the demineralizing agents with a sterile cotton pellet ( h ) the dental fragments were prepared for sem analysis as described by braidotti , et al . the roots surfaces were examined for general morphologic characteristics and for the presence of smear layer at magnification of 1,000. the field shown at magnification of 1,000 was also taken as reference for the area in which the number of exposed dentin tubules was counted . the field shown at magnification of 2,000 was taken as reference for the total area ( 266 , 240 m ) in which the percentage of area occupied by the enlarged dentin tubules was calculated . the selected teeth had to meet the following inclusion criteria : 1 ) no history of scaling and root planing in the previous 6 months ; 2 ) proximal attachment loss of 5 mm or more ; 3 ) absence of decay lesions or restorations near the cementoenamel junction ( cej ) . the radicular fragments were randomly divided into 5 groups of 10 fragments each according to the treatment given to the scaled surface as follows : 1 ) ca : demineralization with a liquid solution of citric acid at 50% and ph1 ( pharmcia specfica ; farmcias e drogarias , bauru , sp , brazil ) for 3 min ; 2 ) tc - hcl : demineralization with a liquid solution of tetracycline hydrochloride at 50 mg / ml ( laboratrio teuto , anpolis , go , brazil ) for 3 min ; 3 ) edta : demineralization by a gel of edta at 24% ( prefgel ; biora ab , malm , sweden ) for 3 min ; 4 ) pa : demineralization with a liquid solution of 37% phosphoric acid ( pharmcia specfica ; farmcias e drogarias ) for 3 min ; 5 ) control : treatment with saline solution for 3 min . a : extracted teeth in which the diseased part of the root was identified by the absence of periodontal ligament remnants ( * ) ; b : tooth crown being transversely cut at the cementoenamel with a water - cooled high - speed bur ; c : radicular dish obtained by sectioning the root 2 mm away from the first cut in the apical direction ; d : pencil marks made 4 mm apart from each other on the mesial and distal surfaces of the dishes where grooves were made ( e ) , delimiting an area of 2 mm x 4 mm approximately ; f : scaling of the area determined in e ; g : mesial and distal halves of the dishes separated before receiving the burnishing of the demineralizing agents with a sterile cotton pellet ( h ) the dental fragments were prepared for sem analysis as described by braidotti , et al . the roots surfaces were examined for general morphologic characteristics and for the presence of smear layer at magnification of 1,000. the field shown at magnification of 1,000 was also taken as reference for the area in which the number of exposed dentin tubules was counted . the field shown at magnification of 2,000 was taken as reference for the total area ( 266 , 240 m ) in which the percentage of area occupied by the enlarged dentin tubules was calculated . dentin tubules were totally exposed at the 1,000 field and the average percentage of the area occupied by tubules was 0.12% ( ranging from 8.67 m to 159 m at the magnification of 2,000 ( figure 2b ) . a : representative specimen of the ca group in which no smear layer can be noted and dentin tubules are totally exposed ( 1,000 ) ; b : 2,000 magnification of the central part of a showing dentin tubules widened ; c : representative specimen of the tc - hcl group at 1,000 showing absence of smear layer and exposed dentin tubules ; d : 2,000 magnification of the central part of c showing widened dentin tubules ; e : representative specimen of edta group presenting smear layer and few exposed tubules ( 1,000 ) ; f : 2,000 magnification of the central part of e ; g : representative specimen of pa group at 1,000 of magnification covered by smear layer and some exposed tubules ; h : 2,000 magnification of the central part of g showing discrete widening of the tubules ; i : representative specimen of the control group at 1,000 showing smear layer without evident tubule exposure ; j : 2,000 magnification of the central part of i in which the tubules are not enlarged enough to measure their corresponding area even though all the tubules were exposed in 8 specimens of the tc - hcl group at the 1,000 field ( figure 2c ) , residual smear layer and some debris were present in 33.3% of them . the mean percentage of area occupied by exposed tubules was 0.08% ( ranging from 0 m to 48.18 m ) at the magnification of 2,000 ( figure 2d ) . the majority of the specimens of edta group presented with smear layer ( 80% ) and there were no exposed tubules 5 of them . the mean number of exposed tubules in this group was 4.4 at the 1,000 field ( figure 2e ) , occupying an area equivalent to 0.01% of the total ( ranging from 0 m to 5.82 m ) at the 2,000 of magnification ( figure 2f ) . although all specimens of this group showed smear layer , open tubules could be seen in 2 of them which were responsible for the mean number of 2.3 tubules for this group at the 1,000 magnification field ( figure 2i ) . the mean number of exposed dentin tubules by the different treatments decreased according to the following order : tc - hcl > ca > pa > edta nevertheless , statistically significant differences were only found when compared the groups tc - hcl , and c ; tc - hcl and edta ; ca and c and ca and edta ( table 1 ) . mean number of exposed dentin tubules and corresponding area after conditioning with the four different demineralizing agents same uppercase or lowercase letter in the same line means no statistically significant difference ( p>0.05 ) . when comparing the area occupied by dentin tubule openings , ca produced the greatest tubule widening followed by tc - hcl , pa , edta and saline ( table 1 ) . there was no statistically significant difference between ca and tc - hcl groups , and ca was different from all other groups . tc - hcl group differed only from group c. the exposure of dentin tubules in group c did not result in widening . dentin tubules were totally exposed at the 1,000 field and the average percentage of the area occupied by tubules was 0.12% ( ranging from 8.67 m to 159 m at the magnification of 2,000 ( figure 2b ) . a : representative specimen of the ca group in which no smear layer can be noted and dentin tubules are totally exposed ( 1,000 ) ; b : 2,000 magnification of the central part of a showing dentin tubules widened ; c : representative specimen of the tc - hcl group at 1,000 showing absence of smear layer and exposed dentin tubules ; d : 2,000 magnification of the central part of c showing widened dentin tubules ; e : representative specimen of edta group presenting smear layer and few exposed tubules ( 1,000 ) ; f : 2,000 magnification of the central part of e ; g : representative specimen of pa group at 1,000 of magnification covered by smear layer and some exposed tubules ; h : 2,000 magnification of the central part of g showing discrete widening of the tubules ; i : representative specimen of the control group at 1,000 showing smear layer without evident tubule exposure ; j : 2,000 magnification of the central part of i in which the tubules are not enlarged enough to measure their corresponding area even though all the tubules were exposed in 8 specimens of the tc - hcl group at the 1,000 field ( figure 2c ) , residual smear layer and some debris were present in 33.3% of them . the mean percentage of area occupied by exposed tubules was 0.08% ( ranging from 0 m to 48.18 m ) at the magnification of 2,000 ( figure 2d ) . the majority of the specimens of edta group presented with smear layer ( 80% ) and there were no exposed tubules 5 of them . the mean number of exposed tubules in this group was 4.4 at the 1,000 field ( figure 2e ) , occupying an area equivalent to 0.01% of the total ( ranging from 0 m to 5.82 m ) at the 2,000 of magnification ( figure 2f ) . although all specimens of this group showed smear layer , open tubules could be seen in 2 of them which were responsible for the mean number of 2.3 tubules for this group at the 1,000 magnification field ( figure 2i ) . the mean number of exposed dentin tubules by the different treatments decreased according to the following order : tc - hcl > ca > nevertheless , statistically significant differences were only found when compared the groups tc - hcl , and c ; tc - hcl and edta ; ca and c and ca and edta ( table 1 ) . mean number of exposed dentin tubules and corresponding area after conditioning with the four different demineralizing agents same uppercase or lowercase letter in the same line means no statistically significant difference ( p>0.05 ) . when comparing the area occupied by dentin tubule openings , ca produced the greatest tubule widening followed by tc - hcl , pa , edta and saline ( table 1 ) . there was no statistically significant difference between ca and tc - hcl groups , and ca was different from all other groups . tc - hcl group differed only from group c. the exposure of dentin tubules in group c did not result in widening . this study compared the 4 most commonly used chemical agents for root conditioning as adjunctive therapy for teeth affected by periodontitis . the presented data suggest that citric acid and tetracycline - hcl , in this particular way of use , are more effective in removing smear layer and in exposing and widening dentin tubules than phosphoric acid and edta . since register and burdick ( 1975 ) compared root conditioning with citric acid ( ph 1 for 2 - 3 min ) and other chemical substances and found optimal cementogenesis and connective tissue new attachment , several investigators have devoted considerable time studying conditioning agents to improve periodontal regeneration . tetracycline became one of the most widely used and studied demineralizing agents since in vitro studies of terranova , et al . ( 2000 ) concluded that concentrations between 50 mg / ml and 125 mg / ml might alter dentin surfaces by removing the smear layer and also maximize tubule openings in a short period if repeated applications are performed . it has also been shown that burnishing the demineralizing agent on the root surface results in efficient removal of smear layer and exposure of the underlying tubules due to demineralization action of fresh acid solution . the low ph of the saturated solutions of citric acid and tetracycline - hcl were suggested as one of the reasons for the reduced cellular insertion and for the unpredictability of the results , once it could denature the organic matrix of dentin . thus , edta at 12%-24% , neutral ph for 30 s to 3 min was introduced aiming at removing smear layer and widening dentin tubules without damaging biological structures . ( 2007 ) presented reliable findings using monoclonal antibodies and field emission in - lens scanning electron microscopy ( feisem ) showing that both citric acid and edta treatments are able to etch and expose collagen fibrils and proteoglycans without any degradation of dentin collagen matrix . five specimens ( 50% ) from the edta group had none of their dentin tubules exposed and only 2 were totally clean from smear layer . it must be emphasized that even though ca and tc - hcl did not differ significantly on tubule number and widening evaluations , tc - hcl could not be considered different from the other groups . another interesting finding was that ca and pa exposed similar numbers of dentin tubules , but ca produced greater enlargement as reflected by the area measurements . this occurrence can be attributed to the extended time of acid contact with the roots ( 3 min ) . fibrin clot adhesion was better supported by the ca - treated than edta - treated dentin surfaces and forces produced by three 5-min rinses in pbs under agitation on a rotary shaker table partially removed the fibrin clot from edta - treated surfaces but not from ca - treated surfaces . the number and diameter of exposed dentin tubules in laser - conditioned roots were calculated in sem micrographs by herrero , et al . ( 2009 ) who related increased numbers and diameters to better root conditioning , once widening tubules openings causes higher exposure of the underlying dentin favoring connective tissue attachment . gamal and mailhot ( 2003 ) also observed that periodontal ligament fibroblasts adhere and differentiate on edta - conditioned roots surfaces that were free from smear layer and presented exposed round to oval dentin tubule orifices . nevertheless , there is no study comparing the number of exposed dentin tubules and their corresponding area after conditioning with several conditioners for comparison with our data . before extrapolation of data to the clinical conditions can be done , the correspondence between capacity for tubule exposure / widening and intensity of connective tissue attachment should be further investigated in in vivo surveys . the comparison among four of the most frequently used chemical root conditioners according to their efficiency on smear layer removal and dentin tubule widening showed that citric acid was the most effective followed by tetracycline - hcl , phosphoric acid and edta . this information can be of value as an extra parameter for choosing one of them for root conditioning .

prostaglandins ( pgs ) are found in virtually all tissues where they exert a wide variety of functions including modulating smooth muscle activity , haemostasis and cytoprotection . it has been known for nearly half a century that , in the bladder , pgs are released into the general circulation by the bladder in response to distension . it has been established that the bladder pgs originate in both the urothelial and muscle layers [ 25 ] . the precise role of this endogenous pg is not known but it is well - documented that exogenous pgs alter bladder activity both in vivo and in vitro influencing voiding and smooth muscle contractility . this occurs in different species : human [ 69 ] , rat [ 1012 ] , guinea pig [ 1315 ] , rabbit [ 1619 ] and monkey [ 20 , 21 ] . the underlying mechanisms , which involve the pgs to alter voiding patterns and induce smooth muscle contraction , are not known . regarding pg - induced changes in voiding frequency it was envisaged that they might act directly on the afferent nerves to modulate firing and so trigger micturition at lower bladder volumes [ 2224 ] . with regard to a direct action on the muscle , it was pointed out that they can be co - released with acetylcholine at efferent nerve endings and so directly contribute to muscle excitation [ 20 , 21 ] . alternatively , they might act indirectly on pre - synaptic motor terminals to affect the release of excitatory transmitters [ 11 , 12 ] . it was considered they might also inhibit acetylcholine esterase or enhance myogenic bladder activity [ 6 , 22 ] . in other organs , pg production in fibroblasts resulted in a decrease in collagen production . it has also been reported that prostaglandin production by fibroblasts plays a role in tumour necrosis . in the intestine , powell et al . found that interstitial cells in the subepithelial space of the intestine are important in the organogenesis of the intestine , and secretion of prostaglandins by these interstitial cells is an important factor of this mechanism . therefore , it must be taken into consideration that , next to effects on contractility , pgs might have additional functions in the bladder , e.g. a role in cell proliferation . recently , a concept was proposed which attempts to integrate the effects of pg on the sensory elements of bladder control with its motor actions . using the isolated bladder it has been argued previously that autonomous activity is part of a motor / sensory system operating within the bladder wall whereby localized contractions of the bladder wall stimulate firing in afferent nerves contributing to sensation [ 29 , 30 ] . pg - induced modulation of the autonomous activity would thus increase bladder sensation and so modify voiding activity . it has been hypothesized that it is generated within and distributed by a network of specialized cells in the bladder wall : interstitial cells [ 30 , 31 ] . the precise identification and definition of what is an interstitial cell in the bladder is , at present under discussion . the initial description of interstitial cells was based on their ability to show a rise in cgmp in response to nitric oxide donors [ 31 , 32 ] . different sub - types of cgmp interstitial cell have been identified lying principally in the sub - urothelial layer and in the outer muscle layer . finally , the stem cell factor receptor ckit , which identifies interstitial cells in the gut , has been reported to mark cells in the bladder . however direct evidence for the physiological role of interstitial cells is , so far , limited and comes from experiments which show that nitric oxide ( no ) , which generates a rise in cgmp in the interstitial cells , also abolishes autonomous activity . since exogenous pg modulates autonomous activity further insights into the origin of this activity and possible links to interstitial cells may be got from a detailed understanding of where pgs are synthesized and where they act in the bladder . pgs are synthesized by the two distinct enzymes : cyclo - oxygenase type i ( cox i ) and cyclo - oxygenase type ii ( cox ii ) . cox i is associated with cell somata in the urothelium and with a network of cells running over the muscle bundles . a detailed description of the different cell types expressing cox - i is not yet available . therefore , the present experiments were done to characterize the cells expressing cox i in the normal bladder and to determine the relationship between these cells to the networks of interstitial cells . the distribution and characteristics of cox ii expression in the bladder is the subject of a separate paper . guinea pigs ( 7 male , weight 270350 g ) were killed by stunning and exsanguinations . the urinary bladder was removed and placed in ice - cold kreb s solution containing 121.1 mm nacl , 1.87 mm kcl , 1.2 mm cacl2 , 1.15 mm mgso4 , 25 mm nahco3 , 1.17 mm kh2po4 , 11.0 mm glucose , bubbled with 5% co2 and 95% o2 . the bladder wall was cut into 3 or 4 sections from the base to the dome . the procedures for isolation , stimulation with the no donor diethylamine - nonoate ( deano sigma - aldrich ) , and detection of cgmp - immunoreactivity were as described previously [ 28 , 31 ] . preparation of the cryostat sections and immunocytochemical procedures were as described before [ 28 , 31 ] . specificity studies on the sheep or rabbit anti - formaldehyde - fixed - cgmp antisera have been published before and preabsorbtion studies to ascertain the specificity of the cox i antiserum have been published before also . primary antibodies used were goat polyclonal antibody to cox i ( 1:2000 ; santa cruz biotech , santa cruz , ca , usa ) , rabbit anti - nnos antiserum ( 1:3000 , diasorin , diasorin s.p.a . , saluggia , italy ) , mouse anti - vimentin antiserum ( 1:5000 ; sigma / aldrich ) . secondary antibodies were alexa fluor 488 donkey anti - sheep igg ( h+l , 1:100 ) and donkey anti - mouse conjugate ( molecular probes , 1:100 ) ; alexa fluor 594 donkey anti - rabbit igg conjugate ( molecular probes , 1:100 ) ; cy3 donkey anti - goat igg conjugate ( jackson immunoresearch , newmarket , uk ; 1:800 ) . sections were analysed using an olympus ax70 fluorescence microscope , equipped with a narrow band - pass mniba - filter for the detection of alexa 488 , and for detection of cy3 and alexa 594 we used a filter with a narrow excitation band , the u - m41007a filter ( both from chroma technologies , rockingham , vt , usa ) . the microscope was equipped with a cooled charge - coupled device , the olympus digital video camera f - view . images were stored digitally as 16 bits images by using the computer program cell ( soft imaging systems , olympus , germany ) . colour images were produced by combination of the original grey values photographs using the cell - p program . images were arranged using the program adobe photoshop 7.0.1 ( san jose , ca , usa ) without further processing . the key basic observations regarding the distribution of cox i - ir in the bladder wall are illustrated in fig . cox i - ir cells are seen within the urothelium and also associated with a network of cells , which extends from the lamina propria into the inner muscle layers . note that there are fewer cox i - ir cells associated with the outer muscle layers . these observations are illustrated in greater detail in panels 1 and 2 , which show the sub - urothelial region ( 1 ) and outer muscle layer ( 2 ) at higher magnification . the localization of cox i - ir , vimentin cells and no - responsive cgmp cells in the guinea pig bladder . ( a ) , ( b ) and ( c ) show , respectively , images stained with antibodies to cox i , vimentin and cgmp . in each panel , two regions are identified ( 1 and 2 ) located in the outer and inner regions of the bladder wall . for ( a c ) , these identified regions are shown at greater magnification in the adjacent panels . more cox i - ir cells are seen in the inner regions of the bladder . in ( b ) , vimentin cells are seen in both the outer and inner layers . in ( c ) , the presence of cgmp cells is clearly seen . no cgmp cells are seen associated with the inner muscle layer but the umbrella cells and sub - urothelial cells are cgmp . calibrations bars : 150 m in ( a ) and 40 m in ( b ) . cox i - ir is therefore associated with two distinct structures : the urothelium and a network of cells in the lamina propria and around the muscle . the patter of distribution of these network cells bears a strong resemblance to the distribution of vimentin positive ( vim ) cells in the muscle layers bladder wall but not the distribution of no - responsive cgmp interstitial cells ( fig . note that the cgmp interstitial cells are found in the outer muscle layers where there is considerably less cox i - ir . these observations on cox i - ir in the urothelium and associated with the networks of interstitial cells are described in more detail below . figure 2a illustrates a section showing the urothelium and sub - urothelial region , double stained for cox i ( red ) and cgmp ( green ) . figure 2b shows a similar section but from a different bladder illustrating the combined colour image ( top ) and the component cox i ( middle ) and cgmp ( lower ) images . as reported previously , the umbrella cells lying on the surface of the urothelium and the sub - urothelial interstitial cell layer closes to the urothelium are sensitive to exogenous no and demonstrate a rise in cgmp ( cgmp ) . it is also clear that cox i - ir is associated with intense staining in the intermediate and basal layers of the urothelium ( buc ) and weekly in cells in the lamina propria ( lamina propria interstitial cells ( lp - ics ) ) . figure 2b shows , in greater detail that , in this region of the bladder wall , the majority of the sub - urothelial cgmp interstitial cells do not have cox i - ir . cgmp- and cox i - ir associated with the urothelium in the guinea pig bladder . panel ( a ) shows an image of the urothelium of the lateral wall stained for cox i - ir ( red ) and cgmp ( green ) . the preparation was stimulated with a no donor to elevate cgmp levels in responsive cells prior to fixation . the umbrella cells on the surface of the urothelium ( umb ) and cells in the sub - urothelial space , the sub - urothelial interstitial cells ( su - ics ) are cgmp . cells in the basal regions of the urothelium ( basal urothelial cells : buc ) are stained intensely with the cox i antibody . in addition , cox i - ir cells are also found in the lamina propria interstitial cells ( ip - ics ) . ( b ) illustrates a section from a different bladder processed and stained in the same way as section ( a ) . the middle and lower panels show cox i - ir and cgmp - ir , respectively . these two panels illustrate that the cgmp su - ics are negative for cox i - ir . they also show that the cox i - ir cells in the buc are separated from the cox i - ir ip - ics . calibration bars 50 m in ( a ) and 30 m in ( b ) . the cells of the basal layer of the urothelium express neuronal nitric oxide synthase ( nnos ) . the relationship between these cells and those with cox i - ir is shown in fig . the sections were double stained with nnos ( green ) and cox i ( red ) . these cells are also cox i - ir ( showing green / yellow in the combined sections ) . note that in the intermediate urothelial layer the cells are cox i - ir but not nnos ( fig . thus , there are two types of cox i - ir cell in the urothelium : basal cox i - ir / nnos and intermediate cox i - ir / nnos . the identification of cells expressing neuronal nitric oxide synthase ( nnos ) and cox i - ir in the urothelial layer of the guinea pig bladder . sections were co - stained with antibodies to nnos ( green ) and cox i ( red ) . ( a ) shows an example of the staining pattern associated with the urothelium . nnos cells ( # ) are seen to be located within a single layer in the basal urothelium ( buc ) . note that the cox i - ir extends into further cell layers within the urothelium ( ) . ( b ) shows a further example from a different bladder . the combined image and the individually stained images are shown . the location of nnos to a single layer in the basal urothelium is clearly seen while the more diffuse location of the cox i - ir is apparent . calibration bars : 30 m in ( a ) and ( b ) . the layer of densely packed cells lying immediately below the urothelium , the sub - urothelial interstitial cells ( su - ics ) , are responsive to no with an elevation in cgmp . figure 4 illustrates a section double labelled to show these cgmp cells ( green ) and the vimentin cells ( red ) . cgmp umbrella cells are seen on the surface of the urothelium but there were no vim+ cells in any cell type within the urothelium . in contrast , the cells immediately below the urothelium , the su - ics , were vim and cgmp . vimentin staining revealed a network of intracellular fibres , whereas the cgmp staining was most intense within the cell bodies ( fig . processes of these cells were seen to contain vim fibres but these structures were often not strongly cgmp indicating that there are intracellular gradients of cgmp . vim cells were seen in the layer of the lamina propria between the su - ic layer and the muscle layer ( fig . these cells did not demonstrate a cgmp signal and can be described as lamina propria interstitial cells ( lp - ics ) . thus , there appear to be different types of interstitial cell within the lamina propria : a dense region of cgmp / vim and sparse cgmp / vim cells . the location of vimentin structures in the urothelium and sub - urothelial space . sections were double labelled with antibodies to show cgmp ( green ) and vimentin ( red ) . panel ( a ) shows a section from the lateral wall of the bladder illustrating the urothelium and sub - urothelial space . the umbrella cells ( umb ) and suburothelial interstitial cells ( su - ics ) are cgmp . intense staining is seen associated with the cells immediately below the urothelium while structures laying deeper within the lamina propria are vimentin but more defuse . ( b ) and ( c ) illustrate examples of the cells in the sub - urothelial layer at higher magnification , showing the combined colour image ( upper panel : cgmp - green , vimentin - red ) , the cgmp image alone ( middle panel ) and vimentin image alone ( lower panel ) . vimentin staining is associated with the cell bodies of these cells being highly concentrated around the cell nuclei ( # ) . calibration bars 80 m in ( a ) and 20 m in ( b ) and ( c ) . an additional feature of the cox i / vimentin double staining becomes apparent when studying in detail the diffuse network of vim cells in the region of the lamina propria immediately below the su - ic and above the muscle layer ( fig . these cells appear to form a distinct network of inter - connecting cells contiguous with the su - ic layer and with vim cells within the muscle layer ( see below ) . in this section , but , cox i - ir is seen within the cell bodies of the vim lp - ics ( fig . 5b ) . identification of cox i - ir and vimentin in the lamina propria . sections were double labelled with antibodies to cox i ( red ) and vimentin ( green ) . ( a ) shows a low power image showing the urothelium , sub - urothelial layer and the lamina propria . the cox i - ir cells are clearly visible within the urothelium ( uro ) , the suburothelial interstitial cells ( su - ics ) and the lamina propria ( ip - ics ) . below the su - ics , there is a network of interstitial cells within the lamina propria ( ip - ics ) . structures positive for vimentin and with cox i - ir are seen within this layer . ( b ) shows a section of the image in ( a ) at higher magnification . the panel on the left shows the doubled labelled image ( vimentin - green , cox i - red ) , the middle panel shows the vimentin image and the right panel shows the cox - i image . the basal urothelial layer ( 1 ) , sub - urothelial layer ( 2 ) the staining pattern of the vimentin cells in the lp is highly suggestive for a network of vimentin structures ( 4 ) . in addition , there appear to be cox i - ir cells ( 3).these cells are spindle shaped and are weakly positive for vimentin . calibration bars 40 m in ( a ) and 20 m in ( b ) . regions of the lateral wall , particularly towards the bladder base , could be found in which the density of lp - ics was high ( figs . 6 and 7 ) . in these regions , two different cell types were readily identified based on the expression of vimentin and cox i - ir . small bipolar cells with round cell bodies , which were cox i - ir but did not stain strongly for vimentin ( * ) ( fig . 6b ) and larger complex cells with multiple processes which showed cox i - ir and a diffuse network of vimentin fibres ( ( ) and fig . the sections were stained with antibodies to cox i ( red ) and vimentin ( green ) . the cox i - ir of the urothelium ( uro ) and spindle cells in the lamina propria are seen ( * ) ( see also fig . 5 ) . large irregular cells are also seen which are cox i - ir and which have an extensive diffuse network of vimentin fibres ( ) . ( b ) and ( c ) show selected areas of the image in ( a ) with the individual images of cox i ( middle panel ) and vimentin ( lower panel ) . in ( b ) , the cox i - ir cells are seen to have little or no vimentin staining . the edges of the cox i - ir cells are indicated by the arrows and ( c ) . ( c ) illustrates the larger cells with the diffuse vimentin ' network in the lamina propria . calibration bars 20 m in ( a ) , ( b ) and ( c ) . sections were double stained for cox i ( red ) and vimentin ( green ) . the cox i - ir cells in the urothelium ( uro ) and lamina propria ( ip - ics ) are seen . the vimentin fibres of the sub - urothelial cell layer ( su - ics ) and within the cell network in the lamina propria are also apparent . ( a ) shows a network of vimentin fibres which are associated with the muscle bundles . collections of cox i - ir cells are seen between the muscle bundles ( n ) . nodes. ( b ) and ( c ) show examples of the cox i - ir cells and their relationship to the vimentin fibres . ( b ) and ( c ) showing combined colour image ( upper panel : vimentin - green , cox i - red ) , the cox i image alone ( middle panel ) and the vimentin image alone ( lower panel ) . the cox i - ir cells ( 1 ) are weakly vimentin and are associated with the vimentin processes ( 2 ) of other cells . calibration bars 240 m in ( a ) and 10 m in ( b ) and ( c ) . the network of vim fibres was observed to continue from the lamina propria into the inner smooth muscle layer where they run primarily on the surface of the muscle bundles ( fig . 7 ) . here , the vim cells appear on the surface of the muscle bundles and so can be described as surface muscle interstitial cell ( sm - ics ) . within this network , cell bodies were apparent , which were cox i - ir but that did not stain strongly for vimentin . examples of such cells are shown in fig . 7b and c. at the junction of the lamina propria and inner smooth muscle cell layer , small clusters of cox i - ir cell bodies were often observed ( fig . the cell bodies in these clusters did not stain strongly for vimentin but were in close proximity to vimentin positive cell processes ( fig . these collections of cell bodies , which have the appearance of nodes , were also apparent , although fewer in number and with fewer cells , lying between the muscle bundles of the inner muscle layer ( see fig . sections were double stained with antibodies cox i ( red ) and vimentin ( green ) . collections of cox i - ir structures ( nodes ) are seen associated with collections of vimentin fibres . ( b ) and ( c ) show selected areas of the image in ( a ) with the associated individual images to cox i ( middle panel ) and vimentin ( lower panel ) . calibration bars 50 m in ( a ) and 30 m in ( b ) and ( c ) . the network of vim sm - ics was observed to extend into the outer muscle layers of the bladder wall ( figs . 1 and 9 ) . however , in this outer region there are few cell bodies that are cox i - ir . this points out that there must be different types of sm - ic associated with the inner and outer muscle layers . 10 , which shows sections double labelled for cgmp ( green ) and vimentin ( red ) . as has been reported previously , the sm - ics of the outer muscle layers respond to no with a rise in cgmp . the cells bodies of these outer sm - ics are clearly seen but these are not cox i - ir . in contrast , there are few cgmp cells in the inner muscle layers ( see also fig . no - responsive cells producing cgmp are also found within the muscle bundles of the outer muscle layer : intramuscular interstitial cells ( im - ics ) ) . the distribution of cox i - ir and cgmp cells in the outer muscle layers . ( a ) shows an image double labelled with antibodies to cgmp ( green ) and cox i ( red ) . cgmp cells are seen in the outer muscle layer ( oml ) while there are no cells associated with the inner muscle layer ( iml ) . cgmp cells ( * ) and cox i - ir cells ( # ) are identified . ( b ) and ( c ) show regions of the image in ( a ) of the outer and inner layers respectively at higher magnification , showing the combined colour image ( upper panel : cgmp - green , cox i - red ) , the cgmp image alone ( middle panel ) and cox i image alone ( lower panel ) . in ( b ) , cgmp cells are clearly seen ( * ) while cox i - ir cells are scarce ( # ) . ( c ) shows the network of cox i - ir cells in the iml and the absence of cgmp staining . calibration bars 120 m in ( a ) and 40 m in ( b ) and ( c ) . the sections are double labelled with antibodies to cgmp ( green ) and vimentin ( red ) . ( a ) shows a low - power image identifying the outer ( oml ) and inner muscle layers ( iml ) . the network of cgmp ics associated with the outer layer and the network of vimentin cells in the inner ( * ) and outer layers are readily seen . the different types of cgmp ics are indicated by the arrows : muscle coat interstitial cells ( mc - ics ) , surface muscle interstitial cells ( sm - ics ) and intra - muscular interstitial cells ( im - ics ) . ( b ) , ( c ) and ( d ) illustrate regions of the image in ( a ) at higher magnification and showing combined colour image ( left panel : cgmp - green , vimentin - red ) , the cgmp image alone ( middle panel ) and vimentin image alone ( left panel ) . ( d ) illustrates that there is a variation on the intensity of cgmp and vimentin staining . all im - ics express vimentin . however , there are vimentin cells which are strongly cgmp ( 1 ) while others have little cgmp immunostaining ( 2 and 3 ) . calibration bars , 100 m in ( a ) , 40 m in ( b ) and 20 m in ( c ) and ( d ) . thus , these data support the idea that there are different sub - types of interstitial cell associated with the lamina propria and inter - muscular spaces in the guinea pig bladder . the different cell types which have been identified and described above , based on the staining for cgmp , vimentin , cox i and nnos , are summarized in table 1 . location of cgmp , vimentin , cox i and nnos in the bladder ip - ics : lamina propria interstitial cells - lp - ics#1 : lamina propria interstitial cells type # 1 ( only vimentin positive ip - ics ) - ip - ics#2 : lamina propria interstitial cells type # 2 ( only cox i positive ip - ics ) - lp - ics#3 : lamina propria interstitial cells type # 3 ( ip - ics that are both vimentin and cox i positive ) - sm - ics : surface muscle interstitial cells - sm - ics#1 : surface muscle interstitial cells type # 1 ( vimentin positive sm - ics ) - sm - ics#2 : surface muscle interstitial cells type # 2 ( cox i positive sm - ics ) - im - ics : intramuscular interstitial cells - mc - ics : muscle coat interstitial cells figure 2a illustrates a section showing the urothelium and sub - urothelial region , double stained for cox i ( red ) and cgmp ( green ) . figure 2b shows a similar section but from a different bladder illustrating the combined colour image ( top ) and the component cox i ( middle ) and cgmp ( lower ) images . as reported previously , the umbrella cells lying on the surface of the urothelium and the sub - urothelial interstitial cell layer closes to the urothelium are sensitive to exogenous no and demonstrate a rise in cgmp ( cgmp ) . it is also clear that cox i - ir is associated with intense staining in the intermediate and basal layers of the urothelium ( buc ) and weekly in cells in the lamina propria ( lamina propria interstitial cells ( lp - ics ) ) . figure 2b shows , in greater detail that , in this region of the bladder wall , the majority of the sub - urothelial cgmp interstitial cells do not have cox i - ir . cgmp- and cox i - ir associated with the urothelium in the guinea pig bladder . panel ( a ) shows an image of the urothelium of the lateral wall stained for cox i - ir ( red ) and cgmp ( green ) . the preparation was stimulated with a no donor to elevate cgmp levels in responsive cells prior to fixation . the umbrella cells on the surface of the urothelium ( umb ) and cells in the sub - urothelial space , the sub - urothelial interstitial cells ( su - ics ) are cgmp . cells in the basal regions of the urothelium ( basal urothelial cells : buc ) are stained intensely with the cox i antibody . in addition , cox i - ir cells are also found in the lamina propria interstitial cells ( ip - ics ) . ( b ) illustrates a section from a different bladder processed and stained in the same way as section ( a ) . the middle and lower panels show cox i - ir and cgmp - ir , respectively . these two panels illustrate that the cgmp su - ics are negative for cox i - ir . they also show that the cox i - ir cells in the buc are separated from the cox i - ir ip - ics . calibration bars 50 m in ( a ) and 30 m in ( b ) . the cells of the basal layer of the urothelium express neuronal nitric oxide synthase ( nnos ) . the relationship between these cells and those with cox i - ir is shown in fig . the sections were double stained with nnos ( green ) and cox i ( red ) . these cells are also cox i - ir ( showing green / yellow in the combined sections ) . note that in the intermediate urothelial layer the cells are cox i - ir but not nnos ( fig . thus , there are two types of cox i - ir cell in the urothelium : basal cox i - ir / nnos and intermediate cox i - ir / nnos . the identification of cells expressing neuronal nitric oxide synthase ( nnos ) and cox i - ir in the urothelial layer of the guinea pig bladder . sections were co - stained with antibodies to nnos ( green ) and cox i ( red ) . nnos cells ( # ) are seen to be located within a single layer in the basal urothelium ( buc ) . note that the cox i - ir extends into further cell layers within the urothelium ( ) . ( b ) shows a further example from a different bladder . the combined image and the individually stained images are shown . the location of nnos to a single layer in the basal urothelium is clearly seen while the more diffuse location of the cox i - ir is apparent . the layer of densely packed cells lying immediately below the urothelium , the sub - urothelial interstitial cells ( su - ics ) , are responsive to no with an elevation in cgmp . figure 4 illustrates a section double labelled to show these cgmp cells ( green ) and the vimentin cells ( red ) . cgmp umbrella cells are seen on the surface of the urothelium but there were no vim+ cells in any cell type within the urothelium . in contrast , the cells immediately below the urothelium , the su - ics , were vim and cgmp . vimentin staining revealed a network of intracellular fibres , whereas the cgmp staining was most intense within the cell bodies ( fig . processes of these cells were seen to contain vim fibres but these structures were often not strongly cgmp indicating that there are intracellular gradients of cgmp . vim cells were seen in the layer of the lamina propria between the su - ic layer and the muscle layer ( fig . 4a ) . these cells did not demonstrate a cgmp signal and can be described as lamina propria interstitial cells ( lp - ics ) . thus , there appear to be different types of interstitial cell within the lamina propria : a dense region of cgmp / vim and sparse cgmp / vim cells . the location of vimentin structures in the urothelium and sub - urothelial space . sections were double labelled with antibodies to show cgmp ( green ) and vimentin ( red ) . panel ( a ) shows a section from the lateral wall of the bladder illustrating the urothelium and sub - urothelial space . the umbrella cells ( umb ) and suburothelial interstitial cells ( su - ics ) are cgmp . intense staining is seen associated with the cells immediately below the urothelium while structures laying deeper within the lamina propria are vimentin but more defuse . ( b ) and ( c ) illustrate examples of the cells in the sub - urothelial layer at higher magnification , showing the combined colour image ( upper panel : cgmp - green , vimentin - red ) , the cgmp image alone ( middle panel ) and vimentin image alone ( lower panel ) . vimentin staining is associated with the cell bodies of these cells being highly concentrated around the cell nuclei ( # ) . calibration bars 80 m in ( a ) and 20 m in ( b ) and ( c ) . an additional feature of the cox i / vimentin double staining becomes apparent when studying in detail the diffuse network of vim cells in the region of the lamina propria immediately below the su - ic and above the muscle layer ( fig . these cells appear to form a distinct network of inter - connecting cells contiguous with the su - ic layer and with vim cells within the muscle layer ( see below ) . in this section , no cox i - ir cells are visible within the su - ic layer . but , cox i - ir is seen within the cell bodies of the vim lp - ics ( fig . 5b ) . identification of cox i - ir and vimentin in the lamina propria . sections were double labelled with antibodies to cox i ( red ) and vimentin ( green ) . ( a ) shows a low power image showing the urothelium , sub - urothelial layer and the lamina propria . the cox i - ir cells are clearly visible within the urothelium ( uro ) , the suburothelial interstitial cells ( su - ics ) and the lamina propria ( ip - ics ) . below the su - ics , there is a network of interstitial cells within the lamina propria ( ip - ics ) . structures positive for vimentin and with cox i - ir are seen within this layer . ( b ) shows a section of the image in ( a ) at higher magnification . the panel on the left shows the doubled labelled image ( vimentin - green , cox i - red ) , the middle panel shows the vimentin image and the right panel shows the cox - i image . the basal urothelial layer ( 1 ) , sub - urothelial layer ( 2 ) the staining pattern of the vimentin cells in the lp is highly suggestive for a network of vimentin structures ( 4 ) . in addition , there appear to be cox i - ir cells ( 3).these cells are spindle shaped and are weakly positive for vimentin . calibration bars 40 m in ( a ) and 20 m in ( b ) . regions of the lateral wall , particularly towards the bladder base , could be found in which the density of lp - ics was high ( figs . 6 and 7 ) . in these regions , two different cell types were readily identified based on the expression of vimentin and cox i - ir . small bipolar cells with round cell bodies , which were cox i - ir but did not stain strongly for vimentin ( * ) ( fig . 6b ) and larger complex cells with multiple processes which showed cox i - ir and a diffuse network of vimentin fibres ( ( ) and fig . the sections were stained with antibodies to cox i ( red ) and vimentin ( green ) . the cox i - ir of the urothelium ( uro ) and spindle cells in the lamina propria are seen ( * ) ( see also fig large irregular cells are also seen which are cox i - ir and which have an extensive diffuse network of vimentin fibres ( ) . ( b ) and ( c ) show selected areas of the image in ( a ) with the individual images of cox i ( middle panel ) and vimentin ( lower panel ) . in ( b ) , the cox i - ir cells are seen to have little or no vimentin staining . the edges of the cox i - ir cells are indicated by the arrows and ( c ) . ( c ) illustrates the larger cells with the diffuse vimentin ' network in the lamina propria . calibration bars 20 m in ( a ) , ( b ) and ( c ) . a network of vimentin processes around the muscle bundles of the inner muscle layer . sections were double stained for cox i ( red ) and vimentin ( green ) . the cox i - ir cells in the urothelium ( uro ) and lamina propria ( ip - ics ) are seen . the vimentin fibres of the sub - urothelial cell layer ( su - ics ) and within the cell network in the lamina propria are also apparent . ( a ) shows a network of vimentin fibres which are associated with the muscle bundles . collections of cox i - ir cells are seen between the muscle bundles ( n ) . nodes. ( b ) and ( c ) show examples of the cox i - ir cells and their relationship to the vimentin fibres . ( b ) and ( c ) showing combined colour image ( upper panel : vimentin - green , cox i - red ) , the cox i image alone ( middle panel ) and the vimentin image alone ( lower panel ) . the cox i - ir cells ( 1 ) are weakly vimentin and are associated with the vimentin processes ( 2 ) of other cells . calibration bars 240 m in ( a ) and 10 m in ( b ) and ( c ) . the network of vim fibres was observed to continue from the lamina propria into the inner smooth muscle layer where they run primarily on the surface of the muscle bundles ( fig . 7 ) . here , the vim cells appear on the surface of the muscle bundles and so can be described as surface muscle interstitial cell ( sm - ics ) . within this network , cell bodies were apparent , which were cox i - ir but that did not stain strongly for vimentin . examples of such cells are shown in fig . 7b and c. at the junction of the lamina propria and inner smooth muscle cell layer , small clusters of cox i - ir cell bodies were often observed ( fig . the cell bodies in these clusters did not stain strongly for vimentin but were in close proximity to vimentin positive cell processes ( fig . these collections of cell bodies , which have the appearance of nodes , were also apparent , although fewer in number and with fewer cells , lying between the muscle bundles of the inner muscle layer ( see fig . sections were double stained with antibodies cox i ( red ) and vimentin ( green ) . collections of cox i - ir structures ( nodes ) are seen associated with collections of vimentin fibres . ( b ) and ( c ) show selected areas of the image in ( a ) with the associated individual images to cox i ( middle panel ) and vimentin ( lower panel ) . calibration bars 50 m in ( a ) and 30 m in ( b ) and ( c ) . the network of vim sm - ics was observed to extend into the outer muscle layers of the bladder wall ( figs . 1 and 9 ) . however , in this outer region there are few cell bodies that are cox i - ir . this points out that there must be different types of sm - ic associated with the inner and outer muscle layers . 10 , which shows sections double labelled for cgmp ( green ) and vimentin ( red ) . as has been reported previously , the sm - ics of the outer muscle layers respond to no with a rise in cgmp . the cells bodies of these outer sm - ics are clearly seen but these are not cox i - ir . in contrast , there are few cgmp cells in the inner muscle layers ( see also fig . no - responsive cells producing cgmp are also found within the muscle bundles of the outer muscle layer : intramuscular interstitial cells ( im - ics ) ) . the distribution of cox i - ir and cgmp cells in the outer muscle layers . ( a ) shows an image double labelled with antibodies to cgmp ( green ) and cox i ( red ) . cgmp cells are seen in the outer muscle layer ( oml ) while there are no cells associated with the inner muscle layer ( iml ) . cgmp cells ( * ) and cox i - ir cells ( # ) are identified . ( b ) and ( c ) show regions of the image in ( a ) of the outer and inner layers respectively at higher magnification , showing the combined colour image ( upper panel : cgmp - green , cox i - red ) , the cgmp image alone ( middle panel ) and cox i image alone ( lower panel ) . in ( b ) , cgmp cells are clearly seen ( * ) while cox i - ir cells are scarce ( # ) . ( c ) shows the network of cox i - ir cells in the iml and the absence of cgmp staining . calibration bars 120 m in ( a ) and 40 m in ( b ) and ( c ) . the sections are double labelled with antibodies to cgmp ( green ) and vimentin ( red ) . ( a ) shows a low - power image identifying the outer ( oml ) and inner muscle layers ( iml ) . the network of cgmp ics associated with the outer layer and the network of vimentin cells in the inner ( * ) and outer layers are readily seen . the different types of cgmp ics are indicated by the arrows : muscle coat interstitial cells ( mc - ics ) , surface muscle interstitial cells ( sm - ics ) and intra - muscular interstitial cells ( im - ics ) . ( b ) , ( c ) and ( d ) illustrate regions of the image in ( a ) at higher magnification and showing combined colour image ( left panel : cgmp - green , vimentin - red ) , the cgmp image alone ( middle panel ) and vimentin image alone ( left panel ) . ( d ) illustrates that there is a variation on the intensity of cgmp and vimentin staining . all im - ics express vimentin . however , there are vimentin cells which are strongly cgmp ( 1 ) while others have little cgmp immunostaining ( 2 and 3 ) . calibration bars , 100 m in ( a ) , 40 m in ( b ) and 20 m in ( c ) and ( d ) . thus , these data support the idea that there are different sub - types of interstitial cell associated with the lamina propria and inter - muscular spaces in the guinea pig bladder . the different cell types which have been identified and described above , based on the staining for cgmp , vimentin , cox i and nnos in the bladder ip - ics : lamina propria interstitial cells - lp - ics#1 : lamina propria interstitial cells type # 1 ( only vimentin positive ip - ics ) - ip - ics#2 : lamina propria interstitial cells type # 2 ( only cox i positive ip - ics ) - lp - ics#3 : lamina propria interstitial cells type # 3 ( ip - ics that are both vimentin and cox i positive ) - sm - ics : surface muscle interstitial cells - sm - ics#1 : surface muscle interstitial cells type # 1 ( vimentin positive sm - ics ) - sm - ics#2 : surface muscle interstitial cells type # 2 ( cox i positive sm - ics ) - im - ics : intramuscular it has been known for over 30 years that pgs are released from the bladder in response to stretch , the pgs coming from both the urothelium and muscle layers [ 19 , 24 , 35 , 36 ] . found that intravesically administrated ketoprofen , a non - selective cox inhibitor , reduced detrusor instability . based on these findings , it has been argued that the pgs play central roles not only in bladder physiology but also in the generation of bladder patho - physiology [ 25 , 3840 ] . this led to the trial of cyclo - oxygenase inhibitors for the treatment of bladder over - activity [ 37 , 41 ] . one indication as to the possible role of pgs has come from experiments involving the infusion of pg into the bladder lumen . when this is done , it gives rise to an increase in micturition frequency and the incidence of non - voiding phasic contractions between voiding episodes [ 42 , 43 ] . bachteeva et al . reported that pgs play a role in the osmotic water permeability of the frog urinary bladder . it has been reported that the urothelium contains both the prostaglandin receptor subtype e2 ( ep2 ) , which plays a role in osmoregulation , as well as the ep1 receptor , which plays a role in the micturation reflex . schrder et al . reported that the ep1 receptor has a role in the development of detrusor overactivity caused by pge2 and outlet obstruction . the details of the mechanisms underlying this action of pgs on voiding frequency are not known . one idea is that the pgs have a direct effect on bladder afferent nerve fibres . by increasing afferent nerve activity support for the idea comes indirectly from experiments in which the bladder was treated with capsaicin to remove the afferent contribution of c fibres . after functionally removing the c fibres , the pg - induced increase in micturition frequency of micturition was reduced . a different mechanism involving an indirect action of pgs on afferent nerves has recently been proposed . it is known that small localized contractions occur in the bladder wall of many species . this complex activity is hypothesized to be the motor component of a motor / sensory system involved in the generation of afferent firing and bladder sensation [ 29 , 31 ] . using the isolated guinea pig bladder it has been shown that pgs increase the frequency of this phasic motor activity . thus , the increased phasic motor activity could result in an increase in afferent discharges and in so doing influence the point at which voiding is triggered . the present observations extend this broad observation and demonstrate specifically that the expression of cox i - ir predominates within two general cell systems in the bladder wall : ( i ) cells within the basal and intermediate layers of the urothelium and ( ii ) within a population of small cells that are closely associated with a network of vimentin positive cells . these vimentin positive cells are present through out the sub - urothelial space of the lamina propria and extend over the surface of the muscle bundles , which make up the inner layers . it is interesting and important to note that no cox i - ir was seen within the muscles indicating that , in the guinea pig , it is not the smooth muscle that is producing pg but the cells associated with the vimentin network . these observations raise intriguing questions regarding the mechanisms of action of pgs in the guinea pig bladder . one hypothesis might be that the cox i in the basal urothelial cell layers is activated by bladder distension . the pgs produced here could diffuse the relatively short distance to the sub - urothelial space in which sensory afferent nerve fibres are found . thus , this arrangement would represent the site where there is a pg - induced direct modulation of afferent nerves . however , the abundance of cox i - ir in the urothelium of the lateral wall and the relative paucity of afferent nerves indicate that this is not the only role for pgs produced by the urothelium in the lateral wall . the present data also show that the cox i - ir cells in the base of the urothelium also express nnos . like pg , it is also known , on other cell systems , that pg production is influenced by no and , conversely , that no production is influenced by pgs [ 51 , 52 ] . specifically , atp has been shown to be released and one of its actions is to influence afferent nerve firing . also , there are reports that acetylcholine is released from the urothelium . thus , we conclude that there is a complex and inter - related release of signalling substances from the urothelium in response to mechanical deformation . to add to this complexity , the cell layer immediately below the urothelium , the sub - urothelial interstitial cell ( su - ic ) layer , can be a possible location for further integration of urothelial derived signals . these su - ics , in the guinea pig and human , respond to both exogenous and endogenous no demonstrating a rise in cgmp [ 30 , 32 ] . these cells are also immuno - reactive for antibodies to the type 3 muscarinic receptor ( m3 ) ( unpublished observation ) , purinergic receptor and the type 2 prostaglandin receptor ( ep2 ) ( unpublished observation ) . thus , the integrated output of pg , no , atp and cholinergic stimuli from the urothelium can be further integrated and modulated on the su - ics . the specific function of the su - ics cells is , at present , not known . indeed , there is indirect support for such a possible integration on the sub - urothelial cells . the su - ics are in close relation with the network of interstitial cells of the lamina propria and on the surface of the inner muscles . the autonomous bladder hypothesis suggests that , based on the observations of lagou et al . that the muscle interstitial cells are involved in the activation and co - ordination of complex phasic activity ( autonomous activity ) within the smooth muscle and it is this activity that is the motor component of the motor sensory system . the amplitude and frequency of the autonomous activity are increased by cholinergic agonists [ 58 , 59 ] and atp . if this type of activity is generated within a network of interstital cells this points out that there are m3/atp - activated pacemakers linked to a distributed network . it has also been shown that bladder distension alters autonomous activity : an increase in bladder volume increases autonomous activity , whereas a decrease in bladder volume inhibits it . the complex scheme outlined above might provide an explanation for these volume - related events . mechanical deformation of the urothelium in the lateral wall triggers a complex cascade of interacting signals within the urothelial epithelium resulting in the release of signals into the sub - urothelial space . activity in the su - ics is then distributed to the muscle via the vimentin positive interstitial cell network resulting in the excitatory and inhibitory effects on the phasic activity ( see fig . summary diagram illustrating the arrangement of cells found in the wall of the guinea pig bladder and their hypothesized interaction . the substances released act on afferent nerves but may also act upon the sub - urothelial interstitial cells . this network is in direct contact with a network of vimentin positive interstitial cells which run in the sub - urothelial space of the lamina propria and extend over the surface of the inner muscle bundles . the mechanisms connecting cells in this network can be via gap junctions ( black squares ) . cox i - ir cells ( satellite cells ) are associated with the vimentin interstitial cell network where they influence the activity of the network . the precise cell types and their location have not been considered in detail ; it was simply assumed that it originated in smooth muscle cells . the present observations put forward a new idea that , at least in the guinea pig , it is the cox i - ir cells associated with the network of vimentin positive interstitial cells on the surface of the muscle bundles that are responsible for synthesizing pg in this layer . as already discussed , one of the possible roles of this network of muscle interstitial cells is to generate and distribute signals leading to phasic contractions in the bladder wall [ 34 , 57 , 62 ] . thus , the micro - anatomical arrangement of cells in the muscle layer appears to involve cox i - ir ( pg producing ) cells contacting vimentin interstitial cells in close apposition to smooth muscle . functionally , it can be hypothesized that this arrangement involves a pg regulation of activity in the vimentin network and consequently an input to the muscle . the actions of pg on the muscle would thus be indirect being interceded by the network of interstitial cells ( see fig . the present observations also demonstrate a difference in the interstitial cells in the outer and inner muscle layers of the bladder wall , specifically the reduction in number of cox i - ir cells in the outer layers . however , the functional roles for these apparently different types of muscle interstitial cell are not known . what is clear is that the network of interstitial cells is complex and may sub - serve several different functions . in conclusion , it is now quite clear that there are several complex signalling systems operating within the bladder wall . in addition , it is clear that these signals act within cell - systems that interact between the urothelium , interstitial cells , muscle , sensory fibres and intra - mural ganglia . it is a major challenge for the future to unravel this complexity not only in relation to the physiology and pharmacology of the bladder but also in relation to the origins and treatment of bladder pathology .

localized phasic contractions in the bladder wall ( autonomous activity ) have been hypothesized to be an integral part of a motor / sensory system contributing to bladder sensation . the sites responsible for generating this activity , the mechanisms involved in its propagation and modulation remain unknown . this phasic motor activity is modulated by exogenous prostaglandins . therefore , analysis of the sites of prostaglandin production and action within the bladder wall may shed light on the mechanisms of generation and modulation of this phasic activity . in this paper we report the localization of immuno - reactivity indicative of the expression of cyclo - oxygenase enzyme type i ( cox i - ir ) within the bladder wall . basically , three types of cox i - ir cell were identified : epithelial cells in the basal and intermediate layers of the urothelium , complex vimentin - positive and cox i - ir cells in the lamina propria and vimentin - negative cox i - ir cells in the lamina propria and on the surface of the inner muscle bundles . these vimentin - negative / cox i - ir cells appear to be in close apposition to a continuous network of vimentin - positive cells , which extends from the lamina propria into the inner muscle layers and subsequently into the outer muscle layers . however , the interstitial cells in this region might form a distinctly different sub - type . first , the interstitial cells in this region differ from those in the inner layer by their responsiveness to no with a rise in cgmp . two subtypes have been identified : cells on the surface of the muscle bundles and within the muscle bundles . second , cox i - ir cells are not associated with the interstitial cells in the outer layers . the physiological significance for these apparent differences in the interstitial cell network is not clear . however , such differences are likely to reflect differences in the processes involved in their activation , modulation and control .

Introduction Materials and methods Results COX I-IR in the urothelium and sub-urothelial layer Outer muscle layers Discussion

using the isolated bladder it has been argued previously that autonomous activity is part of a motor / sensory system operating within the bladder wall whereby localized contractions of the bladder wall stimulate firing in afferent nerves contributing to sensation [ 29 , 30 ] . different sub - types of cgmp interstitial cell have been identified lying principally in the sub - urothelial layer and in the outer muscle layer . however direct evidence for the physiological role of interstitial cells is , so far , limited and comes from experiments which show that nitric oxide ( no ) , which generates a rise in cgmp in the interstitial cells , also abolishes autonomous activity . since exogenous pg modulates autonomous activity further insights into the origin of this activity and possible links to interstitial cells may be got from a detailed understanding of where pgs are synthesized and where they act in the bladder . cox i is associated with cell somata in the urothelium and with a network of cells running over the muscle bundles . the key basic observations regarding the distribution of cox i - ir in the bladder wall are illustrated in fig . cox i - ir cells are seen within the urothelium and also associated with a network of cells , which extends from the lamina propria into the inner muscle layers . note that there are fewer cox i - ir cells associated with the outer muscle layers . the localization of cox i - ir , vimentin cells and no - responsive cgmp cells in the guinea pig bladder . more cox i - ir cells are seen in the inner regions of the bladder . cox i - ir is therefore associated with two distinct structures : the urothelium and a network of cells in the lamina propria and around the muscle . note that the cgmp interstitial cells are found in the outer muscle layers where there is considerably less cox i - ir . these observations on cox i - ir in the urothelium and associated with the networks of interstitial cells are described in more detail below . as reported previously , the umbrella cells lying on the surface of the urothelium and the sub - urothelial interstitial cell layer closes to the urothelium are sensitive to exogenous no and demonstrate a rise in cgmp ( cgmp ) . it is also clear that cox i - ir is associated with intense staining in the intermediate and basal layers of the urothelium ( buc ) and weekly in cells in the lamina propria ( lamina propria interstitial cells ( lp - ics ) ) . figure 2b shows , in greater detail that , in this region of the bladder wall , the majority of the sub - urothelial cgmp interstitial cells do not have cox i - ir . cgmp- and cox i - ir associated with the urothelium in the guinea pig bladder . the umbrella cells on the surface of the urothelium ( umb ) and cells in the sub - urothelial space , the sub - urothelial interstitial cells ( su - ics ) are cgmp . cells in the basal regions of the urothelium ( basal urothelial cells : buc ) are stained intensely with the cox i antibody . in addition , cox i - ir cells are also found in the lamina propria interstitial cells ( ip - ics ) . they also show that the cox i - ir cells in the buc are separated from the cox i - ir ip - ics . thus , there are two types of cox i - ir cell in the urothelium : basal cox i - ir / nnos and intermediate cox i - ir / nnos . the layer of densely packed cells lying immediately below the urothelium , the sub - urothelial interstitial cells ( su - ics ) , are responsive to no with an elevation in cgmp . cgmp umbrella cells are seen on the surface of the urothelium but there were no vim+ cells in any cell type within the urothelium . ( b ) and ( c ) illustrate examples of the cells in the sub - urothelial layer at higher magnification , showing the combined colour image ( upper panel : cgmp - green , vimentin - red ) , the cgmp image alone ( middle panel ) and vimentin image alone ( lower panel ) . an additional feature of the cox i / vimentin double staining becomes apparent when studying in detail the diffuse network of vim cells in the region of the lamina propria immediately below the su - ic and above the muscle layer ( fig . these cells appear to form a distinct network of inter - connecting cells contiguous with the su - ic layer and with vim cells within the muscle layer ( see below ) . in this section , but , cox i - ir is seen within the cell bodies of the vim lp - ics ( fig . identification of cox i - ir and vimentin in the lamina propria . the cox i - ir cells are clearly visible within the urothelium ( uro ) , the suburothelial interstitial cells ( su - ics ) and the lamina propria ( ip - ics ) . in addition , there appear to be cox i - ir cells ( 3).these cells are spindle shaped and are weakly positive for vimentin . in these regions , two different cell types were readily identified based on the expression of vimentin and cox i - ir . the cox i - ir of the urothelium ( uro ) and spindle cells in the lamina propria are seen ( * ) ( see also fig . the cox i - ir cells in the urothelium ( uro ) and lamina propria ( ip - ics ) are seen . the vimentin fibres of the sub - urothelial cell layer ( su - ics ) and within the cell network in the lamina propria are also apparent . collections of cox i - ir cells are seen between the muscle bundles ( n ) . the network of vim fibres was observed to continue from the lamina propria into the inner smooth muscle layer where they run primarily on the surface of the muscle bundles ( fig . here , the vim cells appear on the surface of the muscle bundles and so can be described as surface muscle interstitial cell ( sm - ics ) . 7b and c. at the junction of the lamina propria and inner smooth muscle cell layer , small clusters of cox i - ir cell bodies were often observed ( fig . the network of vim sm - ics was observed to extend into the outer muscle layers of the bladder wall ( figs . as has been reported previously , the sm - ics of the outer muscle layers respond to no with a rise in cgmp . no - responsive cells producing cgmp are also found within the muscle bundles of the outer muscle layer : intramuscular interstitial cells ( im - ics ) ) . the distribution of cox i - ir and cgmp cells in the outer muscle layers . cgmp cells are seen in the outer muscle layer ( oml ) while there are no cells associated with the inner muscle layer ( iml ) . ( c ) shows the network of cox i - ir cells in the iml and the absence of cgmp staining . the network of cgmp ics associated with the outer layer and the network of vimentin cells in the inner ( * ) and outer layers are readily seen . thus , these data support the idea that there are different sub - types of interstitial cell associated with the lamina propria and inter - muscular spaces in the guinea pig bladder . location of cgmp , vimentin , cox i and nnos in the bladder ip - ics : lamina propria interstitial cells - lp - ics#1 : lamina propria interstitial cells type # 1 ( only vimentin positive ip - ics ) - ip - ics#2 : lamina propria interstitial cells type # 2 ( only cox i positive ip - ics ) - lp - ics#3 : lamina propria interstitial cells type # 3 ( ip - ics that are both vimentin and cox i positive ) - sm - ics : surface muscle interstitial cells - sm - ics#1 : surface muscle interstitial cells type # 1 ( vimentin positive sm - ics ) - sm - ics#2 : surface muscle interstitial cells type # 2 ( cox i positive sm - ics ) - im - ics : intramuscular interstitial cells - mc - ics : muscle coat interstitial cells figure 2a illustrates a section showing the urothelium and sub - urothelial region , double stained for cox i ( red ) and cgmp ( green ) . as reported previously , the umbrella cells lying on the surface of the urothelium and the sub - urothelial interstitial cell layer closes to the urothelium are sensitive to exogenous no and demonstrate a rise in cgmp ( cgmp ) . it is also clear that cox i - ir is associated with intense staining in the intermediate and basal layers of the urothelium ( buc ) and weekly in cells in the lamina propria ( lamina propria interstitial cells ( lp - ics ) ) . figure 2b shows , in greater detail that , in this region of the bladder wall , the majority of the sub - urothelial cgmp interstitial cells do not have cox i - ir . cgmp- and cox i - ir associated with the urothelium in the guinea pig bladder . the umbrella cells on the surface of the urothelium ( umb ) and cells in the sub - urothelial space , the sub - urothelial interstitial cells ( su - ics ) are cgmp . cells in the basal regions of the urothelium ( basal urothelial cells : buc ) are stained intensely with the cox i antibody . in addition , cox i - ir cells are also found in the lamina propria interstitial cells ( ip - ics ) . they also show that the cox i - ir cells in the buc are separated from the cox i - ir ip - ics . thus , there are two types of cox i - ir cell in the urothelium : basal cox i - ir / nnos and intermediate cox i - ir / nnos . the location of nnos to a single layer in the basal urothelium is clearly seen while the more diffuse location of the cox i - ir is apparent . the layer of densely packed cells lying immediately below the urothelium , the sub - urothelial interstitial cells ( su - ics ) , are responsive to no with an elevation in cgmp . cgmp umbrella cells are seen on the surface of the urothelium but there were no vim+ cells in any cell type within the urothelium . ( b ) and ( c ) illustrate examples of the cells in the sub - urothelial layer at higher magnification , showing the combined colour image ( upper panel : cgmp - green , vimentin - red ) , the cgmp image alone ( middle panel ) and vimentin image alone ( lower panel ) . an additional feature of the cox i / vimentin double staining becomes apparent when studying in detail the diffuse network of vim cells in the region of the lamina propria immediately below the su - ic and above the muscle layer ( fig . these cells appear to form a distinct network of inter - connecting cells contiguous with the su - ic layer and with vim cells within the muscle layer ( see below ) . in this section , no cox i - ir cells are visible within the su - ic layer . identification of cox i - ir and vimentin in the lamina propria . the cox i - ir cells are clearly visible within the urothelium ( uro ) , the suburothelial interstitial cells ( su - ics ) and the lamina propria ( ip - ics ) . the basal urothelial layer ( 1 ) , sub - urothelial layer ( 2 ) the staining pattern of the vimentin cells in the lp is highly suggestive for a network of vimentin structures ( 4 ) . in addition , there appear to be cox i - ir cells ( 3).these cells are spindle shaped and are weakly positive for vimentin . in these regions , two different cell types were readily identified based on the expression of vimentin and cox i - ir . the cox i - ir of the urothelium ( uro ) and spindle cells in the lamina propria are seen ( * ) ( see also fig large irregular cells are also seen which are cox i - ir and which have an extensive diffuse network of vimentin fibres ( ) . a network of vimentin processes around the muscle bundles of the inner muscle layer . the cox i - ir cells in the urothelium ( uro ) and lamina propria ( ip - ics ) are seen . the vimentin fibres of the sub - urothelial cell layer ( su - ics ) and within the cell network in the lamina propria are also apparent . collections of cox i - ir cells are seen between the muscle bundles ( n ) . the network of vim fibres was observed to continue from the lamina propria into the inner smooth muscle layer where they run primarily on the surface of the muscle bundles ( fig . here , the vim cells appear on the surface of the muscle bundles and so can be described as surface muscle interstitial cell ( sm - ics ) . 7b and c. at the junction of the lamina propria and inner smooth muscle cell layer , small clusters of cox i - ir cell bodies were often observed ( fig . these collections of cell bodies , which have the appearance of nodes , were also apparent , although fewer in number and with fewer cells , lying between the muscle bundles of the inner muscle layer ( see fig . the network of vim sm - ics was observed to extend into the outer muscle layers of the bladder wall ( figs . as has been reported previously , the sm - ics of the outer muscle layers respond to no with a rise in cgmp . the distribution of cox i - ir and cgmp cells in the outer muscle layers . cgmp cells are seen in the outer muscle layer ( oml ) while there are no cells associated with the inner muscle layer ( iml ) . ( c ) shows the network of cox i - ir cells in the iml and the absence of cgmp staining . the network of cgmp ics associated with the outer layer and the network of vimentin cells in the inner ( * ) and outer layers are readily seen . thus , these data support the idea that there are different sub - types of interstitial cell associated with the lamina propria and inter - muscular spaces in the guinea pig bladder . the different cell types which have been identified and described above , based on the staining for cgmp , vimentin , cox i and nnos in the bladder ip - ics : lamina propria interstitial cells - lp - ics#1 : lamina propria interstitial cells type # 1 ( only vimentin positive ip - ics ) - ip - ics#2 : lamina propria interstitial cells type # 2 ( only cox i positive ip - ics ) - lp - ics#3 : lamina propria interstitial cells type # 3 ( ip - ics that are both vimentin and cox i positive ) - sm - ics : surface muscle interstitial cells - sm - ics#1 : surface muscle interstitial cells type # 1 ( vimentin positive sm - ics ) - sm - ics#2 : surface muscle interstitial cells type # 2 ( cox i positive sm - ics ) - im - ics : intramuscular it has been known for over 30 years that pgs are released from the bladder in response to stretch , the pgs coming from both the urothelium and muscle layers [ 19 , 24 , 35 , 36 ] . this complex activity is hypothesized to be the motor component of a motor / sensory system involved in the generation of afferent firing and bladder sensation [ 29 , 31 ] . the present observations extend this broad observation and demonstrate specifically that the expression of cox i - ir predominates within two general cell systems in the bladder wall : ( i ) cells within the basal and intermediate layers of the urothelium and ( ii ) within a population of small cells that are closely associated with a network of vimentin positive cells . these vimentin positive cells are present through out the sub - urothelial space of the lamina propria and extend over the surface of the muscle bundles , which make up the inner layers . it is interesting and important to note that no cox i - ir was seen within the muscles indicating that , in the guinea pig , it is not the smooth muscle that is producing pg but the cells associated with the vimentin network . however , the abundance of cox i - ir in the urothelium of the lateral wall and the relative paucity of afferent nerves indicate that this is not the only role for pgs produced by the urothelium in the lateral wall . the present data also show that the cox i - ir cells in the base of the urothelium also express nnos . the su - ics are in close relation with the network of interstitial cells of the lamina propria and on the surface of the inner muscles . that the muscle interstitial cells are involved in the activation and co - ordination of complex phasic activity ( autonomous activity ) within the smooth muscle and it is this activity that is the motor component of the motor sensory system . activity in the su - ics is then distributed to the muscle via the vimentin positive interstitial cell network resulting in the excitatory and inhibitory effects on the phasic activity ( see fig . this network is in direct contact with a network of vimentin positive interstitial cells which run in the sub - urothelial space of the lamina propria and extend over the surface of the inner muscle bundles . cox i - ir cells ( satellite cells ) are associated with the vimentin interstitial cell network where they influence the activity of the network . the present observations put forward a new idea that , at least in the guinea pig , it is the cox i - ir cells associated with the network of vimentin positive interstitial cells on the surface of the muscle bundles that are responsible for synthesizing pg in this layer . as already discussed , one of the possible roles of this network of muscle interstitial cells is to generate and distribute signals leading to phasic contractions in the bladder wall [ 34 , 57 , 62 ] . thus , the micro - anatomical arrangement of cells in the muscle layer appears to involve cox i - ir ( pg producing ) cells contacting vimentin interstitial cells in close apposition to smooth muscle . the present observations also demonstrate a difference in the interstitial cells in the outer and inner muscle layers of the bladder wall , specifically the reduction in number of cox i - ir cells in the outer layers .

medical technology includes all medical equipment used by health organizations for diagnosis , therapy , monitoring , rehabilitation , and care . effective medical device management is required to ensure high - quality patient care [ 1 , 2 ] . accomplished medical device management will greatly assist in the reduction of adverse incidents and medical device - related accidents . for medical technology management , hospitals must have activities for maintaining , inspecting , and testing all medical equipment in the inventory . these activities must be performed within the scope of a program called maintenance program . the medicine and healthcare products regulatory agency declares that maintenance activities and their intervals should be planned in accordance with the manufacturers ' recommendations or strategies listed in an alternative equipment maintenance program . a maintenance program , generated by considering the characteristics and failures of medical equipment , is important with regard to usability and efficiency . however , it is inefficient to use the same strategies for the management of older technology devices and newer high - tech devices because of their different characteristics . the new high - tech devices functional control activities planned in accordance with the manufacturers ' recommendations and daily programmed self - tests should be done . these devices are tested against their specifications presented by their manufacturers . according to the 2007/47/ec directive the directive states that the instructions for use must contain details of the nature and frequency of the maintenance and calibration needed to ensure that the devices operate properly and safely at all times . for this reason , daily checks , including visual controls and specific device tests , are described in the user guide and carried out by users . unlike new high - tech devices , the manufacturers ' recommendations for older technology devices are not applicable because of the long usage time and device age . generally , in developing countries , such as turkey , older technology equipment mainly receives corrective maintenance . this study investigated whether older technology devices could be included in maintenance strategies similar to those used for high - tech devices . the quality of older technology medical devices can be ensured through periodical performance verification and safety testing ( pvst ) in accordance with international standards . pvst uses a standard measurement system with known accuracy to measure the accuracy of medical equipment [ 57 ] . pvst which includes qualitative and quantitative tests is performed by qualified biomedical personnel . during pvst , if a device is identified as not compatible with international standards , the hidden failures are determined and recorded by the biomedical staff . these failures are repaired by the hospital 's biomedical staff or service technicians employed by manufacturers . all test results indicate causation , a tremendously important factor in the prevention of adverse incidents and generation of an effective maintenance program . valuable lessons can be learned from an analysis of failures and these can be applied to maintenance programs . recently , the demand for medical device management is increasing as the number of medical devices increases . therefore , the development of more effective maintenance programs has achieved prominence . the initial purpose of this study was to generate a maintenance program comprising two different maintenance strategies , one each for older technology devices and newer high - tech devices , utilizing the manufacturers ' recommendations and pvst results , respectively , and to determine the success rate of this program using the indicators . accordingly , old technology devices will be included in the maintenance systems through separate testing . the food and drug administration maintains a database of medical equipment failures and has conducted some preventive studies for all medical sectors . however , very little information about hidden failures was available in the literature when the collection of hidden failure data was initiated for this study . wallace and kuhn presented an analysis of software - related medical device failures that led to manufacturer recalls , but they caused no deaths or injuries . in addition , bliznakov et al . reported medical device recalls due to software failures . the authors collected data related to software failures and performed an analysis via failure classification . taghipour et al . also described a periodic inspection optimization model for complex repairable systems [ 14 , 15 ] . the present study differs from previous studies because it presents a maintenance program created using two different strategies . the first strategy incorporates daily checks for new high - tech devices , whereas the second implements pvst as the sole performance measurement for older technology devices . to the best of our knowledge , no previous study has focused on hidden medical device failures determined during the pvst of medical devices . this study included a total of 723 high - risk medical devices maintained by the medical faculty of istanbul university . the high - risk devices were classified as older technology devices and newer high - tech devices . this classification was performed because of the lack of service or user manuals , and corresponding lack of manufacturer recommendations for many older technology devices . this lack makes it impossible to apply the same procedures to old technology and new high - tech devices . thus , different procedures were applied to old technology and new high - tech devices in order to develop maintenance . as seen in figure 1 , old technology devices ( 589 devices ) comprised of electrocardiography ( ecg ) devices , pulse oximetry devices , sphygmomanometers , infant incubators , phototherapy units , defibrillators , surgical aspirators , and electrosurgical units . these devices were tested by applying pvst . the second group ( 134 devices ) , which mostly contained imaging devices , comprised computerized tomography ( ct ) , angiography , mammography , c - arm radiography , magnetic resonance ( mr ) , and positron emission tomography and gamma cameras . the devices in the first group , excluding those used in intensive care and emergency departments , had 8-hour workloads . the groups were investigated separately and two different maintenance strategies were developed : predictive maintenance for newer high - tech devices and preventive maintenance for older technology devices . the development of preventive maintenance for older technology devices required a long procedural duration , whereas predictive maintenance for newer high - tech devices was developed in accordance with the manufacturers ' recommendations . a predictive maintenance program for newer high - tech devices was developed by applying maintenance time schedules created according to the manufacturers ' recommendations . under this program , predictive maintenance was conducted for each device through a contract with the manufacturer 's technical service and cooperation of the biomedical department of the hospital . after each service session , maintenance reports were delivered to the biomedical staff by the manufacturer 's technical service department . they were trained in the performance of daily checks through user training provided by the manufacturer . the most important point in this section was the collection of regular feedback from all device users . although the exact training success and feedback rates were not determined , an increase in feedback was observed . in addition , failures occurring during work hours were reported to the manufacturer 's technical service by the hospital 's biomedical personnel . a preventive maintenance program for older technology devices was developed via analysis of the pvst results of the equipment . the following pvst steps were performed in sequence by the hospital 's biomedical personnel : determination of the pvst intervals , application of the pvst , interpretation of the pvst results according to the acceptance criteria stated in international standards . determination of the pvst intervals , application of the pvst , interpretation of the pvst results according to the acceptance criteria stated in international standards . pvst intervals were determined by calculating the equipment management number ( emn ) , which is described in the clinical equipment management standards of the technology and safety management series developed by the joint commission . given the lack of the manufacturers ' recommendation for the old technology devices investigated in this study , the emn was used with a general approach to determine the initial pvst interval . pvst intervals accepted by industry could have been used if the manufacturers ' recommendations were present . the emn technique , introduced by fennigkoh and smith , classifies equipment using three parameters : function , risk , and maintenance requirements . a numerical value is assigned to each device type by classifying the above - mentioned parameters . the scores used to calculate emn can be seen in table 1 . specifically , the emn is the sum of the equipment function score , equipment risk score , and maintenance requirement score . pvst intervals range from 6 to 12 months , depending on the emn . according to the standards if the calculated number is 12 or higher , the equipment is incorporated into the annual pvst plan . in addition , if the emn is greater than 17 , the device must be controlled every 6 months . the calculated emn and pvst intervals determined for the medical equipment investigated in this study are shown in table 2 . pvst was performed according to the procedures of inspection and preventive maintenance ( ipm ) , prepared by the emergency care research institute . in this study , testing parameters for medical devices measured during pvst were determined from ipm procedures . the procedures comprised both quantitative and qualitative tests . the qualitative test evaluated the device 's physical parameters ( e.g. , connectors , battery , and electrodes ) . although the qualitative test was general , the quantitative test was specific for each device . the quantitative test parameters measured for each device in this study are listed in table 2 . tests were performed with a low level of uncertainty , which was calculated using the procedures declared in the guide to the expression of uncertainty in measurement ( gum ) . to interpret the pvst results , the acceptance criteria in the ipm procedures were considered . medical devices for which measurement results fell within the acceptable range were considered appropriate with respect to international standards and were labeled with green stickers . ( p ) in documentation and the database . medical devices for which measurement results fell outside of the acceptable range were considered inappropriate with respect to international standards and were labeled with red stickers . the red sticker indicated the presence of failures and stated that the device should not be used . clinical staffs were trained with regard to their responses to each label color . according to the procedure applied for red label devices , staffs were prohibited from using the device , which was sent to the clinical engineering department to identify and remove any hidden failures that did not completely disable the primary device functions . after correction and a second pvst , all data regarding information about the equipment , such as the equipment name , location , serial number , interpretation results ( passed or failed ) , and failure definition , were entered in the operation page . pvst , when performed at a determined interval , provides a large statistical failure dataset that could be used to establish a maintenance interval . hidden failures detected during pvst that could affect device performance were considered during preventive maintenance planning . for example , a 3-month interval was planned for maintenance of the most common hidden failures detected in incubators during pvst . maintenance checklists were prepared and required nondurable parts for continuous medical equipment service were determined . the maintenance process with regard to the qualitative and quantitative device parameters was defined using maintenance checklists . the performance of maintenance strategies for older technology devices and newer high - tech devices was assessed in terms of progress in achieving the expectation defined by the program . a 6-month validation phase was planned to monitor whether the failure rates of old technology devices and new high - tech devices would decrease with the application of the maintenance plan . this phase was selected because defibrillators and electrosurgical units selected pilot devices for the evaluation of the preventive maintenance and have a 6-month pvst interval . no preventive maintenance was conducted in the hospital before this study . however , medical devices were subjected to pvst before the study . the hidden failures of all medical devices were recorded . to evaluate preventive maintenance , the results of pvst during preventive maintenance were compared with the results of pvst before maintenance . in addition , the predictive maintenance results were evaluated by comparing the failures that occurred within 6 months of prepredictive maintenance and those that occurred during predictive maintenance . data of failures that occurred before predictive maintenance were extracted from each device 's failure history which was available in the hospital documentation . a reporting system was planned in which an archive of all devices ' failure histories would be created . this reporting system enabled the monitoring of all medical device information . to this end , biomedical personnel collected user checklists and technical service forms from the manufacturers ' technical services . data in these documents were entered on device information cards to generate a failure history for each device . hence , this method provided a reporting system comprising the collection of data from checklists . parameters related to failures , such as the failure definition , repair time , and replaced parts , were followed easily . in particular , unwanted data , such as the maximum repair time and more frequent failure rate , were identified . these data were reported to the decision - maker to explain the overall situation . for this , a one - page report was designed to supporting decision - makers in the allocation of an increased budget for technology procurement , new maintenance contracts , or more biomedical personnel . a sample report form for decision - makers is shown as follows : medical devices predictive and preventative maintenance report number of total medical device : number of total medical device subject to preventative maintenance : number of total medical device subject to predictive maintenance : number of total medical device : number of total medical device subject to preventative maintenance : number of total medical device subject to predictive maintenance : ( 1 ) failed devices often location device manufacturer model serial number ( 2 ) the failed devices in the warranty period location device manufacturer model serial number ( 3 ) failed end - of - support devices location device manufacturer model serial number ( 4 ) parts which were need to be replaced but not included in the annual maintenance contract location device manufacturer model serial number ( 5 ) devices which could not be found their non - durable parts and could not be repaired location device manufacturer model serial number ( 6 ) non - durable parts which have been changed again in the warranty period although they had been changed before location device manufacturer model serial number ( 7 ) the devices which can not get technical service from their contracted firm location device manufacturer model serial number this report will help decision - makers to define short- and long - term priority plans for technology investments based on safety aspects . this study planned predictive maintenance for 134 newer high - tech devices and preventive maintenance for 589 older technology devices . planned predictive maintenance involved the usage of daily checklists created by the manufacturers . these daily checklists which included simple , mandatory pre- and post - use tasks , maintenance time schedules were planned according to the manufacturers ' recommendations provided in the user guides . table 4 shows a sample maintenance time schedule , including each device brand and model . the devices were also subjected to monthly maintenance , in addition to the recommended maintenance period . since annual maintenance fee payments are divided into 12 months in accordance with the turkish currency system , the contracted company must visually maintain the device every month . this visual maintenance comprises short - term maintenance , especially device cleaning . for older technology devices , accordingly , failures were detected in 126 ( 22% ) of the 589 medical devices from different departments in the medical faculty at istanbul university ; they were marked as failed , and the remaining 463 were marked as passed . when the failed devices were analyzed according to their errors , several technical hidden failures were observed . pvst results were used to plan a preventive maintenance time schedule for old technology devices . this preventive maintenance time schedule indicates the maintenance interval for the device . the adequate interval for effective maintenance was determined for each device and nondurable part . the pvst analysis revealed that nondurable parts ( e.g. , ecg patient electrodes , oximeter probes , cuffs , defibrillator batteries , and ultraviolet lamps ) must be stocked for each piece of medical equipment . both the number and features of the nondurable parts required for each type of medical device were determined . however indicators such as failure rates per old technology devices and per new technology devices were determined to evaluate the equipment reliability . the success of preventive maintenance was evaluated by analyzing the results of pvst performed after preventive maintenance . since preventive maintenance includes old technology devices , the failure rate of sample devices provides information about the failure rate of old technology devices . the defibrillator and electrosurgical units were selected as pilot medical devices . before preventive maintenance , 86 defibrillators and 52 electrosurgical units were inspected , and their results were analyzed to develop preventive maintenance . after maintenance , the devices were inspected again after a period of 6 months . figures 3(a ) and 3(b ) show the results of the qualitative and quantitative tests performed before and after preventive maintenance , respectively . in addition , pvst results obtained after preventive maintenance indicated that the minor and major defects detected during pvst were largely rectified . traditional preventive maintenance ( tpm ) of some components was also performed . for example , batteries were replaced before complete depletion , and paddles were lubricated to increase conductivity . therefore , in contrast to the results of pvst performed before preventive maintenance , fewer failed components were identified after preventive maintenance . the patient 's life is at risk if the device fails completely or does not provide sufficient energy to the patient when in use . in particular , the quantitative parameters such as output energy , charge time , and energy after 60 sec are important parameters that may pose a serious threat to the patient . during the evaluation of preventive maintenance , although they are considered to have a lesser impact on patient safety , quantitative parameters are also important because they are directly related to defibrillator function . this is one reason why the defibrillator was used as a pilot device during preventive maintenance evaluation . the above - described situation is also valid for the second pilot device , the electrosurgical unit . the other devices investigated in this study , such as ecg , pulse oximeter , and aspirator , might cause some inconvenience to the patient , but they do not pose a serious risk . in these devices , the determined failures were hidden and indicated deviations from the devices ' functional performance specifications . hidden failure repair is required to prevent serious failures and to ensure standard service from the device but is not essential for the patient safety . for both pilot devices , problems related to quantitative parameters that were determined by pvst before only problems related to the synchronizers of two defibrillators could not be resolved by the hospital 's biomedical staff and required manufacturer 's technical service . in addition , an issue with the output energy of one defibrillator was also not resolved during preventive maintenance ( figure 3(a ) ) and the device was sent to the manufacturer 's technical service . similar to the defibrillators , all hidden failures in the electrosurgical units were resolved during preventive maintenance , except for a bipolar power - related problem ( figure 3(b ) ) . the success of predictive maintenance was evaluated by analyzing medical equipment failure reports after predictive maintenance . their failure rates could be assumed to represent the failure rate of new high - tech devices . failures occurring within 6 months of pre- and postpredictive maintenance were extracted from the devices ' failure histories , which were available in the hospital documentation . figures 4 and 5 present the failures of gamma cameras and ventilators occurring pre- and postpredictive maintenance , respectively . the failures were classified into two categories : those reported during daily checks and those occurring during work . in contrast , the former group was generally noticed during daily checks and generally related to the device 's physical condition or software . as shown in figures 4 and 5 , a greater number of failures were reported during daily checks after predictive maintenance . this might be attributed to failures being ignored by users during daily checks prior to predictive maintenance . the increased reporting of failures during daily checks indicates the user support of predictive maintenance . in contrast , a greater number of failures occurring during work were reported before predictive maintenance . this report describes the formation of a maintenance program using the pvst results for older technology medical devices and the manufacturers ' recommendations for newer high - tech devices . the first point is that older technology devices and newer high - tech devices were investigated separately . this led to the use of two different methodologies : pvst for older technology devices and the manufacturers ' recommendations for newer high - tech devices . it is important to overcome such failures before they occur and thus avoid harming the patient . accordingly , use of the pvst results was preferred for the development of a maintenance program for older technology devices . the second point is that this study examined hidden medical equipment failures arising from noncompliance with international standards . although other studies investigated hardware or software failures of medical devices , the present study addressed hidden failures that affect the quality of the medical device and patient safety . wallace and kuhn and bliznakov et al . also presented an analysis of failures . but , these failures were related to software and resulted in device recalls by the manufacturers . in the present study , hidden failures were analyzed to develop a preventive maintenance protocol , and failures detected during daily checks and usages were analyzed to develop a predictive maintenance protocol . calls related to failures were classified as follows : user - related calls , accessory- or connectivity - related calls , physical - stress - related calls , environmental - stress - related calls , human - interference - related calls . accessory- or connectivity - related calls , physical - stress - related calls , environmental - stress - related calls , human - interference - related calls . after classification , the authors recommended user training , a well - managed battery - care program , availability of the proper accessories , and maintenance of the environmental conditions specified by the equipment manufacturer . in this study , failures were classified and analyzed in terms of technology levels ( older technology devices and newer high - tech devices ) . since failures were detected through daily checks , the failures used to develop predictive maintenance were related to user competence , accessories , connectivity , or environmental stress . in addition , since hidden failures were detected during pvst , failures used to develop preventive maintenance were related to accessories , connectivity , and environmental stress . therefore , it can be stated that both preventive and predictive maintenance require a plan for maintaining the availability of proper accessories , as recommended by ridgway et al . however , in the present study , the battery - care program was included in accessories planning rather than as a separate plan . in addition , biomedical staff competence with regard to pvst is more important than user competence in our model . the authors determined the maintenance activity intervals of devices in terms of intensity of use . they reported that devices with a high intensity of use require maintenance more frequently than devices with a low intensity of use . in the present study , the present study and a small component of a study conducted by taghipour are only similar in terms of the pvst results analysis . taghipour analyzed the pvst results of infusion pumps before and after preventive maintenance and noted that medical devices included in a maintenance program have smaller errors than other devices . similarly , as shown in figures 3(a ) and 3(b ) in the present study , fewer hidden failures were identified after preventive maintenance . as mentioned above , the present study included two different maintenance strategies : predictive and preventive maintenance . programmed maintenance based on the manufacturers ' recommendations for new high - tech medical devices represented the predictive maintenance strategy . predictive maintenance is known as time - based maintenance and is defined as a maintenance strategy wherein maintenance activities are performed at scheduled time intervals recommended by manufacturers . in contrast , programmed maintenance based on an analysis of the performance inspection results of old technology medical devices represented the preventive maintenance strategy . preventive maintenance is also known as condition - based maintenance and is defined as a maintenance strategy that involves periodic and continuous equipment condition monitoring to detect equipment degradation . the information obtained from pvst results was used to determine the maintenance requirements and maintenance time schedule . for example , decision regarding filter replacement before the manufacturer 's recommended replacement interval was based on equipment pvst results . whereas the predictive maintenance strategy was applied to individual components of new high - tech devices in consideration of the equipment brand and model , the preventive maintenance strategy predictive maintenance requires a budget to facilitate contracts with the manufacturer technical services , whereas preventive maintenance requires sensors and special equipment to conduct pvst . in the predictive maintenance program , if the user reports a problem , it will be added to the database . however , biomedical staffs conduct the pvst of equipment included in a preventive maintenance program . however , using the preventive maintenance program , it is possible to track medical devices ' hidden failures and to determine the most appropriate maintenance in terms of required nondurable parts and elapsed time . the replacement of components with failures was included in this model , as devices with nondurable parts fail if those parts are not replaced or restored . these parts are supplied with a storage period of 1 year . for nondurable parts that are replaced twice yearly , a stock of two should be kept and a stock of three should be kept for parts replaced thrice yearly . otherwise , maintaining a supply of expired nondurable parts extends the maintenance process and disrupts preventive maintenance . as mentioned above , the current program helps to prevent problems prior to medical equipment failure and to maintain a stock of required nondurable parts . medical equipment can be better tracked by repeating pvst throughout the year , as the database of equipment failures will contain pvst histories of older technology devices . preventive maintenance involves relatively old technology . since the number of older technology devices in a hospital is greater than the number of new high - tech devices , the scope of preventive maintenance is more extensive than that of predictive maintenance . the distribution of failures might change because some old devices will be removed from use . by adding new devices to the inventory , the scope of preventive maintenance will become narrower , and the scope of predictive maintenance will become broader . however , new high - tech devices are also more expensive than older technology devices . this study has some limitations , because it was limited to high - risk devices in terms of patient safety and cost . devices that pose risks to patients and users , old devices , and complex devices such as radiology devices are frequently considered for maintenance . accordingly , the study did not include low - risk devices such as nebulizers and flow meters . the scope of the study could be extended to include other high - risk devices . for example , anesthesia units and vaporizers might be included in the preventive maintenance category . although all endoscopy systems , including colonoscopy , gastroscopy , bronchoscopy , and laryngoscopy devices , could be incorporated into a preventive maintenance strategy , they do not undergo pvst . operating tables and electrical patient beds requiring only electrical safety measurements may be incorporated into preventive maintenance . in addition , all newly acquired equipment will be included in a maintenance program after considering its technology level . the other limitation of the proposed model is that the criteria suggested by fennigkoh and smith were used to determine the pvst interval . the emn was used because this parameter has been accepted as a supervision criterion by the ministry of health in turkey . this study predicted that the reliability and failure patterns of a device would be affected by external factors such as the expertise level of users and biomedical staffs . accordingly , the users were trained in the performance of daily checks through user training provided by the manufacturer . in addition , the clinical staffs were also trained about their responses to the different colored label on the devices after pvst . this paper proposes two different maintenance strategies : preventive maintenance for old technology devices and predictive maintenance for new high - tech devices . although preventive and predictive maintenance strategies differ in many ways , a maintenance program comprising both strategies yielded positive results . the maintenance strategy evaluation demonstrated that strategies based on pvst results and the manufacturers ' recommendations led to a significant reduction in equipment failures and a significant increase in corrective maintenance . the usage of different maintenance strategies for older devices and newer high - tech technology devices to develop maintenance strategies is important in terms of its consequences . firstly , the older technology devices that applied only corrective maintenance in developing countries will be included in the maintenance strategies like newer high - tech devices . secondly , the inclusion of both old and new technology devices to the maintenance system provides a wider range of maintenance that covers all medical devices in hospitals with many old technology devices . thirdly , the performance verification and safety testing earn importance to develop maintenance strategies for devices without manufacturer recommendations . lastly , considering carefully all outcomes of the medical equipment failures and existence of a detailed history for every device help decision - makers to manage medical equipment . the next plan is to continue the study of failures of other medical devices excluded from this initial study .

a maintenance program generated through the consideration of characteristics and failures of medical equipment is an important component of technology management . however , older technology devices and newer high - tech devices can not be efficiently managed using the same strategies because of their different characteristics . this study aimed to generate a maintenance program comprising two different strategies to increase the efficiency of device management : preventive maintenance for older technology devices and predictive maintenance for newer high - tech devices . for preventive maintenance development , 589 older technology devices were subjected to performance verification and safety testing ( pvst ) . for predictive maintenance development , the manufacturers ' recommendations were used for 134 high - tech devices . these strategies were evaluated in terms of device reliability . this study recommends the use of two different maintenance strategies for old and new devices at hospitals in developing countries . thus , older technology devices that applied only corrective maintenance will be included in maintenance like high - tech devices .

1. Introduction 2. Methods 3. Results 4. Discussion 5. Conclusion

medical technology includes all medical equipment used by health organizations for diagnosis , therapy , monitoring , rehabilitation , and care . effective medical device management is required to ensure high - quality patient care [ 1 , 2 ] . accomplished medical device management will greatly assist in the reduction of adverse incidents and medical device - related accidents . for medical technology management , hospitals must have activities for maintaining , inspecting , and testing all medical equipment in the inventory . these activities must be performed within the scope of a program called maintenance program . the medicine and healthcare products regulatory agency declares that maintenance activities and their intervals should be planned in accordance with the manufacturers ' recommendations or strategies listed in an alternative equipment maintenance program . a maintenance program , generated by considering the characteristics and failures of medical equipment , is important with regard to usability and efficiency . however , it is inefficient to use the same strategies for the management of older technology devices and newer high - tech devices because of their different characteristics . the new high - tech devices functional control activities planned in accordance with the manufacturers ' recommendations and daily programmed self - tests should be done . unlike new high - tech devices , the manufacturers ' recommendations for older technology devices are not applicable because of the long usage time and device age . generally , in developing countries , such as turkey , older technology equipment mainly receives corrective maintenance . this study investigated whether older technology devices could be included in maintenance strategies similar to those used for high - tech devices . the quality of older technology medical devices can be ensured through periodical performance verification and safety testing ( pvst ) in accordance with international standards . pvst uses a standard measurement system with known accuracy to measure the accuracy of medical equipment [ 57 ] . all test results indicate causation , a tremendously important factor in the prevention of adverse incidents and generation of an effective maintenance program . recently , the demand for medical device management is increasing as the number of medical devices increases . the initial purpose of this study was to generate a maintenance program comprising two different maintenance strategies , one each for older technology devices and newer high - tech devices , utilizing the manufacturers ' recommendations and pvst results , respectively , and to determine the success rate of this program using the indicators . accordingly , old technology devices will be included in the maintenance systems through separate testing . the food and drug administration maintains a database of medical equipment failures and has conducted some preventive studies for all medical sectors . however , very little information about hidden failures was available in the literature when the collection of hidden failure data was initiated for this study . the present study differs from previous studies because it presents a maintenance program created using two different strategies . the first strategy incorporates daily checks for new high - tech devices , whereas the second implements pvst as the sole performance measurement for older technology devices . to the best of our knowledge , no previous study has focused on hidden medical device failures determined during the pvst of medical devices . this study included a total of 723 high - risk medical devices maintained by the medical faculty of istanbul university . the high - risk devices were classified as older technology devices and newer high - tech devices . this classification was performed because of the lack of service or user manuals , and corresponding lack of manufacturer recommendations for many older technology devices . this lack makes it impossible to apply the same procedures to old technology and new high - tech devices . thus , different procedures were applied to old technology and new high - tech devices in order to develop maintenance . as seen in figure 1 , old technology devices ( 589 devices ) comprised of electrocardiography ( ecg ) devices , pulse oximetry devices , sphygmomanometers , infant incubators , phototherapy units , defibrillators , surgical aspirators , and electrosurgical units . these devices were tested by applying pvst . the groups were investigated separately and two different maintenance strategies were developed : predictive maintenance for newer high - tech devices and preventive maintenance for older technology devices . the development of preventive maintenance for older technology devices required a long procedural duration , whereas predictive maintenance for newer high - tech devices was developed in accordance with the manufacturers ' recommendations . a predictive maintenance program for newer high - tech devices was developed by applying maintenance time schedules created according to the manufacturers ' recommendations . a preventive maintenance program for older technology devices was developed via analysis of the pvst results of the equipment . pvst intervals were determined by calculating the equipment management number ( emn ) , which is described in the clinical equipment management standards of the technology and safety management series developed by the joint commission . given the lack of the manufacturers ' recommendation for the old technology devices investigated in this study , the emn was used with a general approach to determine the initial pvst interval . pvst intervals accepted by industry could have been used if the manufacturers ' recommendations were present . specifically , the emn is the sum of the equipment function score , equipment risk score , and maintenance requirement score . according to the standards if the calculated number is 12 or higher , the equipment is incorporated into the annual pvst plan . in addition , if the emn is greater than 17 , the device must be controlled every 6 months . the calculated emn and pvst intervals determined for the medical equipment investigated in this study are shown in table 2 . although the qualitative test was general , the quantitative test was specific for each device . tests were performed with a low level of uncertainty , which was calculated using the procedures declared in the guide to the expression of uncertainty in measurement ( gum ) . to interpret the pvst results , the acceptance criteria in the ipm procedures were considered . medical devices for which measurement results fell within the acceptable range were considered appropriate with respect to international standards and were labeled with green stickers . medical devices for which measurement results fell outside of the acceptable range were considered inappropriate with respect to international standards and were labeled with red stickers . according to the procedure applied for red label devices , staffs were prohibited from using the device , which was sent to the clinical engineering department to identify and remove any hidden failures that did not completely disable the primary device functions . maintenance checklists were prepared and required nondurable parts for continuous medical equipment service were determined . the performance of maintenance strategies for older technology devices and newer high - tech devices was assessed in terms of progress in achieving the expectation defined by the program . a 6-month validation phase was planned to monitor whether the failure rates of old technology devices and new high - tech devices would decrease with the application of the maintenance plan . this phase was selected because defibrillators and electrosurgical units selected pilot devices for the evaluation of the preventive maintenance and have a 6-month pvst interval . no preventive maintenance was conducted in the hospital before this study . however , medical devices were subjected to pvst before the study . the hidden failures of all medical devices were recorded . to evaluate preventive maintenance , the results of pvst during preventive maintenance were compared with the results of pvst before maintenance . in addition , the predictive maintenance results were evaluated by comparing the failures that occurred within 6 months of prepredictive maintenance and those that occurred during predictive maintenance . data of failures that occurred before predictive maintenance were extracted from each device 's failure history which was available in the hospital documentation . to this end , biomedical personnel collected user checklists and technical service forms from the manufacturers ' technical services . data in these documents were entered on device information cards to generate a failure history for each device . a sample report form for decision - makers is shown as follows : medical devices predictive and preventative maintenance report number of total medical device : number of total medical device subject to preventative maintenance : number of total medical device subject to predictive maintenance : number of total medical device : number of total medical device subject to preventative maintenance : number of total medical device subject to predictive maintenance : ( 1 ) failed devices often location device manufacturer model serial number ( 2 ) the failed devices in the warranty period location device manufacturer model serial number ( 3 ) failed end - of - support devices location device manufacturer model serial number ( 4 ) parts which were need to be replaced but not included in the annual maintenance contract location device manufacturer model serial number ( 5 ) devices which could not be found their non - durable parts and could not be repaired location device manufacturer model serial number ( 6 ) non - durable parts which have been changed again in the warranty period although they had been changed before location device manufacturer model serial number ( 7 ) the devices which can not get technical service from their contracted firm location device manufacturer model serial number this report will help decision - makers to define short- and long - term priority plans for technology investments based on safety aspects . this study planned predictive maintenance for 134 newer high - tech devices and preventive maintenance for 589 older technology devices . planned predictive maintenance involved the usage of daily checklists created by the manufacturers . these daily checklists which included simple , mandatory pre- and post - use tasks , maintenance time schedules were planned according to the manufacturers ' recommendations provided in the user guides . the devices were also subjected to monthly maintenance , in addition to the recommended maintenance period . since annual maintenance fee payments are divided into 12 months in accordance with the turkish currency system , the contracted company must visually maintain the device every month . for older technology devices , accordingly , failures were detected in 126 ( 22% ) of the 589 medical devices from different departments in the medical faculty at istanbul university ; they were marked as failed , and the remaining 463 were marked as passed . pvst results were used to plan a preventive maintenance time schedule for old technology devices . , ecg patient electrodes , oximeter probes , cuffs , defibrillator batteries , and ultraviolet lamps ) must be stocked for each piece of medical equipment . both the number and features of the nondurable parts required for each type of medical device were determined . however indicators such as failure rates per old technology devices and per new technology devices were determined to evaluate the equipment reliability . the success of preventive maintenance was evaluated by analyzing the results of pvst performed after preventive maintenance . since preventive maintenance includes old technology devices , the failure rate of sample devices provides information about the failure rate of old technology devices . after maintenance , the devices were inspected again after a period of 6 months . figures 3(a ) and 3(b ) show the results of the qualitative and quantitative tests performed before and after preventive maintenance , respectively . in addition , pvst results obtained after preventive maintenance indicated that the minor and major defects detected during pvst were largely rectified . traditional preventive maintenance ( tpm ) of some components was also performed . for example , batteries were replaced before complete depletion , and paddles were lubricated to increase conductivity . therefore , in contrast to the results of pvst performed before preventive maintenance , fewer failed components were identified after preventive maintenance . during the evaluation of preventive maintenance , although they are considered to have a lesser impact on patient safety , quantitative parameters are also important because they are directly related to defibrillator function . this is one reason why the defibrillator was used as a pilot device during preventive maintenance evaluation . the above - described situation is also valid for the second pilot device , the electrosurgical unit . the other devices investigated in this study , such as ecg , pulse oximeter , and aspirator , might cause some inconvenience to the patient , but they do not pose a serious risk . in these devices , the determined failures were hidden and indicated deviations from the devices ' functional performance specifications . for both pilot devices , problems related to quantitative parameters that were determined by pvst before only problems related to the synchronizers of two defibrillators could not be resolved by the hospital 's biomedical staff and required manufacturer 's technical service . the success of predictive maintenance was evaluated by analyzing medical equipment failure reports after predictive maintenance . their failure rates could be assumed to represent the failure rate of new high - tech devices . in contrast , the former group was generally noticed during daily checks and generally related to the device 's physical condition or software . as shown in figures 4 and 5 , a greater number of failures were reported during daily checks after predictive maintenance . the increased reporting of failures during daily checks indicates the user support of predictive maintenance . this report describes the formation of a maintenance program using the pvst results for older technology medical devices and the manufacturers ' recommendations for newer high - tech devices . the first point is that older technology devices and newer high - tech devices were investigated separately . this led to the use of two different methodologies : pvst for older technology devices and the manufacturers ' recommendations for newer high - tech devices . accordingly , use of the pvst results was preferred for the development of a maintenance program for older technology devices . the second point is that this study examined hidden medical equipment failures arising from noncompliance with international standards . although other studies investigated hardware or software failures of medical devices , the present study addressed hidden failures that affect the quality of the medical device and patient safety . but , these failures were related to software and resulted in device recalls by the manufacturers . in the present study , hidden failures were analyzed to develop a preventive maintenance protocol , and failures detected during daily checks and usages were analyzed to develop a predictive maintenance protocol . after classification , the authors recommended user training , a well - managed battery - care program , availability of the proper accessories , and maintenance of the environmental conditions specified by the equipment manufacturer . in this study , failures were classified and analyzed in terms of technology levels ( older technology devices and newer high - tech devices ) . since failures were detected through daily checks , the failures used to develop predictive maintenance were related to user competence , accessories , connectivity , or environmental stress . therefore , it can be stated that both preventive and predictive maintenance require a plan for maintaining the availability of proper accessories , as recommended by ridgway et al . however , in the present study , the battery - care program was included in accessories planning rather than as a separate plan . the authors determined the maintenance activity intervals of devices in terms of intensity of use . in the present study , the present study and a small component of a study conducted by taghipour are only similar in terms of the pvst results analysis . taghipour analyzed the pvst results of infusion pumps before and after preventive maintenance and noted that medical devices included in a maintenance program have smaller errors than other devices . similarly , as shown in figures 3(a ) and 3(b ) in the present study , fewer hidden failures were identified after preventive maintenance . as mentioned above , the present study included two different maintenance strategies : predictive and preventive maintenance . programmed maintenance based on the manufacturers ' recommendations for new high - tech medical devices represented the predictive maintenance strategy . predictive maintenance is known as time - based maintenance and is defined as a maintenance strategy wherein maintenance activities are performed at scheduled time intervals recommended by manufacturers . in contrast , programmed maintenance based on an analysis of the performance inspection results of old technology medical devices represented the preventive maintenance strategy . preventive maintenance is also known as condition - based maintenance and is defined as a maintenance strategy that involves periodic and continuous equipment condition monitoring to detect equipment degradation . whereas the predictive maintenance strategy was applied to individual components of new high - tech devices in consideration of the equipment brand and model , the preventive maintenance strategy predictive maintenance requires a budget to facilitate contracts with the manufacturer technical services , whereas preventive maintenance requires sensors and special equipment to conduct pvst . in the predictive maintenance program , if the user reports a problem , it will be added to the database . however , biomedical staffs conduct the pvst of equipment included in a preventive maintenance program . however , using the preventive maintenance program , it is possible to track medical devices ' hidden failures and to determine the most appropriate maintenance in terms of required nondurable parts and elapsed time . the replacement of components with failures was included in this model , as devices with nondurable parts fail if those parts are not replaced or restored . for nondurable parts that are replaced twice yearly , a stock of two should be kept and a stock of three should be kept for parts replaced thrice yearly . otherwise , maintaining a supply of expired nondurable parts extends the maintenance process and disrupts preventive maintenance . as mentioned above , the current program helps to prevent problems prior to medical equipment failure and to maintain a stock of required nondurable parts . medical equipment can be better tracked by repeating pvst throughout the year , as the database of equipment failures will contain pvst histories of older technology devices . since the number of older technology devices in a hospital is greater than the number of new high - tech devices , the scope of preventive maintenance is more extensive than that of predictive maintenance . by adding new devices to the inventory , the scope of preventive maintenance will become narrower , and the scope of predictive maintenance will become broader . however , new high - tech devices are also more expensive than older technology devices . this study has some limitations , because it was limited to high - risk devices in terms of patient safety and cost . devices that pose risks to patients and users , old devices , and complex devices such as radiology devices are frequently considered for maintenance . for example , anesthesia units and vaporizers might be included in the preventive maintenance category . although all endoscopy systems , including colonoscopy , gastroscopy , bronchoscopy , and laryngoscopy devices , could be incorporated into a preventive maintenance strategy , they do not undergo pvst . operating tables and electrical patient beds requiring only electrical safety measurements may be incorporated into preventive maintenance . in addition , all newly acquired equipment will be included in a maintenance program after considering its technology level . the other limitation of the proposed model is that the criteria suggested by fennigkoh and smith were used to determine the pvst interval . this study predicted that the reliability and failure patterns of a device would be affected by external factors such as the expertise level of users and biomedical staffs . accordingly , the users were trained in the performance of daily checks through user training provided by the manufacturer . in addition , the clinical staffs were also trained about their responses to the different colored label on the devices after pvst . this paper proposes two different maintenance strategies : preventive maintenance for old technology devices and predictive maintenance for new high - tech devices . although preventive and predictive maintenance strategies differ in many ways , a maintenance program comprising both strategies yielded positive results . the maintenance strategy evaluation demonstrated that strategies based on pvst results and the manufacturers ' recommendations led to a significant reduction in equipment failures and a significant increase in corrective maintenance . the usage of different maintenance strategies for older devices and newer high - tech technology devices to develop maintenance strategies is important in terms of its consequences . firstly , the older technology devices that applied only corrective maintenance in developing countries will be included in the maintenance strategies like newer high - tech devices . secondly , the inclusion of both old and new technology devices to the maintenance system provides a wider range of maintenance that covers all medical devices in hospitals with many old technology devices . thirdly , the performance verification and safety testing earn importance to develop maintenance strategies for devices without manufacturer recommendations . the next plan is to continue the study of failures of other medical devices excluded from this initial study .

the retrospective study cohort was derived from the two sets of the cross - sectional surveys conducted in the community ( 65% white and 35% black subjects ) of bogalusa , louisiana , involving five cross - sectional surveys of children during 19811994 ( n = 13,444 ; 40% black and 50% female ) and three cross - sectional surveys of adults during 19952000 who remained in the community and participated in the study ( n = 3,640 ) . subjects ( n = 1,120 ; 36% black and 60% female ) who participated in their childhood and adulthood and had fasting blood samples on both examinations were included in the study . at the baseline examination , the children with a history of the treatment of diabetes or who had a fasting glucose level 100 mg / dl ( 5.6 these subjects were 418 years of age ( means sd age 11.6 3.6 years ) at baseline and 1939 years of age at follow - up ( mean sd age in adulthood was 28.3 5.1 years ) . with respect to age , race , sex , overall adiposity ( bmi z score ) and lipid , glucose , and insulin profile , the baseline childhood characteristics of the study cohort , which represented 8% of the original ascertained childhood population , were similar to the characteristics of the subjects who did not participate in the follow - up survey as adults ( data not shown ) . according to the american diabetes association criteria ( 12 ) , adult subjects were classified as normoglycemic ( n = 1,058 ) if they had a fasting glucose level < 100 mg / dl ( 5.6 mmol / l ) , pre - diabetic ( n = 37 ) if they had a fasting glucose level between 100 and 125 mg / dl ( 5.66.9 mmol / l ) , and diabetic ( n = 25 ) if they had a fasting glucose level 126 mg / dl ( 7 mmol / l ) or were taking medication for diabetes . informed consent was obtained from all participants , and the study was approved by the institutional review board of the tulane university health sciences center . participants were instructed to fast for 12 h before the venipuncture , and compliance was ascertained by an interview on the day of examination . information on personal health history ( e.g. , hypertension , dyslipidemia , or diabetes and medical treatment for these conditions ) was obtained by questionnaires . bmi ( in kg / m = weight in kilograms divided by the square of height in meters ) was used as a measure of overall adiposity ; waist circumference was used as an indicator of abdominal visceral fat . bmi z scores for childhood were calculated from the 2000 centers for disease control and prevention ( cdc ) growth charts to account for the differences in bmis by sex and age ( 14 ) . these growth charts express the bmis of children in the current study relative to their sex- and age - matched peers in the u.s . between 1963 and 1980 ; bmis of 5 year olds in the cdc growth charts also include data from 1988 to 1994 . bmis for adulthood were standardized based on age- and sex - specific means and sds . right upper - arm length and circumference were used to select the cuff size for blood pressure measurements with mercury sphygmomanometers . two randomly assigned nurses measured blood pressure ( three replicates each ) while subjects were in a relaxed , sitting position . systolic and diastolic blood pressures were recorded at the first and fourth ( children ) or fifth ( adults ) korotkoff , respectively . mean arterial pressure ( map ) , calculated as diastolic blood pressure plus one - third pulse pressure , was used in the analysis . cholesterol and triglyceride levels were initially measured using chemical procedures on a technicon autoanalyzer ii ( technicon instruments ) according to the laboratory manual of the lipid research clinics program . later , these variables were determined by enzymatic procedures on the abbott vp instrument ( abbott laboratories ) between 1987 and 1996 and on the hitachi 902 automatic analyzer ( roche diagnostics ) afterward . both chemical and enzymatic procedures met the performance requirements of the lipid standardization program of the cdc , which has routinely monitored the precision and accuracy of cholesterol , triglycerides , and hdl cholesterol measurements since the beginning of this study . serum lipoprotein cholesterol levels were analyzed by using a combination of heparin - calcium precipitation and agar agarose gel electrophoresis procedures ( 15 ) . the intraclass correlation coefficients between the blind duplicate ( 10% random sample ) values ranged from 0.86 to 0.98 for hdl cholesterol , 0.86 to 0.98 for ldl cholesterol , and 0.88 to 0.99 for triglycerides . from 1976 to 1991 , plasma glucose was measured initially by a glucose oxidase method using a beckman glucose analyzer ( beckman instruments ) . since then , it has been measured enzymatically as part of a multichemistry ( sma20 ) profile . plasma immunoreactive insulin levels were measured by a commercial radioimmunoassay kit ( phadebas , pharmacia diagnostics ) . the intraclass correlation coefficients between blind duplicate values ranged from 0.94 to 0.98 for insulin and 0.86 to 0.98 for glucose . in addition , an index of insulin resistance was calculated according to the homeostasis model assessment ( homa ) formula : homa of insulin resistance ( homa - ir ) = ( insulin [ u / ml ] glucose [ mmol / l]/22.5 ) . metabolic syndrome risk factors in adults were identified if subjects were centrally obese ( waist circumference > 102 cm for male subjects or > 88 cm for female subjects ) , were dyslipidemic ( ldl cholesterol 160 mg / dl [ 4.14 mmol / l ] , triglycerides 150 mg / dl [ 2.26 mmol / l ] , hdl cholesterol < 40 mg / dl [ 1.03 mmol / l ] for male or 50 mg / dl [ 1.29 mmol / l ] for female subjects , or on medication for dyslipidemia ) , were hyperglycemic ( fasting glucose 100 mg / dl [ 5.6 mmol / l ] or on treatment for diabetes ) , or were hypertensive ( systolic blood pressure 130 mmhg or diastolic blood pressure 85 mmhg or on antihypertensive medication ) ( 16 ) . metabolic syndrome was defined as coexistence of three or more of the above risk factors ( 16 ) . all of the statistical analyses were performed with sas version 9.1 ( sas institute ) . values of triglycerides , glucose , insulin , and homa - ir variables used in the analyses were log - transformed to improve normality . to evaluate the persistence or tracking of elevated levels of glucose , insulin , and homa index from childhood to adulthood , the baseline ( childhood ) age- , race- , and sex - specific top quintile for each of these variables was used as a cutoff point to classify children as having abnormal glucose homeostasis variables and to examine the distribution of such children among the corresponding adulthood quintiles 17 years later . models assessing the independent relations between childhood cardiometabolic risk factor variables and follow - up ( adulthood ) levels of glucose , insulin , or homa - ir were constructed using a stepwise multiple linear regression . the childhood independent variables initially included in these models were age , race , sex , bmi z score , bmi z score change from childhood to adulthood , map , ratio of total cholesterol to hdl cholesterol , as well as glucose ( for model 1 ) , insulin ( for model 2 ) , and homa - ir ( for model 3 ) . since waist circumference in childhood was not measured , bmi z score values were used as a childhood measure of obesity . the ratio of total to hdl cholesterol was chosen as a measure of dyslipidemia because it is a marker of insulin resistance characteristic of the metabolic syndrome . a stepwise logistic regression analysis including childhood age , race , sex , bmi z score , bmi z score change over time , map , and total - to - hdl cholesterol ratio was then used to determine the odds ratio and 95% ci of developing pre - diabetes and diabetes in adulthood on the basis of childhood levels ( top decile versus the rest ) of glucose and insulin ( model 1 ) and homa - ir ( model 2 ) . collinearity was checked in the fixed model . to assess the overall fit of the logistic regression model , a hosmer - lemeshow goodness - of - fit test was performed . because there was no interaction effect between childhood race ( or sex ) and glucose ( or insulin and homa - ir ) levels , the race - sex groups were combined to increase statistical power and to simplify the presentation . since the prevalence of both pre - diabetes and diabetes in the study cohort was low ( < 3.5% ) and the alternate analysis using the cox proportional hazard model gave essentially identical results , only the results of logistic regression analysis estimating the relative risk of diabetes status are presented . finally , the prevalence of cardiometabolic risk factors in adulthood was examined according to childhood levels ( top quintile versus the rest , specific for age , race , and sex ) of glucose and insulin . significant differences in the prevalence of the metabolic syndrome and its cardiometabolic risk factors in adulthood by childhood glucose , insulin , and homa - ir status were tested by the pearson 's test . participants were instructed to fast for 12 h before the venipuncture , and compliance was ascertained by an interview on the day of examination . information on personal health history ( e.g. , hypertension , dyslipidemia , or diabetes and medical treatment for these conditions ) was obtained by questionnaires . bmi ( in kg / m = weight in kilograms divided by the square of height in meters ) was used as a measure of overall adiposity ; waist circumference was used as an indicator of abdominal visceral fat . bmi z scores for childhood were calculated from the 2000 centers for disease control and prevention ( cdc ) growth charts to account for the differences in bmis by sex and age ( 14 ) . these growth charts express the bmis of children in the current study relative to their sex- and age - matched peers in the u.s . between 1963 and 1980 ; bmis of 5 year olds in the cdc growth charts also include data from 1988 to 1994 . bmis for adulthood were standardized based on age- and sex - specific means and sds . right upper - arm length and circumference were used to select the cuff size for blood pressure measurements with mercury sphygmomanometers . two randomly assigned nurses measured blood pressure ( three replicates each ) while subjects were in a relaxed , sitting position . systolic and diastolic blood pressures were recorded at the first and fourth ( children ) or fifth ( adults ) korotkoff , respectively . mean arterial pressure ( map ) , calculated as diastolic blood pressure plus one - third pulse pressure , was used in the analysis . cholesterol and triglyceride levels were initially measured using chemical procedures on a technicon autoanalyzer ii ( technicon instruments ) according to the laboratory manual of the lipid research clinics program . later , these variables were determined by enzymatic procedures on the abbott vp instrument ( abbott laboratories ) between 1987 and 1996 and on the hitachi 902 automatic analyzer ( roche diagnostics ) afterward . both chemical and enzymatic procedures met the performance requirements of the lipid standardization program of the cdc , which has routinely monitored the precision and accuracy of cholesterol , triglycerides , and hdl cholesterol measurements since the beginning of this study . serum lipoprotein cholesterol levels were analyzed by using a combination of heparin - calcium precipitation and agar agarose gel electrophoresis procedures ( 15 ) . the intraclass correlation coefficients between the blind duplicate ( 10% random sample ) values ranged from 0.86 to 0.98 for hdl cholesterol , 0.86 to 0.98 for ldl cholesterol , and 0.88 to 0.99 for triglycerides . from 1976 to 1991 , plasma glucose was measured initially by a glucose oxidase method using a beckman glucose analyzer ( beckman instruments ) . since then , it has been measured enzymatically as part of a multichemistry ( sma20 ) profile . plasma immunoreactive insulin levels were measured by a commercial radioimmunoassay kit ( phadebas , pharmacia diagnostics ) . the intraclass correlation coefficients between blind duplicate values ranged from 0.94 to 0.98 for insulin and 0.86 to 0.98 for glucose . in addition , an index of insulin resistance was calculated according to the homeostasis model assessment ( homa ) formula : homa of insulin resistance ( homa - ir ) = ( insulin [ u / ml ] glucose [ mmol / l]/22.5 ) . metabolic syndrome risk factors in adults were identified if subjects were centrally obese ( waist circumference > 102 cm for male subjects or > 88 cm for female subjects ) , were dyslipidemic ( ldl cholesterol 160 mg / dl [ 4.14 mmol / l ] , triglycerides 150 mg / dl [ 2.26 mmol / l ] , hdl cholesterol < 40 mg / dl [ 1.03 mmol / l ] for male or 50 mg / dl [ 1.29 mmol / l ] for female subjects , or on medication for dyslipidemia ) , were hyperglycemic ( fasting glucose 100 mg / dl [ 5.6 mmol / l ] or on treatment for diabetes ) , or were hypertensive ( systolic blood pressure 130 mmhg or diastolic blood pressure 85 mmhg or on antihypertensive medication ) ( 16 ) . metabolic syndrome was defined as coexistence of three or more of the above risk factors ( 16 ) . all of the statistical analyses were performed with sas version 9.1 ( sas institute ) . values of triglycerides , glucose , insulin , and homa - ir variables used in the analyses were log - transformed to improve normality . to evaluate the persistence or tracking of elevated levels of glucose , insulin , and homa index from childhood to adulthood , the baseline ( childhood ) age- , race- , and sex - specific top quintile for each of these variables was used as a cutoff point to classify children as having abnormal glucose homeostasis variables and to examine the distribution of such children among the corresponding adulthood quintiles 17 years later . models assessing the independent relations between childhood cardiometabolic risk factor variables and follow - up ( adulthood ) levels of glucose , insulin , or homa - ir were constructed using a stepwise multiple linear regression . the childhood independent variables initially included in these models were age , race , sex , bmi z score , bmi z score change from childhood to adulthood , map , ratio of total cholesterol to hdl cholesterol , as well as glucose ( for model 1 ) , insulin ( for model 2 ) , and homa - ir ( for model 3 ) . since waist circumference in childhood was not measured , bmi z score values were used as a childhood measure of obesity . the ratio of total to hdl cholesterol was chosen as a measure of dyslipidemia because it is a marker of insulin resistance characteristic of the metabolic syndrome . a stepwise logistic regression analysis including childhood age , race , sex , bmi z score , bmi z score change over time , map , and total - to - hdl cholesterol ratio was then used to determine the odds ratio and 95% ci of developing pre - diabetes and diabetes in adulthood on the basis of childhood levels ( top decile versus the rest ) of glucose and insulin ( model 1 ) and homa - ir ( model 2 ) . collinearity was checked in the fixed model . to assess the overall fit of the logistic regression model , a hosmer - lemeshow goodness - of - fit test was performed . because there was no interaction effect between childhood race ( or sex ) and glucose ( or insulin and homa - ir ) levels , the race - sex groups were combined to increase statistical power and to simplify the presentation . since the prevalence of both pre - diabetes and diabetes in the study cohort was low ( < 3.5% ) and the alternate analysis using the cox proportional hazard model gave essentially identical results , only the results of logistic regression analysis estimating the relative risk of diabetes status are presented . finally , the prevalence of cardiometabolic risk factors in adulthood was examined according to childhood levels ( top quintile versus the rest , specific for age , race , and sex ) of glucose and insulin . significant differences in the prevalence of the metabolic syndrome and its cardiometabolic risk factors in adulthood by childhood glucose , insulin , and homa - ir status were tested by the pearson 's test . the persistence ( tracking ) of levels of glucose homeostasis variables ( fasting glucose , insulin , and homa - ir ) from childhood to adulthood was examined in terms of persistence of ranking in highest quintiles of the distribution over a 17-year period . if there is no persistence , 20% of those in a given quintile at baseline would persist in that ranking at the follow - up assessment by chance alone . as shown in fig . 1 , > 32.1% of individuals who ranked highest ( in the top quintile ) with respect to glucose in childhood also did so in adulthood ; another 22.5% remained in the next highest ( fourth ) quintile . in other words , 54.6% of individuals who ranked highest in childhood tended to maintain their high ranks by being above the 60th percentile in adulthood . further , these individuals ( trackers ) who maintained their high ranks by being above the 60th percentile in adulthood with respect to glucose , insulin , and homa - ir , compared with the nontracker group ( individuals in top quintile at baseline and below the 60th percentile in adulthood ) , displayed consistently higher bmi z score at baseline and bmi z score change after 17-year follow - up ( p < 0.05 ) , after adjusting for age , race , and sex at baseline ; no consistent trend was observed in other cardiometabolic variables ( data not shown ) . with respect to tracking in the lowest quintiles from childhood to adulthood tracking of glucose , insulin , and homa index over a 17-year period in young adults . the degree of tracking was evaluated in terms of distribution by adulthood quintiles at follow - up of subjects who were in the extreme top quintile specific for age , race , and sex at baseline in childhood . the percentage on the vertical axis denotes the proportion of subjects at baseline in childhood remaining in each quintile at follow - up in adulthood . as shown in table 1 , based on a stepwise multivariate regression analysis , childhood levels of glucose ( model 1 ) , insulin ( model 2 ) , and homa - ir ( model 3 ) were independent predictors of corresponding follow - up adulthood levels 17 years later . however , as shown by the standardized regression coefficients , the best predictors for adult glucose , insulin , and homa - ir levels were the change of bmi z score from childhood to adulthood and baseline bmi z score , in that order . the next best predictors for all these variables were the corresponding childhood level , followed by male sex ( for glucose ad insulin ) , age ( for glucose ) , and total - to - hdl cholesterol ratio ( for glucose , insulin , and homa - ir ) , in that order . overall , these variables accounted for 12.5 , 45.4 , and 43.9% of the variance in glucose , insulin , and homa - ir , respectively . childhood predictors of follow - up levels of glucose , insulin , and homa - ir in young adults after 17 years is the standardized regression coefficient . * stepwise regression model includes age , race , sex , baseline bmi z score , bmi z score change over time , map , total - to - hdl cholesterol ratio , as well as baseline childhood glucose and insulin ( models 1 and 2 , respectively ) and homa - ir ( model 3 ) . as shown in table 2 , among the childhood glucose homeostasis variables , insulin and homa - ir ( top decile versus the rest ) showed an odds ratio of 2.85 ( p < 0.05 ) and 2.55 ( p < 0.05 ) , respectively , for developing pre - diabetes after 17 years . in addition , age , baseline bmi z score , bmi z score change over time , and total - to - hdl cholesterol ratio showed odds ratios of 1.14 , 1.44 , 1.85 , and 1.05 , respectively , for developing pre - diabetes ( p < 0.05 ) . with respect to developing diabetes , the odds ratios were 3.28 ( p < 0.05 ) for glucose , 5.54 ( p = 0.0001 ) for insulin , and 5.84 for homa - ir ( p < 0.0001 ) ; neither bmi z score nor bmi z score change over time predicted the development of overt type 2 diabetes . further , alternate logistic regression models including age , race , sex , bmi z score , and bmi z score change over time , along with either childhood glucose and insulin ( model 1 ) or homa - ir ( model 2 ) , showed glucose homeostasis variables , bmi z score , and bmi z score change over time as significant predictors of adult pre - diabetes and diabetes ( p < 0.05 ) . p values of goodness - of - fit test for all models were > 0.20 ( data not shown ) . odds ratios ( 95% cis ) for developing pre - diabetes and diabetes in adulthood on the basis of childhood levels of glucose , insulin , and homa - ir * stepwise logistic regression model includes age , race , sex , baseline bmi z score , bmi z score change over time , map , total - to - hdl cholesterol ratio , as well as baseline childhood glucose and insulin ( top decile versus the rest ) for model 1 and homa - ir ( top decile versus the rest ) for model 2 . on the basis of glucose homeostasis variable levels ( top quintile versus the rest ) , children were classified into low - risk versus high - risk group and the prevalence rates of obesity , hypertension , dyslipidemia , hyperglycemia , hyperinsulinemia , and metabolic syndrome in adulthood after 17 years of follow - up were compared between the two groups ( supplementary table 1 of the online appendix [ available at http://care.diabetesjournals.org/cgi/content/full/dc09-1635/dc1 ] ) . the prevalence of adulthood metabolic syndrome and its variables in the high - risk versus low - risk group was significantly greater with respect to hyperglycemia , hypertriglyceridemia ( marginal significant in childhood insulin group ) , hypertension ( except childhood glucose group ) , obesity ( except childhood glucose group ) , low hdl cholesterol ( except childhood glucose group ) , high ldl cholesterol ( childhood glucose group only ) , and metabolic syndrome . this community - based study demonstrates that elevated levels of glucose , insulin , and insulin resistance index ( homa - ir ) in childhood track and persist in ranking over a 17-year period . childhood levels relate independently to corresponding adulthood levels and predict pre - diabetes ( except childhood glucose ) and diabetes conditions in adulthood , independent of age , race , sex , change in bmi z score over time , childhood bmi z score , map , and total - to - hdl cholesterol ratio . in addition , childhood high- versus low - risk status ( top quintile versus the rest ) with respect to glucose homeostasis variables was associated with increased prevalences of the metabolic syndrome and its component cardiometabolic risk factors . of particular interest , childhood glucose levels clinically considered within the normal range persist into adulthood and can predict diabetes . the current findings showing the persistence of adverse levels of glucose homeostasis variables since childhood ( 710 ) , and related predictability of adult pre - diabetes and diabetes conditions , are in agreement with previous reports ( 6,17,18 ) . ( 8) have demonstrated that the individuals with relatively high / low insulin levels trended to retain such levels over an 8-year follow - up . of those who had insulin levels ranked in the top quartile at baseline elevations in fasting plasma glucose within the normoglycemic range indeed may track from childhood to adulthood and reflect the progression from normal glucose tolerance before the onset of impaired glucose regulation as a continuous process in the development of diabetes ( 10,18,19 ) . of note , gain in adiposity ( bmi ) , a modifiable risk factor , from childhood to adulthood along with childhood adiposity were the best predictors of the adult glucose homeostasis variables in this study . because obesity is pathologically linked to insulin resistance / hyperinsulinemia , it plays a crucial role as an initiating factor in the development of dysglycemia . this is consistent with earlier observations showing temporal associations between the degree of baseline adiposity and the incidence of hyperinsulinemia ( 20 ) or metabolic syndrome ( 21 ) , independently of baseline insulin levels . studies ( 6,9,18 ) have also shown baseline obesity to be an independent risk factor for type 2 diabetes . the observational nature of the current study can not address the issue of causality but only suggests putative mechanisms for the observed relationships . intra - abdominal and intramyocellular lipid accumulation along with adipocyte - derived cytokines have been involved in the development of insulin resistance and the attendant type 2 diabetes ( 22 ) . it is also apparent from the present study that children with top quintile ( high risk ) of glucose , insulin , and homa - ir levels displayed increased prevalence of metabolic syndrome and are associated with type 2 diabetes . as mentioned earlier , excess adiposity , especially visceral fat , may be the initiating factor in the observed adverse relationships ( 21 ) . excess fat and related insulin resistance / hyperinsulinemia increase triglyceride ( vldls ) levels as a result of abnormal fatty acid metabolism and excess hepatic triglyceride synthesis and/or low clearance of triglycerides from the circulation ( 23 ) . in turn , increases in ldl cholesterol and decreases in hdl cholesterol levels ensue ( 24 ) . with respect to blood pressure , hyperinsulinemia could relate to raises in levels by 1 ) increasing renal sodium retention , 2 ) stimulating the sympathetic nervous system , 3 ) disturbing cell membrane calcium transport , and 4 ) increasing the smooth muscle cell proliferation ( 5,25 ) . alternatively , excess adiposity , per se , increases blood pressure by adversely altering , among others , intravascular volume , cardiac output , renal pressure natriuresis , and the adipose renin - angiotensin - aldosterone system ( 25 ) . taken together , it appears that excess levels of glucose homeostasis variables within the normoglycemic range even in childhood is a biomarker of risk for developing adverse cardiometabolic conditions including diabetes and subtle abnormalities of the cardiovascular system . the present study has certain limitations in that it lacks direct assessments of postchallenge glucose , in vivo insulin action and secretion , glycosylated hemoglobin , and body fat mass and distribution . instead , we used well - established simple surrogate measures of glucose homeostasis that are applicable to population studies . however , it should be mentioned that nonsystematic misclassification of self - reports would actually tend to underestimate the outcome . further , the current findings should be viewed with caution in view of the modest number of events , especially diabetes . in summary , the present findings indicate the importance of even moderately elevated levels of childhood glucose homeostasis variables ( glucose , insulin , and homa - ir ) considered within the normoglycemic range in terms of predicting pre - diabetes , diabetes , and metabolic syndrome and its cardiometabolic risk factors in apparently healthy young adults , with obesity and the change of obesity levels over time being the major contributors . additional longitudinal population - based studies are obviously needed to validate the current findings and to develop the common glucose homeostasis variable cutoff values for type 2 diabetes and other cardiometabolic risk assessment and intervention in pediatric population .

objectivethis study examines the usefulness of childhood glucose homeostasis variables ( glucose , insulin , and insulin resistance index [ homeostasis model assessment of insulin resistance { homa - ir } ] ) in predicting pre - diabetes and type 2 diabetes and related cardiometabolic risk factors in adulthood.research design and methodsthis retrospective cohort study consisted of normoglycemic ( n = 1,058 ) , pre - diabetic ( n = 37 ) , and type 2 diabetic ( n = 25 ) adults aged 1939 years who were followed on average for 17 years since childhood.resultsat least 50% of the individuals who ranked highest ( top quintile ) in childhood for glucose homeostasis variables maintained their high rank by being above the 60th percentile in adulthood . in a multivariate model , the best predictors of adulthood glucose homeostasis variables were the change in bmi z score from childhood to adulthood and childhood bmi z score , followed by the corresponding childhood levels of glucose , insulin , and homa - ir . further , children in the top decile versus the rest for insulin and homa - ir were 2.85 and 2.55 times , respectively , more likely to develop pre - diabetes ; children in the top decile versus the rest for glucose , insulin , and homa - ir were 3.28 , 5.54 , and 5.84 times , respectively , more likely to develop diabetes , independent of change in bmi z score , baseline bmi z score , and total - to - hdl cholesterol ratio . in addition , children with adverse levels ( top quintile versus the rest ) of glucose homeostasis variables displayed significantly higher prevalences of , among others , hyperglycemia , hypertriglyceridemia , and metabolic syndrome.conclusionsadverse levels of glucose homeostasis variables in childhood not only persist into adulthood but also predict adult pre - diabetes and type 2 diabetes and relate to cardiometabolic risk factors .

RESEARCH DESIGN AND METHODS General examination Laboratory analyses Statistical analysis RESULTS CONCLUSIONS Supplementary Material

the retrospective study cohort was derived from the two sets of the cross - sectional surveys conducted in the community ( 65% white and 35% black subjects ) of bogalusa , louisiana , involving five cross - sectional surveys of children during 19811994 ( n = 13,444 ; 40% black and 50% female ) and three cross - sectional surveys of adults during 19952000 who remained in the community and participated in the study ( n = 3,640 ) . subjects ( n = 1,120 ; 36% black and 60% female ) who participated in their childhood and adulthood and had fasting blood samples on both examinations were included in the study . at the baseline examination , the children with a history of the treatment of diabetes or who had a fasting glucose level 100 mg / dl ( 5.6 these subjects were 418 years of age ( means sd age 11.6 3.6 years ) at baseline and 1939 years of age at follow - up ( mean sd age in adulthood was 28.3 5.1 years ) . with respect to age , race , sex , overall adiposity ( bmi z score ) and lipid , glucose , and insulin profile , the baseline childhood characteristics of the study cohort , which represented 8% of the original ascertained childhood population , were similar to the characteristics of the subjects who did not participate in the follow - up survey as adults ( data not shown ) . according to the american diabetes association criteria ( 12 ) , adult subjects were classified as normoglycemic ( n = 1,058 ) if they had a fasting glucose level < 100 mg / dl ( 5.6 mmol / l ) , pre - diabetic ( n = 37 ) if they had a fasting glucose level between 100 and 125 mg / dl ( 5.66.9 mmol / l ) , and diabetic ( n = 25 ) if they had a fasting glucose level 126 mg / dl ( 7 mmol / l ) or were taking medication for diabetes . informed consent was obtained from all participants , and the study was approved by the institutional review board of the tulane university health sciences center . both chemical and enzymatic procedures met the performance requirements of the lipid standardization program of the cdc , which has routinely monitored the precision and accuracy of cholesterol , triglycerides , and hdl cholesterol measurements since the beginning of this study . the intraclass correlation coefficients between blind duplicate values ranged from 0.94 to 0.98 for insulin and 0.86 to 0.98 for glucose . in addition , an index of insulin resistance was calculated according to the homeostasis model assessment ( homa ) formula : homa of insulin resistance ( homa - ir ) = ( insulin [ u / ml ] glucose [ mmol / l]/22.5 ) . metabolic syndrome risk factors in adults were identified if subjects were centrally obese ( waist circumference > 102 cm for male subjects or > 88 cm for female subjects ) , were dyslipidemic ( ldl cholesterol 160 mg / dl [ 4.14 mmol / l ] , triglycerides 150 mg / dl [ 2.26 mmol / l ] , hdl cholesterol < 40 mg / dl [ 1.03 mmol / l ] for male or 50 mg / dl [ 1.29 mmol / l ] for female subjects , or on medication for dyslipidemia ) , were hyperglycemic ( fasting glucose 100 mg / dl [ 5.6 mmol / l ] or on treatment for diabetes ) , or were hypertensive ( systolic blood pressure 130 mmhg or diastolic blood pressure 85 mmhg or on antihypertensive medication ) ( 16 ) . metabolic syndrome was defined as coexistence of three or more of the above risk factors ( 16 ) . values of triglycerides , glucose , insulin , and homa - ir variables used in the analyses were log - transformed to improve normality . to evaluate the persistence or tracking of elevated levels of glucose , insulin , and homa index from childhood to adulthood , the baseline ( childhood ) age- , race- , and sex - specific top quintile for each of these variables was used as a cutoff point to classify children as having abnormal glucose homeostasis variables and to examine the distribution of such children among the corresponding adulthood quintiles 17 years later . models assessing the independent relations between childhood cardiometabolic risk factor variables and follow - up ( adulthood ) levels of glucose , insulin , or homa - ir were constructed using a stepwise multiple linear regression . the childhood independent variables initially included in these models were age , race , sex , bmi z score , bmi z score change from childhood to adulthood , map , ratio of total cholesterol to hdl cholesterol , as well as glucose ( for model 1 ) , insulin ( for model 2 ) , and homa - ir ( for model 3 ) . since waist circumference in childhood was not measured , bmi z score values were used as a childhood measure of obesity . the ratio of total to hdl cholesterol was chosen as a measure of dyslipidemia because it is a marker of insulin resistance characteristic of the metabolic syndrome . a stepwise logistic regression analysis including childhood age , race , sex , bmi z score , bmi z score change over time , map , and total - to - hdl cholesterol ratio was then used to determine the odds ratio and 95% ci of developing pre - diabetes and diabetes in adulthood on the basis of childhood levels ( top decile versus the rest ) of glucose and insulin ( model 1 ) and homa - ir ( model 2 ) . because there was no interaction effect between childhood race ( or sex ) and glucose ( or insulin and homa - ir ) levels , the race - sex groups were combined to increase statistical power and to simplify the presentation . since the prevalence of both pre - diabetes and diabetes in the study cohort was low ( < 3.5% ) and the alternate analysis using the cox proportional hazard model gave essentially identical results , only the results of logistic regression analysis estimating the relative risk of diabetes status are presented . finally , the prevalence of cardiometabolic risk factors in adulthood was examined according to childhood levels ( top quintile versus the rest , specific for age , race , and sex ) of glucose and insulin . significant differences in the prevalence of the metabolic syndrome and its cardiometabolic risk factors in adulthood by childhood glucose , insulin , and homa - ir status were tested by the pearson 's test . both chemical and enzymatic procedures met the performance requirements of the lipid standardization program of the cdc , which has routinely monitored the precision and accuracy of cholesterol , triglycerides , and hdl cholesterol measurements since the beginning of this study . the intraclass correlation coefficients between blind duplicate values ranged from 0.94 to 0.98 for insulin and 0.86 to 0.98 for glucose . in addition , an index of insulin resistance was calculated according to the homeostasis model assessment ( homa ) formula : homa of insulin resistance ( homa - ir ) = ( insulin [ u / ml ] glucose [ mmol / l]/22.5 ) . metabolic syndrome risk factors in adults were identified if subjects were centrally obese ( waist circumference > 102 cm for male subjects or > 88 cm for female subjects ) , were dyslipidemic ( ldl cholesterol 160 mg / dl [ 4.14 mmol / l ] , triglycerides 150 mg / dl [ 2.26 mmol / l ] , hdl cholesterol < 40 mg / dl [ 1.03 mmol / l ] for male or 50 mg / dl [ 1.29 mmol / l ] for female subjects , or on medication for dyslipidemia ) , were hyperglycemic ( fasting glucose 100 mg / dl [ 5.6 mmol / l ] or on treatment for diabetes ) , or were hypertensive ( systolic blood pressure 130 mmhg or diastolic blood pressure 85 mmhg or on antihypertensive medication ) ( 16 ) . metabolic syndrome was defined as coexistence of three or more of the above risk factors ( 16 ) . values of triglycerides , glucose , insulin , and homa - ir variables used in the analyses were log - transformed to improve normality . to evaluate the persistence or tracking of elevated levels of glucose , insulin , and homa index from childhood to adulthood , the baseline ( childhood ) age- , race- , and sex - specific top quintile for each of these variables was used as a cutoff point to classify children as having abnormal glucose homeostasis variables and to examine the distribution of such children among the corresponding adulthood quintiles 17 years later . models assessing the independent relations between childhood cardiometabolic risk factor variables and follow - up ( adulthood ) levels of glucose , insulin , or homa - ir were constructed using a stepwise multiple linear regression . the childhood independent variables initially included in these models were age , race , sex , bmi z score , bmi z score change from childhood to adulthood , map , ratio of total cholesterol to hdl cholesterol , as well as glucose ( for model 1 ) , insulin ( for model 2 ) , and homa - ir ( for model 3 ) . since waist circumference in childhood was not measured , bmi z score values were used as a childhood measure of obesity . the ratio of total to hdl cholesterol was chosen as a measure of dyslipidemia because it is a marker of insulin resistance characteristic of the metabolic syndrome . a stepwise logistic regression analysis including childhood age , race , sex , bmi z score , bmi z score change over time , map , and total - to - hdl cholesterol ratio was then used to determine the odds ratio and 95% ci of developing pre - diabetes and diabetes in adulthood on the basis of childhood levels ( top decile versus the rest ) of glucose and insulin ( model 1 ) and homa - ir ( model 2 ) . because there was no interaction effect between childhood race ( or sex ) and glucose ( or insulin and homa - ir ) levels , the race - sex groups were combined to increase statistical power and to simplify the presentation . since the prevalence of both pre - diabetes and diabetes in the study cohort was low ( < 3.5% ) and the alternate analysis using the cox proportional hazard model gave essentially identical results , only the results of logistic regression analysis estimating the relative risk of diabetes status are presented . finally , the prevalence of cardiometabolic risk factors in adulthood was examined according to childhood levels ( top quintile versus the rest , specific for age , race , and sex ) of glucose and insulin . significant differences in the prevalence of the metabolic syndrome and its cardiometabolic risk factors in adulthood by childhood glucose , insulin , and homa - ir status were tested by the pearson 's test . the persistence ( tracking ) of levels of glucose homeostasis variables ( fasting glucose , insulin , and homa - ir ) from childhood to adulthood was examined in terms of persistence of ranking in highest quintiles of the distribution over a 17-year period . 1 , > 32.1% of individuals who ranked highest ( in the top quintile ) with respect to glucose in childhood also did so in adulthood ; another 22.5% remained in the next highest ( fourth ) quintile . in other words , 54.6% of individuals who ranked highest in childhood tended to maintain their high ranks by being above the 60th percentile in adulthood . further , these individuals ( trackers ) who maintained their high ranks by being above the 60th percentile in adulthood with respect to glucose , insulin , and homa - ir , compared with the nontracker group ( individuals in top quintile at baseline and below the 60th percentile in adulthood ) , displayed consistently higher bmi z score at baseline and bmi z score change after 17-year follow - up ( p < 0.05 ) , after adjusting for age , race , and sex at baseline ; no consistent trend was observed in other cardiometabolic variables ( data not shown ) . with respect to tracking in the lowest quintiles from childhood to adulthood tracking of glucose , insulin , and homa index over a 17-year period in young adults . the degree of tracking was evaluated in terms of distribution by adulthood quintiles at follow - up of subjects who were in the extreme top quintile specific for age , race , and sex at baseline in childhood . as shown in table 1 , based on a stepwise multivariate regression analysis , childhood levels of glucose ( model 1 ) , insulin ( model 2 ) , and homa - ir ( model 3 ) were independent predictors of corresponding follow - up adulthood levels 17 years later . however , as shown by the standardized regression coefficients , the best predictors for adult glucose , insulin , and homa - ir levels were the change of bmi z score from childhood to adulthood and baseline bmi z score , in that order . the next best predictors for all these variables were the corresponding childhood level , followed by male sex ( for glucose ad insulin ) , age ( for glucose ) , and total - to - hdl cholesterol ratio ( for glucose , insulin , and homa - ir ) , in that order . overall , these variables accounted for 12.5 , 45.4 , and 43.9% of the variance in glucose , insulin , and homa - ir , respectively . childhood predictors of follow - up levels of glucose , insulin , and homa - ir in young adults after 17 years is the standardized regression coefficient . * stepwise regression model includes age , race , sex , baseline bmi z score , bmi z score change over time , map , total - to - hdl cholesterol ratio , as well as baseline childhood glucose and insulin ( models 1 and 2 , respectively ) and homa - ir ( model 3 ) . as shown in table 2 , among the childhood glucose homeostasis variables , insulin and homa - ir ( top decile versus the rest ) showed an odds ratio of 2.85 ( p < 0.05 ) and 2.55 ( p < 0.05 ) , respectively , for developing pre - diabetes after 17 years . in addition , age , baseline bmi z score , bmi z score change over time , and total - to - hdl cholesterol ratio showed odds ratios of 1.14 , 1.44 , 1.85 , and 1.05 , respectively , for developing pre - diabetes ( p < 0.05 ) . with respect to developing diabetes , the odds ratios were 3.28 ( p < 0.05 ) for glucose , 5.54 ( p = 0.0001 ) for insulin , and 5.84 for homa - ir ( p < 0.0001 ) ; neither bmi z score nor bmi z score change over time predicted the development of overt type 2 diabetes . further , alternate logistic regression models including age , race , sex , bmi z score , and bmi z score change over time , along with either childhood glucose and insulin ( model 1 ) or homa - ir ( model 2 ) , showed glucose homeostasis variables , bmi z score , and bmi z score change over time as significant predictors of adult pre - diabetes and diabetes ( p < 0.05 ) . odds ratios ( 95% cis ) for developing pre - diabetes and diabetes in adulthood on the basis of childhood levels of glucose , insulin , and homa - ir * stepwise logistic regression model includes age , race , sex , baseline bmi z score , bmi z score change over time , map , total - to - hdl cholesterol ratio , as well as baseline childhood glucose and insulin ( top decile versus the rest ) for model 1 and homa - ir ( top decile versus the rest ) for model 2 . on the basis of glucose homeostasis variable levels ( top quintile versus the rest ) , children were classified into low - risk versus high - risk group and the prevalence rates of obesity , hypertension , dyslipidemia , hyperglycemia , hyperinsulinemia , and metabolic syndrome in adulthood after 17 years of follow - up were compared between the two groups ( supplementary table 1 of the online appendix [ available at http://care.diabetesjournals.org/cgi/content/full/dc09-1635/dc1 ] ) . the prevalence of adulthood metabolic syndrome and its variables in the high - risk versus low - risk group was significantly greater with respect to hyperglycemia , hypertriglyceridemia ( marginal significant in childhood insulin group ) , hypertension ( except childhood glucose group ) , obesity ( except childhood glucose group ) , low hdl cholesterol ( except childhood glucose group ) , high ldl cholesterol ( childhood glucose group only ) , and metabolic syndrome . this community - based study demonstrates that elevated levels of glucose , insulin , and insulin resistance index ( homa - ir ) in childhood track and persist in ranking over a 17-year period . childhood levels relate independently to corresponding adulthood levels and predict pre - diabetes ( except childhood glucose ) and diabetes conditions in adulthood , independent of age , race , sex , change in bmi z score over time , childhood bmi z score , map , and total - to - hdl cholesterol ratio . in addition , childhood high- versus low - risk status ( top quintile versus the rest ) with respect to glucose homeostasis variables was associated with increased prevalences of the metabolic syndrome and its component cardiometabolic risk factors . of particular interest , childhood glucose levels clinically considered within the normal range persist into adulthood and can predict diabetes . the current findings showing the persistence of adverse levels of glucose homeostasis variables since childhood ( 710 ) , and related predictability of adult pre - diabetes and diabetes conditions , are in agreement with previous reports ( 6,17,18 ) . of those who had insulin levels ranked in the top quartile at baseline elevations in fasting plasma glucose within the normoglycemic range indeed may track from childhood to adulthood and reflect the progression from normal glucose tolerance before the onset of impaired glucose regulation as a continuous process in the development of diabetes ( 10,18,19 ) . of note , gain in adiposity ( bmi ) , a modifiable risk factor , from childhood to adulthood along with childhood adiposity were the best predictors of the adult glucose homeostasis variables in this study . intra - abdominal and intramyocellular lipid accumulation along with adipocyte - derived cytokines have been involved in the development of insulin resistance and the attendant type 2 diabetes ( 22 ) . it is also apparent from the present study that children with top quintile ( high risk ) of glucose , insulin , and homa - ir levels displayed increased prevalence of metabolic syndrome and are associated with type 2 diabetes . alternatively , excess adiposity , per se , increases blood pressure by adversely altering , among others , intravascular volume , cardiac output , renal pressure natriuresis , and the adipose renin - angiotensin - aldosterone system ( 25 ) . taken together , it appears that excess levels of glucose homeostasis variables within the normoglycemic range even in childhood is a biomarker of risk for developing adverse cardiometabolic conditions including diabetes and subtle abnormalities of the cardiovascular system . further , the current findings should be viewed with caution in view of the modest number of events , especially diabetes . in summary , the present findings indicate the importance of even moderately elevated levels of childhood glucose homeostasis variables ( glucose , insulin , and homa - ir ) considered within the normoglycemic range in terms of predicting pre - diabetes , diabetes , and metabolic syndrome and its cardiometabolic risk factors in apparently healthy young adults , with obesity and the change of obesity levels over time being the major contributors . additional longitudinal population - based studies are obviously needed to validate the current findings and to develop the common glucose homeostasis variable cutoff values for type 2 diabetes and other cardiometabolic risk assessment and intervention in pediatric population .

pain is defined as an unpleasant sensory and emotional experience arising from any part of the body . it is associated with actual or potential tissue damage or described in terms of such damage by international association for the study of pain . it is an experience and in this respect , it differs from nociception . nociception is called a neural process that provides transduction and transmission of a noxious stimulus to the brain via pain pathways . the pain arises from a complicated interaction between signaling systems , modulation of higher centers , and individual perception . the whole human population experiences pain in varying degrees and daily routine is affected negatively . pain may be occurred acutely or chronically related with various disturbances such as lesions , traumatic injury , tumors , inflammatory diseases , parkinson 's disease , and diabetes . since different mechanisms involve in the pathophysiology of acute and chronic pain and even nociceptive and neuropathic pain , the management strategies and current drug classes also vary . although there are too many analgesic agents , there are certain problems such as tolerability , tolerance , abstinence syndrome , insufficiency , possible drug interactions , and side effects . thereby , the development of new analgesic compounds is still going on . in this respect , the development and the use of imidazoline receptor ligands have gradually drawn attention since the role of imidazoline receptors in pain modulation was identified . for instance , various ligands which bind to imidazoline-2 ( i2 ) receptors , the imidazoline receptor subtype which is predominantly involved in pain modulation , have been synthesized 2-(4,5-dihydroimidazole-2-il ) quinoline hydrochloride ( bu224 ) , 2-(2-benzofuranyl)-2-imidazoline hydrochloride ( 2-bfi ) , 4-chloro-2-(imidazoline-2-yl ) isoindoline ( rs-45041 ) , etc . , in last decades and all of them have been reported to exhibit antinociceptive properties as discussed in this review . whereas the single use of imidazoline receptor ligands is effective in tonic and chronic pain , combined usage of other analgesic drugs such as morphine and clonidine is also effective in the potentiation of both acute and chronic pain conditions such as neuropathic pain . in fact , it is known that the i2 receptor agonism is one the mechanisms of neuropathic pain control , and the ligands that use this mechanism are in the phase 2 and phase 3 studies . in this review , we document the role of i2 receptors and ligands in antinociception and the relevant experimental studies performed by various researchers . although the term imidazoline receptor has not yet been adopted by major professional societies including international union of pharmacology committee on receptor nomenclature and drug classification since these receptors have not yet been cloned and the signaling pathway not characterized , this term is widely used in the literature . therefore , it is also frequently called as imidazoline binding sites ; however , the term imidazoline receptor is employed in this review . the presence of imidazoline receptors , with high affinity for imidazoline subdivision containing compounds , first became apparent in the mid-1970 . the hypotensive effect induced by clonidine , 2-adrenoceptor agonist , and microinjection into the rat brainstem was not mimicked by norepinephrine . the imidazoline receptors are broadly located in the mammalian cells of the central nervous system ( cns ) and peripheral nervous system and contribute to cardiovascular system activity , gastric acid secretion , insulin release , antinociception , alzheimer 's , and parkinson 's disorders . according to a general opinion , there are three main classes of heterogeneous imidazoline receptors , as seen in figure 1 . imidazoline-1 ( i1 ) receptors constitute a family of nonadrenergic high - affinity binding sites for some ligands such as clonidine and idazoxan . they are located in the plasma membranes in the brain , heart , kidney , liver , and pancreas . the i2 receptors bind imidazolines and guanidines and have a lower affinity for 2-aminoimidazolines such as clonidine . the i2 receptors are mitochondrial , not g - protein coupled , and allosteric binding sites , possibly with a modulatory function , on monoamine oxidase - a and -b ( mao - a and -b ) . these enzymes are found in the neurons and astroglia cells and have a critical role in the inactivation of neurotransmitters . thereby , the i2 receptors may be useful as a therapeutic agent in various neurological diseases in which neurodegeneration is observed . besides , it is known that these subtypes contribute to reduce body temperature , exert control over central noradrenergic and hypothalamic - pituitary - adrenal axis activity , and regulate the small intestinal motility . more remarkably , it has been shown that the i2 receptors take part in the antinociception in several acute and chronic pain models and the ligands acting on the i2 receptors may be assessed as novel analgesics . the imidazoline-3 ( i3 ) receptors whose biological importance investigated further , are present in the pancreatic beta islet cells and modulate insulin secretion . we focus on the i2 receptor subtypes in this educational forum because involvement of this subtype in antinociception is well determined . pain modulation is a highly complex process , including numerous interacting peripheral and central mechanisms . the activation of the peripheral nociceptors or mediators that are released by the damaged tissue is required for the perception of pain . the activation - triggered signal is conveyed with the afferent transmission to the spinal cord with a and c nerve fibers and transmission parts through the dorsal horn ( dh ) to the higher centers via parallel ascending pain pathways . the impulses originated from the brain stem nuclei , descend to the spinal level and affect the transmission of pain signals at the dh . the relative balance between descending inhibition and facilitation can be changed by the type and intensity of the stimulus and also by the time following an injury . this complex process is regulated by the interaction of various chemicals and receptors over an extensive network from the periphery to the cns . the rate of participation of the chemicals and receptor types in the modulation depends on the types of pain and noxious stimulus . the imidazoline receptors that are taken part in this extensive receptor networks have gradually gained important in the recent years . when the roles of imidazoline receptors are evaluated in terms of pain , the i2 receptors are distributed ubiquitously throughout the brain regions such as the arcuate nucleus , caudate nucleus , putamen , globus pallidus , substantia nigra , interpeduncular nucleus , area postrema , mammillary peduncle , ependyma , lateral mammillary nucleus , and the pineal gland . low to moderate densities are found in the cerebral cortex , thalamus , hippocampus , amygdala , inferior olivary nucleus , ependymal , and various periphery regions . the i2 receptors are characterized by their high affinity for imidazolines and guanidines and medium affinity for imidazolidines . also , they have two subtypes as i2a and i2b , which differ in terms of their sensitivity to amiloride . the i2 receptor is first located on the outer membranes of mitochondria and as allosteric sites on enzyme mao - a and mao - b . however , the imidazoline binding site on mao is separate from the active domain of the enzyme that recognizes the mechanism - based inhibitors , and it is not equally available in all the tissues . the i2 receptors have the same molecular weight as mao , and the amino acid sequencing of purified i2 receptors is similar to mao . two mao isoforms play a fundamental role in the metabolism of monoamine neurotransmitters such as serotonin , noradrenaline , and dopamine . as the i2 receptor is thought to be a modulatory site on the mao protein , the activity of i2 receptors may involve in several neurological disorders . another fascinating development with i2 receptors is that its effect on nociception since these monoamines appear to play a significant role in specific cns structures implicated in pain modulation spinal cord , cerebral cortex , etc . , and are involved in the antinociception of several drugs such as antidepressants which are commonly used for the management of pain . the ligands binding to the i2 receptors can be considered as inhibitors of mao - a and mao - b since they inhibited monoamine oxidation , and this inhibition could explain some biological effects of imidazoline receptor ligands such as pain modulation . in fact , it is well identified that increased levels of monoamines such as serotonin and noradrenaline contributes to pain control , especially in the cns regions related to pain . the drugs such as mao inhibitors , serotonin noradrenaline reuptake inhibitors , and selective serotonin reuptake inhibitors that provide the enhancement of levels of these neurotransmitters are successfully being used to relieve various pain conditions . some evidence that shows the i2 receptors contribute to the modulation of pain [ figure 1 ] reports that the ligands that affect i2 receptors are effective for tonic pain , neuropathic pain , but little effective for acute pain . moreover , the activation of i2 receptors has been suggested to be a way of enhancing opioid analgesic actions . when they are combined with opioids , the i2 receptor ligands increase the analgesic effects of opioids in both acute and chronic pain . in chronic use , tolerance and addiction to the opioids can develop . however , the i2 receptor ligands can reduce the development of this opioid tolerance or prevent from deprivation syndrome that is triggered by the antagonists in animals . when all of these findings are considered together , ligands that affect the i2 receptors can be beneficial in monotherapy or combination therapy with opioids to overcome pain . therefore , various new selective ligands are being developed for this receptor types . although the i2 receptors and their signaling pathways have not yet been characterized , the majority of the i2 receptors are widely accepted as being allosteric sites on mao . therefore , the i2 receptor ligands are described as allosteric modulators . allosteric modulation is the modulation of a protein via binding a ligand to the domain that is distinct from the active site of the protein . ligands that improves the activity of the protein are referred as allosteric activators or agonists , whereas those that decrease the activity of the protein are referred as allosteric inhibitors or antagonists . in principle , it is not precisely known whether a ligand is an i2 receptor agonist , an antagonist , or an inverse agonist in a constitutively active system . each and every ligand has two properties ( i.e. , affinity and efficacy ) that govern its effects related to certain receptors in a bioassay . it is possible to have a knowledge of the relative efficacy of these ligands by systematic comparison . ligand efficacy values may vary depending upon assays , however , the rank order of their efficacy remains unchanged with few exceptions ( e.g. , functional selectivity ) . idazoxan , agmatine , and clonidine are ligands that bind to the i2 receptors as well as 2 and/or i1 receptors with varying affinities . due to the proven effect of the i2 receptors in pain , further studies are required to discover selective analgesic i2 receptor ligands and to elucidate their physiological functions . over the last few decades , investigators have attempted to synthesize and recognize selective ligands for the i2 binding sites . for instance , the i2 receptor ligand 2-bfi ( pki = 8.47 for i2 in rabbit kidney membrane with [ h ] 2-bfi ) which has high affinity for both i2a and i2b sites has been used to characterize these sites in several species , including humans . it has been proved that quinolone compound bu224 has high affinity ( pki = 8.43 for i2 in rabbit kidney membrane with [ h ] 2-bfi ) for the i2 receptors over i1 receptors and 2-adrenoceptors , among all the compounds of bu series . rs-45041 - 190 is also a selective ligand with a high affinity ( pki = 9.37 for i2 in rabbit kidney membrane with [ h ] idazoxan ) for the i2 site which exhibits comparable data to that observed using 2-bfi in membrane - binding and autoradiographic studies . as mentioned before , the imidazoline receptor ligands are declared to modulate certain processes which involve mao activities in cns . the studies reveal that among all of the imidazoline receptor ligands those mentioned in this review ; 2-bfi , bu224 , 2-phenyl-6-(1h - imidazol-1il ) quinazoline ( cr4056 ) , idazoxan , and rs-45041 more selectively inhibit mao - a while lsl60101 and lsl61122 inhibit mao - b . after this part of the review , several studies will be conducted with the mentioned ligands [ figure 2 ] that have a higher affinity for the i2 receptors and the provided results will be included . the imidazoline imidazoline-2 receptor ligands in pain it should first be noted that clonidine that mediated the discovery of the presence of imidazoline receptors , is a 2-adrenoceptor agonist that binds not only to 2 adrenoceptors but also to imidazoline receptors in particular to i1 receptors compared to i2 receptors ; however , its antinociceptive activity occurs via 2-adrenoceptors rather than imidazoline receptors . agmatine is a nonselective and most extensively studied endogenous imidazoline receptor agonist , and has a moderate affinity to 2-adrenoceptors as well as all subtypes of imidazoline receptors ( pki < 5 for i2 in rabbit kidney membrane with [ h ] 2-bfi ) . it is indicated that this endogenous substance prevents reflex respond to the noxious stimulus through nonadrenergic receptors in mice . it does not show a significant effect on acute phasic pain while it is vice versa in the acute tonic ( differs from acute phasic pain in terms of using chemicals to induce noxious stimuli ) , inflammatory , and neuropathic pain . although agmatine showed weak effectiveness in a few studies , in many others , it was not efficient when administered systemically in acute phasic pain . spinal and supraspinal agmatine administration also does not reduce the thermal threshold , similar to systemic agmatine treatment . ( supraspinal ) agmatine administration did not produce analgesic effects in the tail - flick test in mice . the acute systemic agmatine treatment markedly decreased mechanical allodynia induced by complete freund 's adjuvant ( cfa ) , chronic pain model , in mice . agmatine injections by systemic and supraspinal route have also been demonstrated to be effective in relieving hyperalgesia and/or allodynia in several neuropathic pain models . systemic administration of the i2 receptor ligands as phenyzoline , 2-bfi , 2-(2-benzofuranyl ) imidazole hydrochloride ( lsl 60101 ) ; the analog of 2-bfi , 2-styryl-2-imidazoline ; valldemossine or tracizoline ( lsl 61122 ) also did not produce antinociception in the acute pain model . in addition , researches showed that the i2 receptor ligands such as rs-45041 - 190 , cr4056 , 2-bfi , bu224 were effective in inflammatory and neuropathic pain . rs-45041 - 190 , the first selective and high - affinity ligand , showed significant results on the carrageenan - induced thermal and mechanic hyperalgesia tests when administered systemically ( i.p . ) , not spinally ( i.t . ) in rats . cr4056 ( moderate affinity i2 receptor ligand ( ic50=596 nm , in rat whole brain membrane with [ h ] 2-bfi ) , is a new , highly selective i2 receptor ligand which inhibits mao - a activity more selectively than mao - b . in a study , cr4056 was found active in cfa - induced model of inflammation . in acute capsaicin - evoked pain model this effect was antagonized by idazoxan ( pki = 7.22 for i2 in rabbit kidney membrane with [ h ] 2-bfi ) , which is both a 2-adrenoceptor and i2 receptor antagonist . similarly , in another study , cr4056 oral administration dose - dependently reversed the allodynia in bortezomib - induced peripheral neuropathy model that is painful . , have investigated the antinociception induced by cr4056 in a rat model of postoperative pain and caselli et al . acute administration of cr4056 was found effective in relieving postoperative pain and joint pain as well as inflammatory and neuropathic pain . even naproxen showed low and nonremarkable antinociception compared to antinociception induced by cr4056 in these pain models . in addition , in joint pain model driven by both nociceptive and neuropathic mechanisms , the rats treated for 7 days ( from days 14 to 21 ) after the induction of cartilage degeneration with cr4056 , showed a significant reduction of both basal pain behaviors ( allodynia and hyperalgesia ) . this result demonstrates either a long lasting effect or even an actual symptom modifying effect . the joint pain model is related to osteoarthritis that is driven by both nociceptive and neuropathic mechanisms . shortly , these studies present a new opportunity for the management of inflammatory , neuropathic , postoperative , and joint pain based on the selective interactions with the central i2 receptors . the phase ii trials on neuropathic pain for cr4056 are available and still going on . the effect of bu224 , a high affinity selective i2 receptor ligand , has been assessed electrophysiologically on the nociceptive neurons responses in the spinal dh . route attenuated the nociceptive responses of dh neurons , creating a dose - dependent inhibition of c - fiber - induced responses , a-fibre - evoked responses , post discharge , and winding - up of the cells . idazoxan ( i.t . ) completely and significantly antagonized these effects while the nonselective 2-adrenoceptor antagonist yohimbine and the highly selective 2-adrenoceptor antagonist atipamezole only partially antagonized . when we consider the results , it is possible to say that bu224 has a high affinity for the spinal i2 receptors , as well as it has an insignificant action at the spinal 2-adrenoceptor receptors . it is obvious that the i2 receptor ligands are more effective in chronic pain models than acute phasic pain models as mentioned with agmatine . chronic pain usually means a persistent pain lasting 3 months or more , and pharmacotherapy of chronic pain always comprises repeated dose . a possible result of repeated doses of analgesics is a gradual decrease in analgesic effect , in other words , development of analgesic tolerance . from this point of view , in a research performed by li et al . , antihyperalgesic effects of 2-bfi and cr4056 have also been tested in repeating treatments , in addition to single doses in chronic constriction injury ( cci)-induced neuropathic pain and cfa - induced inflammatory pain models . the antinociceptive tolerance did not develop against repeated administration ( daily for 79 days ) of 2-bfi or cr4056 in cfa - treated or cci rats . it is possible to say that the repeated dose regimen for the i2 receptor ligands in chronic pain treatment may be useful as monotherapy or adjunctive therapy without tolerance and addiction . it has been also shown that three high selective i2 receptor ligands ; 2-bfi , bu224 , and lsl 61122 possess antihyperalgesic effects in the rat models of cfa evoked inflammatory pain in this study . also , the i2 receptor antagonist idazoxan antagonized the antihyperalgesic effects of 2-bfi in cfa - treated and cci rats . these data also support the beneficial roles of the i2 receptor ligands in pain control . although most parts of preclinical pain studies focus on sensorial relieving pain , the sensorial and emotional compounds should be studied together . for instance , the effects of the i2 receptor ligands such as 2-bfi , bu224 , and cr4056 on escape / avoidance behaviors on cfa injected rats were studied connectedly to the affective pain in the same study . this method is suitable to measure the dissociable components of effective pain which is different from the sensory pain . it was observed that 2-bfi , bu224 , and cr4056 increase the escape / avoidance behavior in the hyperalgesia increasing doses . these results exhibit that the i2 receptor ligands may be effective against the affective components of pain . more recently , thorn et al . have studied the antinociception induced by 2-bfi and phenyzoline , high affinity ligand for i2 receptors ( pki = 8.60 in rabbit kidney membrane with [ h]-idazoxan ) , using the von frey filament test in rats with cfa - induced inflammatory pain . providing antinociception by 2-bfi was not surprising , however , phenyzoline also produces antinociception in this chronic model as in acute phasic pain under weak noxious stimulus , as reported previously in a study by sampson et al . even some i2 receptor ligands as agmatine are not effective alone in acute phasic pain , they potentiate morphine antinociception . in a study , performed in the warm water , tail withdrawal procedure in rats by using selective i2 receptor ligand 2-bfi along with agmatine , it was observed that these two ligands increase the antinociceptive effects of morphine and tramadol . in contrast , another selective i2 receptor ligand bu224 failed to increase the antinociceptive efficacy but prevented agmatine and 2-bfi ligands to increase the morphine and tramadol - induced antinociception . the reason that bu224 acts differently from other imidazoline receptor ligands is its lower efficacy in spite of its high affinity . this contradictory situation may be confusing , but it should be noted that affinity and efficacy are distinct terms from each other . a ligand that shows low efficacy may bind its binding site with high affinity . in yet another study , the combination of 2-bfi and morphine produced additive effects on mechanical hyperalgesia in cfa - treated rats . these results suggest that the combination of the i2 receptor ligands and opioids may be effective in chronic pain treatment . however , the previous studies indicated the i2 receptors to contribute potentiation mechanism , and i1 and i2 receptors contribution of potentiation mechanism was not clearly understood . more recently , a study which has been done to understand which subtype contributes to potentiation mechanism showed that a significant oxycodone - induced antinociceptive respond could not be reversed by efaroxan ( i1 receptor antagonist ) but could be reversed by bu224 ( i2 receptor antagonist ) . as a similar way , endothelin eta receptor antagonist 5-(dimethylamino ) -n-(3,4-dimethyl-5- isoxazolyl)-1-naphthalenesulfonamide - induced potentiation of oxycodone antinociception was reversed by bu224 but not efaroxan . so , it is thought that the i2 receptors participate in the potentiation , the i1 receptors do not . in yet another study performed for understanding the importance and mechanisms of potentiation , agmatine , high selective and more powerful substances such as 2-bfi , lsl 60101 ( pki = 6.45 for i2 in rat cerebral cortex with [ h ] idazoxan ) , lsl 61122 ( pki = 8.74 for i2 in rabbit kidney with [ h ] idazoxan ) , and aganodine have been tested . it was observed that central ( i.c.v . ) or peripheral ( s.c . ) administration of the i2 receptor ligands ( but not i1 or 2 adrenoreceptor ) potentiate the morphine - evoked supraspinal antinociception in mice . the enhanced morphine antinociception via the i2 receptor ligands was reversed by idazoxan , bu224 , and isothiocyanatobenzylimidazoline , an irreversible i2 antagonist . in the same study , it has also been shown how the augmentation of morphine antinociception by the i2 agonists changes in mice with pertussis toxin impaired guanosine triphosphate - binding gi - go proteins . the potentiation ability effect was blocked . therefore , the contribution of gi - go transducer proteins in the modulation of morphine antinociception induced by the i2 receptors can not be disrespected . most recently , thorn et al . studied 2-bfi and phenyzoline with oxycodone as combinations , separately . 2-bfi and oxycodone produced additive interactions while phenyzoline and oxycodone produced synergistic interactions for their effects on mechanical hyperalgesia in cfa - treated rats . the imidazoline receptors are also important for the tolerance developed with opioids as well as improvement of opioid analgesia as mentioned before . for instance , agmatine prevents or decreases the tolerance development against morphine or other opioids . additionally , -difluoromethylornithine and aminoguanidine , which may affect the metabolism of endogenous agmatine , were found effective in the inhibition of acute morphine tolerance in tail - flick test . similarly , boronat et al . , have assessed the role of imidazoline receptors in opioid ( morphine and pentazocine ) tolerance in rats by the administration of idazoxan . idazoxan completely prevented the morphine tolerance , but 2-methoxy - idazoxane and rs-15385 - 197 , selective 2-adrenoceptor antagonists , did not and it remarkably reduced tolerance to pentazocine . in contrary , su et al . , showed that idazoxan promoted the development of tolerance to morphine and induced the abstinence syndrome in morphine - dependent mice and rats similar to naloxone . the chronic concurrent administration of 2-bfi , lsl 60101 , and lsl 61122 , selective and potent i2 receptor ligands , and morphine , also prevented or attenuated morphine tolerance . in the light of the positive outcomes , it is supported that the i2 receptor ligands as promising therapeutic coadjuvants in the management of chronic pain with opiate drugs since these agents prevent tolerance development and enhance opioid analgesia . all the studies show us that the i2 receptors are also steady , new drug targets for analgesics . even if the mechanism of the i2 receptor is not well known in the modulation of pain , it is known that it plays a role in tonic and chronic pain but not in the acute phasic pain . additionally , when they are combined with opioids in both acute and chronic pain , the i2 receptor ligands increase the antinociceptive actions of opioids . the development of tolerance and addiction induced by chronic administration of opioids is one of the major problems in the clinic . however , the i2 receptor ligands can reduce the opioid tolerance development or prevent from deprivation syndrome in the combination therapy . they are valuable for the chronic pain treatment and also valuable as therapeutic coadjuvants of opiates , because of the attenuation of opioid tolerance and addiction .

pain is an unpleasant experience and effects daily routine negatively . although there are various drugs , many of them are not entirely successful in relieving pain , since pain modulation is a complex process involving numerous mediators and receptors . therefore , it is a rational approach to identify the factors involved in the complex process and develop new agents that act on these pain producing mechanisms . in this respect , the involvement of the imidazoline receptors in pain modulation has drawn attention in recent years . in this review , it is aimed to focus on the imidazoline receptors and their ligands which contribute to the pain modulation . it is demonstrated that imidazoline-2 ( i2 ) receptors are steady new drug targets for analgesics . even if the mechanism of i2 receptor is not well known in the modulation of pain , it is known that it plays a role in tonic and chronic pain but not in acute phasic pain . moreover , the i2 receptor ligands increase the analgesic effects of opioids in both acute and chronic pain and prevent the development of opioid tolerance . so , they are valuable for the chronic pain treatment and also therapeutic coadjuvants in the management of chronic pain with opiate drugs due to the attenuation of opioid tolerance and addiction . thus , the use of the ligands which bind to the imidazoline receptors is an effective strategy for relieving pain . this educational forum exhibits the role of imidazoline receptors and ligands in pain process by utilizing experimental studies .

Introduction Imidazoline Receptors and Their Biologic Roles Imidazoline-2 Receptors in Pain Modulation Imidazoline-2 Receptor Ligands in Pain Conclusion None Financial Support and Sponsorship Conflicts of Interest

pain is defined as an unpleasant sensory and emotional experience arising from any part of the body . it is associated with actual or potential tissue damage or described in terms of such damage by international association for the study of pain . it is an experience and in this respect , it differs from nociception . the pain arises from a complicated interaction between signaling systems , modulation of higher centers , and individual perception . since different mechanisms involve in the pathophysiology of acute and chronic pain and even nociceptive and neuropathic pain , the management strategies and current drug classes also vary . although there are too many analgesic agents , there are certain problems such as tolerability , tolerance , abstinence syndrome , insufficiency , possible drug interactions , and side effects . thereby , the development of new analgesic compounds is still going on . in this respect , the development and the use of imidazoline receptor ligands have gradually drawn attention since the role of imidazoline receptors in pain modulation was identified . for instance , various ligands which bind to imidazoline-2 ( i2 ) receptors , the imidazoline receptor subtype which is predominantly involved in pain modulation , have been synthesized 2-(4,5-dihydroimidazole-2-il ) quinoline hydrochloride ( bu224 ) , 2-(2-benzofuranyl)-2-imidazoline hydrochloride ( 2-bfi ) , 4-chloro-2-(imidazoline-2-yl ) isoindoline ( rs-45041 ) , etc . , in last decades and all of them have been reported to exhibit antinociceptive properties as discussed in this review . whereas the single use of imidazoline receptor ligands is effective in tonic and chronic pain , combined usage of other analgesic drugs such as morphine and clonidine is also effective in the potentiation of both acute and chronic pain conditions such as neuropathic pain . in fact , it is known that the i2 receptor agonism is one the mechanisms of neuropathic pain control , and the ligands that use this mechanism are in the phase 2 and phase 3 studies . in this review , we document the role of i2 receptors and ligands in antinociception and the relevant experimental studies performed by various researchers . therefore , it is also frequently called as imidazoline binding sites ; however , the term imidazoline receptor is employed in this review . the presence of imidazoline receptors , with high affinity for imidazoline subdivision containing compounds , first became apparent in the mid-1970 . the imidazoline receptors are broadly located in the mammalian cells of the central nervous system ( cns ) and peripheral nervous system and contribute to cardiovascular system activity , gastric acid secretion , insulin release , antinociception , alzheimer 's , and parkinson 's disorders . according to a general opinion , there are three main classes of heterogeneous imidazoline receptors , as seen in figure 1 . they are located in the plasma membranes in the brain , heart , kidney , liver , and pancreas . the i2 receptors are mitochondrial , not g - protein coupled , and allosteric binding sites , possibly with a modulatory function , on monoamine oxidase - a and -b ( mao - a and -b ) . these enzymes are found in the neurons and astroglia cells and have a critical role in the inactivation of neurotransmitters . besides , it is known that these subtypes contribute to reduce body temperature , exert control over central noradrenergic and hypothalamic - pituitary - adrenal axis activity , and regulate the small intestinal motility . more remarkably , it has been shown that the i2 receptors take part in the antinociception in several acute and chronic pain models and the ligands acting on the i2 receptors may be assessed as novel analgesics . the imidazoline-3 ( i3 ) receptors whose biological importance investigated further , are present in the pancreatic beta islet cells and modulate insulin secretion . we focus on the i2 receptor subtypes in this educational forum because involvement of this subtype in antinociception is well determined . pain modulation is a highly complex process , including numerous interacting peripheral and central mechanisms . the activation of the peripheral nociceptors or mediators that are released by the damaged tissue is required for the perception of pain . the relative balance between descending inhibition and facilitation can be changed by the type and intensity of the stimulus and also by the time following an injury . this complex process is regulated by the interaction of various chemicals and receptors over an extensive network from the periphery to the cns . the rate of participation of the chemicals and receptor types in the modulation depends on the types of pain and noxious stimulus . the imidazoline receptors that are taken part in this extensive receptor networks have gradually gained important in the recent years . when the roles of imidazoline receptors are evaluated in terms of pain , the i2 receptors are distributed ubiquitously throughout the brain regions such as the arcuate nucleus , caudate nucleus , putamen , globus pallidus , substantia nigra , interpeduncular nucleus , area postrema , mammillary peduncle , ependyma , lateral mammillary nucleus , and the pineal gland . the i2 receptors are characterized by their high affinity for imidazolines and guanidines and medium affinity for imidazolidines . the i2 receptor is first located on the outer membranes of mitochondria and as allosteric sites on enzyme mao - a and mao - b . however , the imidazoline binding site on mao is separate from the active domain of the enzyme that recognizes the mechanism - based inhibitors , and it is not equally available in all the tissues . the i2 receptors have the same molecular weight as mao , and the amino acid sequencing of purified i2 receptors is similar to mao . as the i2 receptor is thought to be a modulatory site on the mao protein , the activity of i2 receptors may involve in several neurological disorders . another fascinating development with i2 receptors is that its effect on nociception since these monoamines appear to play a significant role in specific cns structures implicated in pain modulation spinal cord , cerebral cortex , etc . , and are involved in the antinociception of several drugs such as antidepressants which are commonly used for the management of pain . the ligands binding to the i2 receptors can be considered as inhibitors of mao - a and mao - b since they inhibited monoamine oxidation , and this inhibition could explain some biological effects of imidazoline receptor ligands such as pain modulation . in fact , it is well identified that increased levels of monoamines such as serotonin and noradrenaline contributes to pain control , especially in the cns regions related to pain . some evidence that shows the i2 receptors contribute to the modulation of pain [ figure 1 ] reports that the ligands that affect i2 receptors are effective for tonic pain , neuropathic pain , but little effective for acute pain . moreover , the activation of i2 receptors has been suggested to be a way of enhancing opioid analgesic actions . when they are combined with opioids , the i2 receptor ligands increase the analgesic effects of opioids in both acute and chronic pain . in chronic use , tolerance and addiction to the opioids can develop . however , the i2 receptor ligands can reduce the development of this opioid tolerance or prevent from deprivation syndrome that is triggered by the antagonists in animals . although the i2 receptors and their signaling pathways have not yet been characterized , the majority of the i2 receptors are widely accepted as being allosteric sites on mao . therefore , the i2 receptor ligands are described as allosteric modulators . allosteric modulation is the modulation of a protein via binding a ligand to the domain that is distinct from the active site of the protein . in principle , it is not precisely known whether a ligand is an i2 receptor agonist , an antagonist , or an inverse agonist in a constitutively active system . it is possible to have a knowledge of the relative efficacy of these ligands by systematic comparison . idazoxan , agmatine , and clonidine are ligands that bind to the i2 receptors as well as 2 and/or i1 receptors with varying affinities . due to the proven effect of the i2 receptors in pain , further studies are required to discover selective analgesic i2 receptor ligands and to elucidate their physiological functions . over the last few decades , investigators have attempted to synthesize and recognize selective ligands for the i2 binding sites . for instance , the i2 receptor ligand 2-bfi ( pki = 8.47 for i2 in rabbit kidney membrane with [ h ] 2-bfi ) which has high affinity for both i2a and i2b sites has been used to characterize these sites in several species , including humans . it has been proved that quinolone compound bu224 has high affinity ( pki = 8.43 for i2 in rabbit kidney membrane with [ h ] 2-bfi ) for the i2 receptors over i1 receptors and 2-adrenoceptors , among all the compounds of bu series . as mentioned before , the imidazoline receptor ligands are declared to modulate certain processes which involve mao activities in cns . the studies reveal that among all of the imidazoline receptor ligands those mentioned in this review ; 2-bfi , bu224 , 2-phenyl-6-(1h - imidazol-1il ) quinazoline ( cr4056 ) , idazoxan , and rs-45041 more selectively inhibit mao - a while lsl60101 and lsl61122 inhibit mao - b . after this part of the review , several studies will be conducted with the mentioned ligands [ figure 2 ] that have a higher affinity for the i2 receptors and the provided results will be included . the imidazoline imidazoline-2 receptor ligands in pain it should first be noted that clonidine that mediated the discovery of the presence of imidazoline receptors , is a 2-adrenoceptor agonist that binds not only to 2 adrenoceptors but also to imidazoline receptors in particular to i1 receptors compared to i2 receptors ; however , its antinociceptive activity occurs via 2-adrenoceptors rather than imidazoline receptors . agmatine is a nonselective and most extensively studied endogenous imidazoline receptor agonist , and has a moderate affinity to 2-adrenoceptors as well as all subtypes of imidazoline receptors ( pki < 5 for i2 in rabbit kidney membrane with [ h ] 2-bfi ) . it is indicated that this endogenous substance prevents reflex respond to the noxious stimulus through nonadrenergic receptors in mice . it does not show a significant effect on acute phasic pain while it is vice versa in the acute tonic ( differs from acute phasic pain in terms of using chemicals to induce noxious stimuli ) , inflammatory , and neuropathic pain . although agmatine showed weak effectiveness in a few studies , in many others , it was not efficient when administered systemically in acute phasic pain . ( supraspinal ) agmatine administration did not produce analgesic effects in the tail - flick test in mice . systemic administration of the i2 receptor ligands as phenyzoline , 2-bfi , 2-(2-benzofuranyl ) imidazole hydrochloride ( lsl 60101 ) ; the analog of 2-bfi , 2-styryl-2-imidazoline ; valldemossine or tracizoline ( lsl 61122 ) also did not produce antinociception in the acute pain model . in addition , researches showed that the i2 receptor ligands such as rs-45041 - 190 , cr4056 , 2-bfi , bu224 were effective in inflammatory and neuropathic pain . rs-45041 - 190 , the first selective and high - affinity ligand , showed significant results on the carrageenan - induced thermal and mechanic hyperalgesia tests when administered systemically ( i.p . ) cr4056 ( moderate affinity i2 receptor ligand ( ic50=596 nm , in rat whole brain membrane with [ h ] 2-bfi ) , is a new , highly selective i2 receptor ligand which inhibits mao - a activity more selectively than mao - b . in acute capsaicin - evoked pain model this effect was antagonized by idazoxan ( pki = 7.22 for i2 in rabbit kidney membrane with [ h ] 2-bfi ) , which is both a 2-adrenoceptor and i2 receptor antagonist . acute administration of cr4056 was found effective in relieving postoperative pain and joint pain as well as inflammatory and neuropathic pain . shortly , these studies present a new opportunity for the management of inflammatory , neuropathic , postoperative , and joint pain based on the selective interactions with the central i2 receptors . the effect of bu224 , a high affinity selective i2 receptor ligand , has been assessed electrophysiologically on the nociceptive neurons responses in the spinal dh . when we consider the results , it is possible to say that bu224 has a high affinity for the spinal i2 receptors , as well as it has an insignificant action at the spinal 2-adrenoceptor receptors . it is obvious that the i2 receptor ligands are more effective in chronic pain models than acute phasic pain models as mentioned with agmatine . chronic pain usually means a persistent pain lasting 3 months or more , and pharmacotherapy of chronic pain always comprises repeated dose . a possible result of repeated doses of analgesics is a gradual decrease in analgesic effect , in other words , development of analgesic tolerance . , antihyperalgesic effects of 2-bfi and cr4056 have also been tested in repeating treatments , in addition to single doses in chronic constriction injury ( cci)-induced neuropathic pain and cfa - induced inflammatory pain models . it is possible to say that the repeated dose regimen for the i2 receptor ligands in chronic pain treatment may be useful as monotherapy or adjunctive therapy without tolerance and addiction . it has been also shown that three high selective i2 receptor ligands ; 2-bfi , bu224 , and lsl 61122 possess antihyperalgesic effects in the rat models of cfa evoked inflammatory pain in this study . also , the i2 receptor antagonist idazoxan antagonized the antihyperalgesic effects of 2-bfi in cfa - treated and cci rats . these data also support the beneficial roles of the i2 receptor ligands in pain control . although most parts of preclinical pain studies focus on sensorial relieving pain , the sensorial and emotional compounds should be studied together . for instance , the effects of the i2 receptor ligands such as 2-bfi , bu224 , and cr4056 on escape / avoidance behaviors on cfa injected rats were studied connectedly to the affective pain in the same study . it was observed that 2-bfi , bu224 , and cr4056 increase the escape / avoidance behavior in the hyperalgesia increasing doses . these results exhibit that the i2 receptor ligands may be effective against the affective components of pain . providing antinociception by 2-bfi was not surprising , however , phenyzoline also produces antinociception in this chronic model as in acute phasic pain under weak noxious stimulus , as reported previously in a study by sampson et al . even some i2 receptor ligands as agmatine are not effective alone in acute phasic pain , they potentiate morphine antinociception . in a study , performed in the warm water , tail withdrawal procedure in rats by using selective i2 receptor ligand 2-bfi along with agmatine , it was observed that these two ligands increase the antinociceptive effects of morphine and tramadol . in contrast , another selective i2 receptor ligand bu224 failed to increase the antinociceptive efficacy but prevented agmatine and 2-bfi ligands to increase the morphine and tramadol - induced antinociception . these results suggest that the combination of the i2 receptor ligands and opioids may be effective in chronic pain treatment . however , the previous studies indicated the i2 receptors to contribute potentiation mechanism , and i1 and i2 receptors contribution of potentiation mechanism was not clearly understood . more recently , a study which has been done to understand which subtype contributes to potentiation mechanism showed that a significant oxycodone - induced antinociceptive respond could not be reversed by efaroxan ( i1 receptor antagonist ) but could be reversed by bu224 ( i2 receptor antagonist ) . so , it is thought that the i2 receptors participate in the potentiation , the i1 receptors do not . administration of the i2 receptor ligands ( but not i1 or 2 adrenoreceptor ) potentiate the morphine - evoked supraspinal antinociception in mice . the enhanced morphine antinociception via the i2 receptor ligands was reversed by idazoxan , bu224 , and isothiocyanatobenzylimidazoline , an irreversible i2 antagonist . in the same study , it has also been shown how the augmentation of morphine antinociception by the i2 agonists changes in mice with pertussis toxin impaired guanosine triphosphate - binding gi - go proteins . therefore , the contribution of gi - go transducer proteins in the modulation of morphine antinociception induced by the i2 receptors can not be disrespected . the imidazoline receptors are also important for the tolerance developed with opioids as well as improvement of opioid analgesia as mentioned before . , have assessed the role of imidazoline receptors in opioid ( morphine and pentazocine ) tolerance in rats by the administration of idazoxan . , showed that idazoxan promoted the development of tolerance to morphine and induced the abstinence syndrome in morphine - dependent mice and rats similar to naloxone . the chronic concurrent administration of 2-bfi , lsl 60101 , and lsl 61122 , selective and potent i2 receptor ligands , and morphine , also prevented or attenuated morphine tolerance . in the light of the positive outcomes , it is supported that the i2 receptor ligands as promising therapeutic coadjuvants in the management of chronic pain with opiate drugs since these agents prevent tolerance development and enhance opioid analgesia . all the studies show us that the i2 receptors are also steady , new drug targets for analgesics . even if the mechanism of the i2 receptor is not well known in the modulation of pain , it is known that it plays a role in tonic and chronic pain but not in the acute phasic pain . additionally , when they are combined with opioids in both acute and chronic pain , the i2 receptor ligands increase the antinociceptive actions of opioids . the development of tolerance and addiction induced by chronic administration of opioids is one of the major problems in the clinic . however , the i2 receptor ligands can reduce the opioid tolerance development or prevent from deprivation syndrome in the combination therapy . they are valuable for the chronic pain treatment and also valuable as therapeutic coadjuvants of opiates , because of the attenuation of opioid tolerance and addiction .

skeletal muscle produces efficient contractile force because they contain thin and thick filaments of well - defined lengths that are organized into regular , symmetric arrays that interdigitate . filament length is an important aspect of muscle function because a muscle generates force in proportion to thin and thick filament overlap . whereas thick filament length is considered a constant 1.6 m , thin filament lengths are fine - tuned at ~1.01.3 m , depending on species and muscle type ( littlefield and fowler , 2008 ) to overlap with thick filaments and to meet the muscle s physiological demands ( granzier et al . , 1991 ; thick filament overlap and impact a muscle s force generating capacity at a given sarcomere length : thus , thin filament length is a key aspect of muscle function . since length is not an intrinsic property of actin filaments ( actin monomers assemble in vitro to highly variable polymer lengths ( pollard and borisy , 2003 ) , thin filament length is likely to be specified in vivo by an actin - binding protein ; for this , nebulin has been considered for a long time a prime candidate ( wang and wright , 1988 ; labeit et al . , 1991 ; labeit and kolmerer , 1995 ) , but critical evidence has been lacking up until recently . nebulin is a giant protein ( mw 700800 kda ) expressed in skeletal muscle , and makes up 23% of the myofibrillar protein mass . immuno - electron microscopy revealed that a single nebulin molecule spans the thin filament with its c - terminus anchored at the z - disk and its n - terminal region directed toward the thin filament pointed - end ( for a schematic representation , see figure 1 ; wang and wright , 1988 ) . the first evidence for nebulin s proposed role in specifying thin filament length came from the analysis of nebulin s cdna sequence . this revealed that the bulk of the molecule is comprised of modules with the centrally located modules , m9 to m162 , each thought to represent individual actin - binding motifs , and organized into seven - module super - repeats that match the repeat of the actin filament ( figure 1 ) . this precise arrangement is thought to allow each nebulin module to interact with a single monomer of the actin filament ( labeit et al . , 1991 ; labeit and kolmerer , 1995 ) , and each nebulin super - repeat to associate with a single tropomyosin ( tm)/troponin ( tn ) complex ( jin and wang , 1991 ; mcelhinny et al . , 2003 ; extreme n - terminal modules m1m3 ( figure 1 ) contain a high - affinity binding site for the thin filament pointed - end capping protein tropomodulin ( mcelhinny et al . , 2001 ) . tropomodulin , in addition to binding nebulin s n - terminus , binds actin and tropomyosin with high - affinity and prevents actin filaments from elongating or shortening at the pointed - end ( dos remedios et al . , 2003 ) . furthermore , earlier studies revealed that the electrophoretic mobility of nebulin from different muscle types correlates with thin filament length ( kruger et al . top : three schematics of the skeletal muscle sarcomere depicting the three proposed models of nebulin in the z - disk : the overlap model in which nebulin penetrates the z - disk creating a ~75 nm wide nebulin overlap zone in the center of the z - disk ; the no - overlap model in which nebulin penetrates the z - disk by only about 25 nm , and nebulin filaments from neighboring sarcomeres do not overlap ; the cross - linking model in which nebulin fully penetrates the z - disk and cross - links thin filaments from adjacent sarcomeres . also note that a single nebulin molecule spans most of the length of the thin filament , but that according to recent studies the thin filament pointed - end is nebulin - free . the length of the nebulin - free thin filament extension is variable ( as indicated in the drawing by the dotted nebulin filament ) and depends on species and muscle type . nebulin has a highly modular structure , with in the central region ( m9m162 ) seven modular repeats arranged into 22 super - repeats . although the findings discussed above were consistent with the hypothesis that nebulin is involved in specifying thin filament length , direct evidence was lacking . more conclusive evidence for a role for nebulin in specifying thin filament length required studies of muscle that lack nebulin . to test the role of nebulin in skeletal muscle in vivo , nebulin ko mouse models were generated ( bang et al . , 2006 ; witt et al . the first work on these models revealed that in nebulin - deficient skeletal muscle the thin filaments are on average shorter , thus supporting a role for nebulin in the in vivo regulation of thin filament length ( bang et al . witt et al . ( 2006 ) performed an immuno - electron microscopy study and reported that thin filament lengths in wildtype tibialis cranialis muscle are a constant 1.2 m , but in nebulin - deficient muscle are on average ~0.8 m , and range from ~0.4 to 1.2 m . that such reduction in thin filament length greatly affects force production was illustrated by ottenheijm et al . ( 2009 ) by plotting force as a function of sarcomere length for both wildtype and nebulin - deficient muscle . in these experiments , skinned muscle fibers were activated by exogenous calcium at various sarcomere lengths and the force response was measured ( note that in skinned fiber preparations factors outside of the myofilaments , e.g. , calcium handling by the sarcoplasmic reticulum , do not contribute to force production ) . the force - sarcomere length relation of wildtype muscle is characterized by a force plateau reflecting optimal thick thin filament overlap , followed by a descending limb at higher sarcomere lengths reflecting the decreased filament overlap . that the descending limb in wildtype muscle starts at a sarcomere length of ~2.6 m and ends at ~4.0 m suggests a thin filament length of ~1.2 m , which is in line with the previously mentioned electron microscopy data . in nebulin - deficient muscle , the shortened thin filaments reduce thin thick filament overlap at a given sarcomere length , impairing force production and resulting in a leftward shift of the force - sarcomere length relation ( see figure 2b ) . furthermore , when thin filaments are non - uniform in length no optimal thick thin filament overlap exists , and therefore the force - sarcomere length relation of nebulin - deficient muscle lacks the characteristic plateau . consistent with these findings on demembranated muscle , studies on intact nebulin - deficient muscle from another nebulin knockout model ( gokhin et al . , 2009 ) , in which muscles were activated at various lengths by electrical field stimulation , also revealed a leftward shift of the force - muscle length relation of nebulin - deficient muscle . thus , the force - length relation of nebulin - deficient muscle is altered in a manner that is consistent with the presence of shorter thin filament lengths . ( a ) force ca characteristics of murine skinned nebulin - deficient muscle fibers from m. tibialis cranialis ( neb - ko , left panel ) , and of skinned muscle fibers from m. quadriceps of patients with nebulin - based nemaline myopathy ( neb - nm , right panel ) . note that the force generated in response to incubation with incremental increase of [ ca ] is significantly decreased in both neb - ko and neb - nm tissue , resulting in a rightward shift of the force ca relationship ( figures adapted from chandra et al . , 2009 ; ottenheijm et al . , ( b ) left : the force - sarcomere length relation of murine wt ( neb - wt ) fibers has a characteristic force plateau followed by a descending limb . the force - sarcomere length relation of neb - ko fibers is shifted leftward compared to control fibers , and the force plateau is absent . right : the force - sarcomere length relation of muscle fibers from nm - neb patients is shifted to the left and is very similar to that found in neb - ko fibers ( figures adapted from ottenheijm et al . , 2008 ) . ( 2006 ) on their nebulin ko model , using confocal microscopy on 1-day - old mice , indicated that in the absence of nebulin thin filament lengths are reduced from ~1.15 to 1.3 m ( depending on muscle type ) in wildtype muscle to a consistent ~1.0 m in all muscles types . these findings led to the proposal ( littlefield and fowler , 2008 ) that a nebulin - independent mechanism specifies uniform thin filament lengths of ~1.0 m in all muscle types , whereas nebulin is responsible for specifying longer thin filament lengths in a muscle - specific manner . ( 2009 ) who used immuno - fluorescence microscopy on rabbit muscle and concluded that nebulin specifies the minimum thin filament length ( ~1.0 m ) with a nebulin - independent mechanism regulating the final length according to the requirements of a particular muscle . ( 2012 ) , showing that human thin filaments have nebulin - free pointed - end extensions that comprise up to 30% of total thin filament length , and by pappas et al . ( 2010 ) who showed that when endogenous nebulin is replaced with a mini - nebulin in skeletal myocytes , thin filaments extend beyond the end of mini - nebulin . detecting differences in thin filament length , especially length gradients , with high precision is challenging and might be hard to accomplish with confocal microscopy , as was done in the two aforementioned studies . in contrast , witt et al . ( 2006 ) used electron microscopy and decorated thin filaments with gold beads ( attached to actin monomers with the actin - binding peptide phalloidin ) . this made it possible to determine thin filament length gradients and showed that thin filaments varied in length and were on average shorter than in wildtype muscle . ( 2006 ) that there is a nebulin - independent mechanism that sets a constant thin filament length of 1.0 m , but is consistent with castillo et al . that there is a nebulin - dependent mechanism that sets a minimum thin filament length . to resolve these discrepancies additional studies on a range of mouse muscle types are needed that measure by electron microscopy thin filament length and the location of nebulin s n - terminus . it is clear , however , from the above referenced studies that nebulin does play a critical role in regulating thin filament length : in its absence the average thin filament length is shorter and force is reduced . to understand the layout of the c - terminal region of nebulin in the z - disk , immunoelectron microscopy ( iem ) has been used on human soleus muscle and nebulin s c - terminus has been labeled with the nebulin - specific anti - sh3 antibody and the more n - terminal m177181 domains with anti - neb177181 ( millevoi et al . , 1998 ) . results showed that the nebulin sh3 domain is located about 25 nm inside the z - disk , and the repeats neb176 to neb181 near to the edge of the z - disk . these results are consistent with two distinct models of the layout of nebulin in the z - disk ( for details , see millevoi et al . , 1998 ; , nebulin penetrates the z - disk by only about 25 nm , and nebulin filaments from neighboring sarcomeres do not overlap ( no - overlap model ) . in the second model , nebulin penetrates the z - disk by about 100 nm and this creates a ~75 nm wide nebulin overlap zone in the center of the z - disk ( overlap model ) . it has been argued that the no - overlap model is more likely to be correct ( millevoi et al . , 1998 ) , but definite experimental evidence for either model is lacking . in a more recently proposed model , nebulin fully penetrates the z - disk and cross - links thin filaments from adjacent sarcomeres ( pappas et al . , 2008 ) . a drawback of this model is that it predicts that the sh3 epitope is further from the center of the z - disk than the m177181 epitope , which is opposite of what has been measured . it also remains to be explained why nebulin leaves one actin filament to which it is anchored and moves to a neighboring filament , and how the thin filament spanning region of nebulin accommodates the myofilament lattice spacing changes that occur during muscle contraction . clearly further work is needed to map the layout of nebulin in the z - disk . z - disks of different muscles can vary greatly in width , from less than 100 nm in fast skeletal muscle to more than 150 nm in slow skeletal muscle ( tonino et al . , 2010 ) . the importance of regulation of z - disk width is illustrated by muscle from patients with nemaline myopathy , which displays greatly widened z - disks , including the characteristic nemaline rods ( wallgren - pettersson et al . previously titin has been suggested to play a role in z - disk assembly ( gautel et al . , 1996 ) . the z - disk region of titin contains a family of differentially expressed repeats , the titin z - repeats ( gautel et al . , 1996 ) . these z - repeats are a family of -actinin - binding motifs , which are differentially expressed in a tissue- and developmental - stage - specific fashion ( gautel et al . , 1996 ) . as previously pointed out , it is unlikely that the differential expression of the titin z - repeats alone can determine the z - disk width , because too few isoforms exist to account for the wide range of different z - disk widths ( millevoi et al . , 1998 ) . cdna sequencing of rabbit nebulin has demonstrated that the z - disk region of nebulin is differentially expressed ( millevoi et al . , 1998 ) . several different isoforms have been identified that result from the skipping of various combinations of seven z - disk domains ( millevoi et al . , 1998 ) , leading to the suggestion that nebulin is one of several proteins that are important for z - disk width regulation ( millevoi et al . , 1998 ) . consistent with this notion , the ultrastructural characterization of the z - disk in neb - ko mice revealed that average z - disk width is increased by 4080 nm depending on muscle type ( bang et al . in addition , inclusion - like bodies of mis - assembled z - disks are detected that resemble the rod bodies that are a hallmark of nemaline myopathy ( bang et al . further support was obtained by a recent study on nebulin in a range of muscle types during postnatal development of the mouse in which transcript studies were performed with a mouse nebulin exon microarray ( buck et al . , 2010 ) . during postnatal development of the soleus muscle major changes in splicing were detected in the z - disk region of nebulin . three differentially spliced z - disk exons were upregulated during postnatal development of soleus muscle and this correlated with a significant increase in z - disk width . the increase in z - disk width of the soleus muscle might reflect the increasing stress exerted on this muscle due to the rapid increase in body weight during postnatal development . together , these findings support a model in which titin and nebulin together specify z - disk width , with titin constructing the central region of the z - disk , including the number and positions of -actinin cross - links and nebulin determining the ending of the z - disk structure and its transition to the i - band , i.e. , nebulin functions as a z - disk terminator . the mechanism by which nebulin terminates the z - disk might involve interaction between nebulin and z - disk - localized proteins , such as capz . capz is a barbed - end actin capping protein that binds near the c - terminus of nebulin ( pappas et al . , 2008 ) . in muscle fibers devoid of nebulin ( witt et al . , 2006 ; pappas et al . , 2008 ) , capz does not localize properly , allowing the barbed ends of thin filaments to continue to grow beyond the z - disk resulting in widened z - disks . when this feature of nebulin is not present , z - disks widen , ultimately culminating in the formation of nemaline rods . in addition to regulating z - disk width and structure , nebulin is suggested to play a role in laterally linking myofibrils at the z - diks . longitudinally , sarcomeres are connected by z - disk lattices that anchor thin filaments and transmit force along the myofibril . in the transverse direction , linkage of myofibrils at the z - disks allows for lateral force transmission and limits the degree to which adjacent myofibrils translocate relative to each other during active contraction or passive stretch , thereby preventing damage to intermyofibrillar membrane systems , such as t - tubules and the sarcoplasmic reticulum . an important protein involved in linking adjacent z - disks is the intermediate filament protein desmin , which forms a network of filaments that surrounds myofibrils at the level of the z - disk ( wang and ramirez - mitchell , 1983 ; capetanaki et al . , 2007 ) . the subunit proteins of desmin filaments are elongated coiled - coils with extensive intermolecular ionic and hydrophobic interactions between individual subunits , giving rise to filaments with high tensile strength as well as plasticity ( costa et al . , 2004 ) . that desmin tethers adjacent z - disks is supported by work on a desmin ko mouse in which z - disk misalignment was shown to occur in stretched muscle ( shah et al . , 2002 ) . in vitro work , using a yeast two - hybrid approach , suggested that desmin binds to the c - terminal region of nebulin ( bang et al . , 2002 ) , which is anchored in the z - disk , and recently it was shown that nebulin modules m160170 are involved in this interaction ( conover et al . , 2009 ; conover and gregorio , 2011 ) . these findings lead bang et al . to speculate that this desmin - nebulin interaction links myofibrillar z - disks to the intermediate filament system , thereby forming a lateral linkage system which maintains adjacent z - disks in register . this role for nebulin in intermyofibrillar connectivity was tested recently by studies using a nebulin ko mouse model ( tonino et al . , 2010 ) . in these studies it was found that upon stretch , myofibrils devoid of nebulin translocate to a much higher degree than wt muscle , resulting in much larger z - disk displacement . although desmin is present in muscle devoid of nebulin , it is reduced in the intermyofibrillar spaces that surround the z - disks , suggesting that nebulin is required for proper localization of desmin at the z - disk . consistent with this , both knockdown of nebulin with sirna and overexpression of m160m170 did not interfere with the formation of normal striation patterns but did prevent desmin localization at the mature z - disk . thus , nebulin is required to laterally link myofibrils at the z - disk by desmin filaments ; in the absence of nebulin myofibrillar connectivity is significantly reduced leading to z - disk displacement . exciting recent work revealed that nebulin s c - terminal sh3 domain is involved in the induction of muscle hypertrophy . ( 2010 ) found that insulin - like growth factor 1 induces skeletal muscle maturation and hypertrophy by forming a complex of nebulin and n - wasp at the z - disks of myofibrils by interfering with glycogen synthase kinase-3. their results convincingly show that n - wasp interacts with nebulin s sh3 domain , and that n - wasp is rapidly recruited to the z - disk after treatment with insulin - like growth factor 1 , which stimulates myofibrillogenesis and suppresses sarcomere breakdown via the pi3k akt pathway . the notion that nebulin is important in maintaining muscle mass is consistent with the previous observation that nebulin - deficient mice display severe muscle fiber atrophy ( bang et al . , 2006 ; witt et al . , 2006 ) although thin filament instability caused by nebulin - deficiency is likely to be involved as well in the mechanisms underlying muscle atrophy . recent work shows that nebulin s role is not merely structural , but that nebulin also regulates contraction . muscle contraction is driven by the cyclic interaction between the myosin - based crossbridges and actin and the level of force a muscle generates is proportional to the force generated per crossbridge and the number of crossbridges in the force generating state . it is generally accepted that this interaction between actin and myosin is regulated through a steric hindrance mechanism in which tropomyosin and troponin control the conversion between interaction permissive and non - permissive states ( gordon et al . , 2000 ) . , 2009 ; chandra et al . , 2009 ) recently identified that nebulin contributes to the regulation of crossbridge cycling kinetics , with one of the two ( chandra et al . , 2009 ) also identifying a role for nebulin in the calcium sensitivity of force generation . during the crossbridge cycle , unbound non - force generating crossbridges move to an actin - bound force generating state followed by atp - driven crossbridge release back to the non - force generating state ( huxley and simmons , 1971 ; lymn and taylor , 1971 ) . ( brenner , 1988 ) proposed an analytical framework in which this transition between force and non - force generating crossbridge states can be described by two apparent rate constants ; one for crossbridge attachment ( fapp ) and one for crossbridge detachment ( gapp ) . these two rate constants determine the fraction of force generating crossbridges during activation , and a change in one or both will affect this fraction and thus force production . gapp is directly proportional to the atp consumption rate normalized to tension generation ( i.e. , tension cost ) , and can therefore be estimated from the simultaneous determination of atp consumption rate and tension in activated muscle fibers . such studies on nebulin - deficient muscle ( chandra et al . , 2009 ) revealed significantly higher tension cost in nebulin - deficient muscle , thus indicating a faster gapp and crossbridge detachment rate when nebulin is absent . likewise , studies on another nebulin ko mouse model ( bang et al . , 2009 ) reported higher velocity of unloaded shortening in nebulin - deficient muscle , also suggesting that gapp is higher when nebulin is absent . it is also important to highlight that these findings are consistent with results of in vitro motility assays in which nebulin fragments were found to reduce the sliding velocity of f - actin over myosin ( root and wang , 1994 ) . in brenner s framework ( brenner , 1988 ) , the rate constant of force redevelopment ( ktr ) is proportional to fapp + gapp , and the fraction of force generating crossbridges to fapp/(fapp + gapp ) . the rate constant of force redevelopment can be estimated by imposing a rapid release - restretch protocol on an activated fiber , mechanically disengaging all bound crossbridges so that force drops to zero and then measuring force redevelopment . such experiments revealed that force redevelopment is slower in nebulin - deficient muscle ( bang et al . thus , the decrease in ktr of nebulin - deficient muscle , together with the notion that gapp is increased , indicates that fapp must be reduced and that the reduction must be larger than the increase in gapp . combined , this leads to the conclusion that the fraction of force generating crossbridges [ fapp/(fapp + gapp ) ] is reduced in nebulin - deficient muscle . furthermore , stiffness measurements indicated that the force per crossbridge was not affected by the absence of nebulin ( bang et al . , recent studies suggest that nebulin increases the rate of crossbridge attachment and reduces the rate of crossbridge detachment , and that as a result the number of force generating crossbridges is increased . although the mechanism by which nebulin affects crossbridge cycling needs further investigation , previous work ( root and wang , 1994 ) has shown that nebulin associates with the actin n - terminus in subdomain 1 , where also the myosin crossbridge binds . thus , the presence of nebulin at or near the s1 binding site might enhance the binding of crossbridges and slow their detachment . chandra et al . ( 2009 ) estimated that the effect of nebulin on crossbridge kinetics enhances a muscle s force generating capacity by ~50% , and increases the economy of contraction by ~35% . ( 2009 ) , and they can largely account for the more pronounced leftward shift of the measured force - sarcomere length relation of nebulin - deficient muscle when compared to the predicted relation based on only thin filament length measurements . clearly , nebulin is a major factor in determining the level of force and the energetic cost of force production in skeletal muscle . in line with this role of nebulin in the regulation of crossbridge cycling kinetics , recent studies on muscle fibers from patients with nemaline myopathy with severely reduced nebulin protein levels revealed that in addition to altered thin filament length , changes in crossbridge cycling kinetics contribute to the muscle weakness observed in these patients ( ottenheijm et al . , 2010 ) . chandra et al . ( 2009 ) also measured active force at a range of calcium levels and the obtained force - pca relations were markedly shifted to the right in nebulin - deficient muscle fibers ( see figure 2a ) , with a 0.16 unit reduction in pca50 ( pca that gives the half - maximal force level ) . to rule out a possible difference in the troponin complex ( isoform composition and posttranslational modification ) between wt and ko fibers , these studies were carried out on wildtype and ko fibers that had been reconstituted by the same recombinant troponin complex . an analysis of expression levels of tropomyosin , myosin heavy chain , myosin light chain did not reveal a significant difference in these proteins between the fibers , suggesting that the absence of nebulin is the most likely explanation for the lower calcium sensitivity of the ko fibers . it is possible that the discrepancy is due to the fact that those studies did not carry out a troponin exchange and that differences in the troponin complex between wt and ko fibers could have negated nebulin s effect on calcium sensitivity . the two studies that did not detect a difference in calcium sensitivity were carried out at long sarcomere lengths [ ~2.5 m ( witt et al . , 2006 ) and ~2.6 m ( bang et al . , 2009 ) ] whereas the study that did show a difference ( chandra et al . , 2009 ) was performed at ~2.0 m . the implication is that nebulin plays a role in the length dependence of activation with a much larger pca50 in the nebulin ko fibers than in wt fibers . it is a well known phenomenon that as sarcomere length increases muscle becomes more calcium sensitive . this length dependence of activation is most prominent in cardiac muscle ( and is thought to underlie the frank starling law of the heart ) but is much less pronounced in skeletal muscle ( konhilas et al . , the presence of nebulin provides an explanation for why skeletal muscle has less length dependence : the presence of nebulin increases calcium sensitivity at short length . the structure and protein binding properties of nebulin support a role in thin filament activation . nebulin contains ~200 domains of ~35 amino acids that are characterized by the actin - binding sequence sdxxyk ; these domains make up seven domain super - repeats characterized by the tm / tn binding motif , wlkgigw ( mcelhinny et al . , 2003 ) . biochemical studies have shown that a single nebulin module interacts with a single actin monomer and that each nebulin super - repeat interacts with a tm / tn complex of the thin filament ( ogut et al . , 2003 ) , binding characteristics that support that nebulin follows the helical path of f - actin . it is interesting that similar to tm , nebulin appears to have different binding sites on f - actin with one site in close proximity to both the strong binding site for myosin and the blocked state of tropomyosin ( lukoyanova et al . , 2002 ) . this leads to the intriguing possibility that nebulin acts in concert with tm and that it promotes the transition of contractile regulatory units ( tm / tn ) from the non - permissive to the permissive states , thereby increasing myofilament calcium sensitivity . it is striking that nebulin - deficient skeletal muscle shares the low calcium sensitivity and low maximal active force with cardiac muscle , where stoichiometric levels of nebulin are absent ( bang et al . cardiac muscle contains the nebulin - homolog nebulette ( moncman and wang , 1995 ) . however , nebulette is much smaller than nebulin ( ~100 vs ~800 kda for nebulin ) and its location is restricted to the z - disk and the near z - disk i - band region ( millevoi et al . , although it can not be ruled out that nebulette induces long - range conformational effects that propagate along the thin filament to affect its function , such effects would have to be first demonstrated in a direct manner , and in absence of such findings we consider it not likely that nebulette is intimately involved in thin filament activation . cardiac muscle has multiple mechanisms for enhancing thin filament activation , such as , enhanced length - dependent activation , and the presence of multiple cardiac - specific phosphorylation sites in various thin and thick filament based protein sites ( solaro and de tombe , 2008 ) , which allow cardiac muscle to grade its force response to different loading conditions . since skeletal muscle lacks the aforementioned features unique to cardiac muscle , nebulin might be essential for tuning thin filament activation for optimal skeletal muscle function . a striking upregulation of sarcolipin ( sln ) , an inhibitor of serca , occurs in neb - ko mice ( bang et al . , 2006 ; gokhin et al . , 2009 ; this upregulation might be viewed as an adaptation in neb - ko mice as an attempt to increase cytosolic calcium levels and counteract reduced myofilament calcium sensitivity . the mechanism by which nebulin - deficiency upregulates sln requires further future studies , as does the functional role of sln upregulation , for example by crossing sln - ko mice and nebulin ko mice . the above discussed studies clearly indicate that nebulin is not merely involved in regulation sarcomere structure , but also acts as a regulator of muscle contraction . dysfunctions of the skeletal muscle thin filament have emerged as important causes of skeletal myopathies of which nemaline myopathy is the most common disease with a previously estimated prevalence of 0.002% ( sanoudou and beggs , 2001 ) . however , because of its heterogeneous nature and often milder phenotypes , the actual frequency of nemaline myopathy in older patients may be considerably higher . genetically , nm is heterogeneous and so far seven genes have been identified as nm - causing , namely alpha - tropomyosin-3 and beta - tropomyosin ( tpm3 and tpm2 ) , neb , actin alpha 1 ( acta1 ) , troponin t type 1 ( tnnt1 ) , cofilin-2 , and kbtbd13 ( laing et al . , 1992 , 1995 ; nowak et al . , 1999 ; , 2007 ; sambuughin et al . , 2010 ) . an unbiased linkage analysis of 45 nm families derived from 10 different countries implicated nebulin mutations as disease causing in 41 of these families . the typical form of nm presents with early onset in infancy , has a non - progressive or slowly progressive course and is caused by mutations in the nebulin gene . these mutations are often missense mutations , but in addition a 2502-bp deletion causing a 33-residue in frame deletion of exon 55 was identified as a prominent recessive disease causing allele initially in the ashkenazi jewish population , and more recently also in non - jewish patients ( anderson et al . , 2004 ; lehtokari et al . , 2006 , 2009 ) . recent studies on muscle fibers from patients with nemaline myopathy due to the deletion of nebulin exon 55 showed that these patients have severely reduced nebulin protein levels and that they show remarkable phenotypic similarities to fibers from nebulin ko mice , i.e. , shorter and non - uniform thin filament lengths ( figure 2b ) and significantly impaired force generating capacity ( ottenheijm et al . , 2009 ) . thus , loss of thin filament length regulation appears to be an important contributor to muscle weakness in patients with nemaline myopathy . in addition to altered thin filament length , changes in crossbridge cycling kinetics and reduced calcium sensitivity of force production contribute to the muscle weakness observed in these patients ( see figure 2a ; ottenheijm et al . , 2010 ) , in line with the role of nebulin in these processes ( chandra et al . , 2009 ; ottenheijm and granzier , 2010 ) . thus , nebulin s role in thin filament length regulation and contraction deduced from work on neb - ko mice provides a mechanism for the first time to explain severe muscle weakness in patients with nemaline myopathy . interestingly , recent work form our lab demonstrates that patients with nemaline myopathy due to mutations in the tropomyosin gene ( tpm3 ) display a contractile phenotype that is distinct from that of patients with neb - based myopathy ( ottenheijm et al . , 2011 ) . whereas both show severe myofilament - based muscle weakness , the contractile dysfunction in tpm3-based myopathy can be largely explained by changes in crossbridge cycling kinetics , whereas dysregulation of thin filament length and altered crossbridge cycling kinetics play a prominent role in neb - based myopathy ( ottenheijm et al . , 2009 ) . furthermore , the loss of force generating capacity in tpm3-based myopathy appears to be partly compensated by enhanced calcium sensitivity of force generation , whereas decreased calcium sensitivity of force generation further depresses the capacity for force production in neb - based myopathy ( ottenheijm et al . , 2010 ) . whereas these findings support the existence of distinct genotype - phenotype correlations in nm , it is yet unclear whether they extrapolate to other patients with mutations in neb or tpm3 , and how they compare to the phenotype of nm patients with mutations in acta1 , tpm2 , tnnt1 , cfl2 , and kbtbd13 . understanding the genotype - phenotype correlations is important , as it allows the development of genotype - targeted treatment strategies . for example , analogous to current clinical studies that address whether contractility during heart failure can be augmented by actomyosin activating small molecules ( malik et al . , 2011 ) 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 sliding filament model of the sarcomere was developed more than half a century ago . this model , consisting only of thin and thick filaments , has been successful in explaining many , but not all , features of skeletal muscle . work during the 1980s revealed the existence of two additional filaments : the giant filamentous proteins titin and nebulin . whereas the role of titin rapidly progressed , nebulin s role in muscle structure and function remained long nebulous . an important feature of muscle structure and function that has remained relatively obscure concerns the mechanisms that are involved in regulating thin filament length . filament length is an important aspect of muscle function as force production is proportional to the amount of overlap between thick and thin filaments . recent advances , due in part to the generation of nebulin ko models , reveal that nebulin plays an important role in the regulation of thin filament length , most likely by stabilizing f - actin assemblies . another structural feature of skeletal muscle that has been incompletely understood concerns the mechanisms involved in maintaining z - disk structure and the regular lateral alignment of adjacent sarcomeres during contraction . recent studies indicate that nebulin is part of a protein complex that mechanically links adjacent myofibrils . in addition to these structural roles in support of myofibrillar force generation , nebulin has been also shown to regulate directly muscle contraction at the level of individual crossbridges : cycling kinetics and the calcium sensitivity of force producing crossbridges is enhanced in the presence of nebulin . thus , these recent data all point to nebulin being important for muscle force optimization . consequently , muscle weakness as the lead symptom develops in the case of patients with nemaline myopathy that have mutations in the nebulin gene . here , we discuss these important novel insights into the role of nebulin in skeletal muscle function .

Nebulin is Involved in Specifying Thin Filament Length Nebulin as Regulator of Z-Disk Structure Nebulin Maintains Intermyofibrillar Connectivity Nebulin and Muscle Hypertrophy Nebulin Regulates Contraction Nebulin and Nemaline Myopathy Conflict of Interest Statement

skeletal muscle produces efficient contractile force because they contain thin and thick filaments of well - defined lengths that are organized into regular , symmetric arrays that interdigitate . filament length is an important aspect of muscle function because a muscle generates force in proportion to thin and thick filament overlap . whereas thick filament length is considered a constant 1.6 m , thin filament lengths are fine - tuned at ~1.01.3 m , depending on species and muscle type ( littlefield and fowler , 2008 ) to overlap with thick filaments and to meet the muscle s physiological demands ( granzier et al . , 1991 ; thick filament overlap and impact a muscle s force generating capacity at a given sarcomere length : thus , thin filament length is a key aspect of muscle function . since length is not an intrinsic property of actin filaments ( actin monomers assemble in vitro to highly variable polymer lengths ( pollard and borisy , 2003 ) , thin filament length is likely to be specified in vivo by an actin - binding protein ; for this , nebulin has been considered for a long time a prime candidate ( wang and wright , 1988 ; labeit et al . nebulin is a giant protein ( mw 700800 kda ) expressed in skeletal muscle , and makes up 23% of the myofibrillar protein mass . immuno - electron microscopy revealed that a single nebulin molecule spans the thin filament with its c - terminus anchored at the z - disk and its n - terminal region directed toward the thin filament pointed - end ( for a schematic representation , see figure 1 ; wang and wright , 1988 ) . the first evidence for nebulin s proposed role in specifying thin filament length came from the analysis of nebulin s cdna sequence . tropomodulin , in addition to binding nebulin s n - terminus , binds actin and tropomyosin with high - affinity and prevents actin filaments from elongating or shortening at the pointed - end ( dos remedios et al . furthermore , earlier studies revealed that the electrophoretic mobility of nebulin from different muscle types correlates with thin filament length ( kruger et al . top : three schematics of the skeletal muscle sarcomere depicting the three proposed models of nebulin in the z - disk : the overlap model in which nebulin penetrates the z - disk creating a ~75 nm wide nebulin overlap zone in the center of the z - disk ; the no - overlap model in which nebulin penetrates the z - disk by only about 25 nm , and nebulin filaments from neighboring sarcomeres do not overlap ; the cross - linking model in which nebulin fully penetrates the z - disk and cross - links thin filaments from adjacent sarcomeres . also note that a single nebulin molecule spans most of the length of the thin filament , but that according to recent studies the thin filament pointed - end is nebulin - free . the length of the nebulin - free thin filament extension is variable ( as indicated in the drawing by the dotted nebulin filament ) and depends on species and muscle type . although the findings discussed above were consistent with the hypothesis that nebulin is involved in specifying thin filament length , direct evidence was lacking . more conclusive evidence for a role for nebulin in specifying thin filament length required studies of muscle that lack nebulin . to test the role of nebulin in skeletal muscle in vivo , nebulin ko mouse models were generated ( bang et al . the first work on these models revealed that in nebulin - deficient skeletal muscle the thin filaments are on average shorter , thus supporting a role for nebulin in the in vivo regulation of thin filament length ( bang et al . that such reduction in thin filament length greatly affects force production was illustrated by ottenheijm et al . in nebulin - deficient muscle , the shortened thin filaments reduce thin thick filament overlap at a given sarcomere length , impairing force production and resulting in a leftward shift of the force - sarcomere length relation ( see figure 2b ) . thus , the force - length relation of nebulin - deficient muscle is altered in a manner that is consistent with the presence of shorter thin filament lengths . ( 2006 ) on their nebulin ko model , using confocal microscopy on 1-day - old mice , indicated that in the absence of nebulin thin filament lengths are reduced from ~1.15 to 1.3 m ( depending on muscle type ) in wildtype muscle to a consistent ~1.0 m in all muscles types . these findings led to the proposal ( littlefield and fowler , 2008 ) that a nebulin - independent mechanism specifies uniform thin filament lengths of ~1.0 m in all muscle types , whereas nebulin is responsible for specifying longer thin filament lengths in a muscle - specific manner . ( 2009 ) who used immuno - fluorescence microscopy on rabbit muscle and concluded that nebulin specifies the minimum thin filament length ( ~1.0 m ) with a nebulin - independent mechanism regulating the final length according to the requirements of a particular muscle . ( 2012 ) , showing that human thin filaments have nebulin - free pointed - end extensions that comprise up to 30% of total thin filament length , and by pappas et al . ( 2010 ) who showed that when endogenous nebulin is replaced with a mini - nebulin in skeletal myocytes , thin filaments extend beyond the end of mini - nebulin . detecting differences in thin filament length , especially length gradients , with high precision is challenging and might be hard to accomplish with confocal microscopy , as was done in the two aforementioned studies . to resolve these discrepancies additional studies on a range of mouse muscle types are needed that measure by electron microscopy thin filament length and the location of nebulin s n - terminus . it is clear , however , from the above referenced studies that nebulin does play a critical role in regulating thin filament length : in its absence the average thin filament length is shorter and force is reduced . to understand the layout of the c - terminal region of nebulin in the z - disk , immunoelectron microscopy ( iem ) has been used on human soleus muscle and nebulin s c - terminus has been labeled with the nebulin - specific anti - sh3 antibody and the more n - terminal m177181 domains with anti - neb177181 ( millevoi et al . results showed that the nebulin sh3 domain is located about 25 nm inside the z - disk , and the repeats neb176 to neb181 near to the edge of the z - disk . these results are consistent with two distinct models of the layout of nebulin in the z - disk ( for details , see millevoi et al . , 1998 ; , nebulin penetrates the z - disk by only about 25 nm , and nebulin filaments from neighboring sarcomeres do not overlap ( no - overlap model ) . in the second model , nebulin penetrates the z - disk by about 100 nm and this creates a ~75 nm wide nebulin overlap zone in the center of the z - disk ( overlap model ) . in a more recently proposed model , nebulin fully penetrates the z - disk and cross - links thin filaments from adjacent sarcomeres ( pappas et al . a drawback of this model is that it predicts that the sh3 epitope is further from the center of the z - disk than the m177181 epitope , which is opposite of what has been measured . clearly further work is needed to map the layout of nebulin in the z - disk . the importance of regulation of z - disk width is illustrated by muscle from patients with nemaline myopathy , which displays greatly widened z - disks , including the characteristic nemaline rods ( wallgren - pettersson et al . previously titin has been suggested to play a role in z - disk assembly ( gautel et al . the z - disk region of titin contains a family of differentially expressed repeats , the titin z - repeats ( gautel et al . as previously pointed out , it is unlikely that the differential expression of the titin z - repeats alone can determine the z - disk width , because too few isoforms exist to account for the wide range of different z - disk widths ( millevoi et al . cdna sequencing of rabbit nebulin has demonstrated that the z - disk region of nebulin is differentially expressed ( millevoi et al . , 1998 ) , leading to the suggestion that nebulin is one of several proteins that are important for z - disk width regulation ( millevoi et al . consistent with this notion , the ultrastructural characterization of the z - disk in neb - ko mice revealed that average z - disk width is increased by 4080 nm depending on muscle type ( bang et al . in addition , inclusion - like bodies of mis - assembled z - disks are detected that resemble the rod bodies that are a hallmark of nemaline myopathy ( bang et al . during postnatal development of the soleus muscle major changes in splicing were detected in the z - disk region of nebulin . the increase in z - disk width of the soleus muscle might reflect the increasing stress exerted on this muscle due to the rapid increase in body weight during postnatal development . together , these findings support a model in which titin and nebulin together specify z - disk width , with titin constructing the central region of the z - disk , including the number and positions of -actinin cross - links and nebulin determining the ending of the z - disk structure and its transition to the i - band , i.e. , 2008 ) , capz does not localize properly , allowing the barbed ends of thin filaments to continue to grow beyond the z - disk resulting in widened z - disks . when this feature of nebulin is not present , z - disks widen , ultimately culminating in the formation of nemaline rods . in addition to regulating z - disk width and structure , nebulin is suggested to play a role in laterally linking myofibrils at the z - diks . longitudinally , sarcomeres are connected by z - disk lattices that anchor thin filaments and transmit force along the myofibril . in the transverse direction , linkage of myofibrils at the z - disks allows for lateral force transmission and limits the degree to which adjacent myofibrils translocate relative to each other during active contraction or passive stretch , thereby preventing damage to intermyofibrillar membrane systems , such as t - tubules and the sarcoplasmic reticulum . an important protein involved in linking adjacent z - disks is the intermediate filament protein desmin , which forms a network of filaments that surrounds myofibrils at the level of the z - disk ( wang and ramirez - mitchell , 1983 ; capetanaki et al . that desmin tethers adjacent z - disks is supported by work on a desmin ko mouse in which z - disk misalignment was shown to occur in stretched muscle ( shah et al . , 2002 ) , which is anchored in the z - disk , and recently it was shown that nebulin modules m160170 are involved in this interaction ( conover et al . although desmin is present in muscle devoid of nebulin , it is reduced in the intermyofibrillar spaces that surround the z - disks , suggesting that nebulin is required for proper localization of desmin at the z - disk . consistent with this , both knockdown of nebulin with sirna and overexpression of m160m170 did not interfere with the formation of normal striation patterns but did prevent desmin localization at the mature z - disk . thus , nebulin is required to laterally link myofibrils at the z - disk by desmin filaments ; in the absence of nebulin myofibrillar connectivity is significantly reduced leading to z - disk displacement . exciting recent work revealed that nebulin s c - terminal sh3 domain is involved in the induction of muscle hypertrophy . ( 2010 ) found that insulin - like growth factor 1 induces skeletal muscle maturation and hypertrophy by forming a complex of nebulin and n - wasp at the z - disks of myofibrils by interfering with glycogen synthase kinase-3. their results convincingly show that n - wasp interacts with nebulin s sh3 domain , and that n - wasp is rapidly recruited to the z - disk after treatment with insulin - like growth factor 1 , which stimulates myofibrillogenesis and suppresses sarcomere breakdown via the pi3k akt pathway . recent work shows that nebulin s role is not merely structural , but that nebulin also regulates contraction . muscle contraction is driven by the cyclic interaction between the myosin - based crossbridges and actin and the level of force a muscle generates is proportional to the force generated per crossbridge and the number of crossbridges in the force generating state . , 2009 ) recently identified that nebulin contributes to the regulation of crossbridge cycling kinetics , with one of the two ( chandra et al . , 2009 ) also identifying a role for nebulin in the calcium sensitivity of force generation . it is also important to highlight that these findings are consistent with results of in vitro motility assays in which nebulin fragments were found to reduce the sliding velocity of f - actin over myosin ( root and wang , 1994 ) . in brenner s framework ( brenner , 1988 ) , the rate constant of force redevelopment ( ktr ) is proportional to fapp + gapp , and the fraction of force generating crossbridges to fapp/(fapp + gapp ) . , recent studies suggest that nebulin increases the rate of crossbridge attachment and reduces the rate of crossbridge detachment , and that as a result the number of force generating crossbridges is increased . thus , the presence of nebulin at or near the s1 binding site might enhance the binding of crossbridges and slow their detachment . ( 2009 ) , and they can largely account for the more pronounced leftward shift of the measured force - sarcomere length relation of nebulin - deficient muscle when compared to the predicted relation based on only thin filament length measurements . clearly , nebulin is a major factor in determining the level of force and the energetic cost of force production in skeletal muscle . in line with this role of nebulin in the regulation of crossbridge cycling kinetics , recent studies on muscle fibers from patients with nemaline myopathy with severely reduced nebulin protein levels revealed that in addition to altered thin filament length , changes in crossbridge cycling kinetics contribute to the muscle weakness observed in these patients ( ottenheijm et al . an analysis of expression levels of tropomyosin , myosin heavy chain , myosin light chain did not reveal a significant difference in these proteins between the fibers , suggesting that the absence of nebulin is the most likely explanation for the lower calcium sensitivity of the ko fibers . it is possible that the discrepancy is due to the fact that those studies did not carry out a troponin exchange and that differences in the troponin complex between wt and ko fibers could have negated nebulin s effect on calcium sensitivity . the implication is that nebulin plays a role in the length dependence of activation with a much larger pca50 in the nebulin ko fibers than in wt fibers . , the presence of nebulin provides an explanation for why skeletal muscle has less length dependence : the presence of nebulin increases calcium sensitivity at short length . the structure and protein binding properties of nebulin support a role in thin filament activation . it is interesting that similar to tm , nebulin appears to have different binding sites on f - actin with one site in close proximity to both the strong binding site for myosin and the blocked state of tropomyosin ( lukoyanova et al . this leads to the intriguing possibility that nebulin acts in concert with tm and that it promotes the transition of contractile regulatory units ( tm / tn ) from the non - permissive to the permissive states , thereby increasing myofilament calcium sensitivity . it is striking that nebulin - deficient skeletal muscle shares the low calcium sensitivity and low maximal active force with cardiac muscle , where stoichiometric levels of nebulin are absent ( bang et al . however , nebulette is much smaller than nebulin ( ~100 vs ~800 kda for nebulin ) and its location is restricted to the z - disk and the near z - disk i - band region ( millevoi et al . cardiac muscle has multiple mechanisms for enhancing thin filament activation , such as , enhanced length - dependent activation , and the presence of multiple cardiac - specific phosphorylation sites in various thin and thick filament based protein sites ( solaro and de tombe , 2008 ) , which allow cardiac muscle to grade its force response to different loading conditions . since skeletal muscle lacks the aforementioned features unique to cardiac muscle , nebulin might be essential for tuning thin filament activation for optimal skeletal muscle function . the above discussed studies clearly indicate that nebulin is not merely involved in regulation sarcomere structure , but also acts as a regulator of muscle contraction . dysfunctions of the skeletal muscle thin filament have emerged as important causes of skeletal myopathies of which nemaline myopathy is the most common disease with a previously estimated prevalence of 0.002% ( sanoudou and beggs , 2001 ) . the typical form of nm presents with early onset in infancy , has a non - progressive or slowly progressive course and is caused by mutations in the nebulin gene . recent studies on muscle fibers from patients with nemaline myopathy due to the deletion of nebulin exon 55 showed that these patients have severely reduced nebulin protein levels and that they show remarkable phenotypic similarities to fibers from nebulin ko mice , i.e. thus , loss of thin filament length regulation appears to be an important contributor to muscle weakness in patients with nemaline myopathy . in addition to altered thin filament length , changes in crossbridge cycling kinetics and reduced calcium sensitivity of force production contribute to the muscle weakness observed in these patients ( see figure 2a ; ottenheijm et al . , 2010 ) , in line with the role of nebulin in these processes ( chandra et al . thus , nebulin s role in thin filament length regulation and contraction deduced from work on neb - ko mice provides a mechanism for the first time to explain severe muscle weakness in patients with nemaline myopathy . interestingly , recent work form our lab demonstrates that patients with nemaline myopathy due to mutations in the tropomyosin gene ( tpm3 ) display a contractile phenotype that is distinct from that of patients with neb - based myopathy ( ottenheijm et al . whereas both show severe myofilament - based muscle weakness , the contractile dysfunction in tpm3-based myopathy can be largely explained by changes in crossbridge cycling kinetics , whereas dysregulation of thin filament length and altered crossbridge cycling kinetics play a prominent role in neb - based myopathy ( ottenheijm et al . furthermore , the loss of force generating capacity in tpm3-based myopathy appears to be partly compensated by enhanced calcium sensitivity of force generation , whereas decreased calcium sensitivity of force generation further depresses the capacity for force production in neb - based myopathy ( ottenheijm et al . whereas these findings support the existence of distinct genotype - phenotype correlations in nm , it is yet unclear whether they extrapolate to other patients with mutations in neb or tpm3 , and how they compare to the phenotype of nm patients with mutations in acta1 , tpm2 , tnnt1 , cfl2 , and kbtbd13 .

over the past decade , graphene has attracted considerable attention because of its wide range of applications , e.g. , as chemical sensors , organic semiconductors , energy storage devices , in spintronics and nonlinear optics . when the graphene sheet is cut into nanosized fragments , therefore , the properties of pahs are closely related to that of nanographene . these pahs are composed of fused aromatic rings , a feature with almost unlimited possibilities that can lead to a rich diversity of compounds . many of the pahs have a closed - shell electronic configuration in their ground state . however , there are types of pahs which possess a high - spin , open - shell radical character in their ground state . for example , phenalenyl ( 1 ) ( chart 1 ) in its neutral ground state contains an odd number of carbon atoms with an odd number of electrons , which makes it a radical . the extension of benzene rings in a triangular fashion can lead to several -conjugated phenalenyl derivatives such as triangulene ( 2 ) , -extended triangulene system ( 3 ) ( see chart 1 ) and even larger systems . an * and n are the number of starred and unstarred atoms . n * and n are the number of starred and unstarred atoms . these molecules are categorized as open - shell non - kekul polynuclear benzenoid molecules where some electrons are unpaired due to the topology of the electron arrangement . the topological effect on the ground state of these molecules can be explained using ovchinnikov s rule which states that the ground - state spin quantum number , s , of a given pah with six - membered rings can be expressed as s = ( n * n)/2 where n * and n are the number of starred and unstarred atoms of alternant systems . as shown in chart 1 , in the case of phenalenyl ( 1 ) the open - shell character of this system can be understood qualitatively by considering no matter what resonance structure is chosen one starred atom will be left without a bonding partner . in the case of 2 , two such empty valences are created , while there are three in the case of 3 . on the other hand , zethrenes starting with heptazethrene ( 4 ) ( chart 2 ) , n - acenes longer than pentacene , and some zigzag - edged graphene nanoribbons ( gnrs ) have a singlet ground state but nevertheless significant biradical or even polyradical character . among the different types of pahs zethrenes are z - shaped pahs where two phenalenyl systems are head - to - head fused with or without a benzenoid spacer . the smallest member is the hexazethrene which contains a total of six condensed rings where the two phenalenyl rings are directly fused . the next one is heptazethrene ( 4 ) where an additional benzene ring is inserted in between the two phenalenyl units . if the terminal naphthalene units are replaced by anthracene , depending on the position of the ring fusion , two different structural isomers with the same chemical compositions are formed : 1,2;9,10-dibenzoheptazethrene ( 5 ) and 5,6;13,14-dibenzoheptazethrene ( 6 ) ( chart 2 ) . athe benzene rings in red represent clar s aromatic sextet rings . the benzene rings in red represent clar s aromatic sextet rings . as an alternative to zethrenes , kubo and co - workers designed a p - quinodimethane - linked bisphenalenyl ( 7 ) that contains p - quinodimethane and two phenalenyl rings ( chart 3 ) . a characteristic feature of the zethrenes and structure 7 is the competition between a closed - shell quinoid kekul form and an open - shell biradical resonance form ( charts 2 and 3 ) . because of the presence of these two resonance structures , two interesting questions arise : which resonance form is dominating in the ground state and , consequently , which isomer contains a larger biradical character ? clar s aromatic sextet rule qualitatively predicts that for benzenoid pahs the molecule with more aromatic sextet rings possesses more aromatic stabilization energy . thus , if in different valence bond ( vb ) structures for a given pah the biradical form contains more aromatic sextet rings than the closed - shell quinoid structure , then , as discussed in ref ( 32 ) and shown in charts 2 and 3 , its enhanced stability should be the source of a greater singlet biradical character . for example , in the case of structures 4 and 5 three aromatic sextets occur in the biradical form , whereas in the closed - shell kekul form only two aromatic sextets are present ; for structure 6 , a total of five sextets can be drawn ( 6c ) when the two radical centers are located at the terminal units . thus , structures 4 and 5 are expected to exhibit significant biradical character , and the even larger number of sextets for structure 6 should help to stabilize the biradical form in this case even more . chart 3 represents the resonance structures between kekul and the biradical forms for structure 7 where in the biradical form the central benzene ring obtains aromatic character . it is expected , therefore , that structure 7 also possesses a significant biradical character . structure 8 in chart 3 represents the non - kekul biradical form of the clar goblet . pahs with radical character possess unique electronic , magnetic , and optical properties . quantum chemical calculations play an important role for understanding the unique electronic characteristics of the pahs . among the methods available , density functional theory ( dft ) would be the first choice because of its computational efficiency and good overall performance . however , due to the partly open - shell character of these compounds , an unrestricted approach has to be used which suffers from the problem of spin - contamination , especially in the case of broken symmetry ( bs ) dft for singlet biradical species or low - spin cases in general . therefore , several other approaches such as spin - flip methods , projected hartree fock theory , active - space variational two - electron reduced - density - matrix ( 2-rdm ) , density matrix renormalization group ( dmrg ) , and coupled cluster with singles , doubles , and noniterative triples ccsd(t ) have been applied successfully to investigate the electronic structure of various pahs . as a systematic and general alternative theory , multireference ( mr ) the multireference averaged quadratic coupled cluster ( mr - aqcc ) approach represents a good option since it allows the inclusion of quasi - degenerate configurations in the reference wave function and of dynamic electron correlation including size - extensivity contributions by considering single and double excitations explicitly . recently , mr - aqcc calculations for the n - acenes and periacenes and several smaller challenging biradicals have been performed successfully . to understand the structural variation of the pahs , their unique properties , and possible applications , it is important to investigate their electronic structure in more detail . though ovchinnikov s rule and clar s aromatic sextet rule give a general idea about the multiplicity , stability , and electronic character of the ground state , it is nevertheless crucial to obtain a reliable quantitative description of these properties as well . phenalenyl , its extended versions , and the combination to form zethrenes or p - quinodimethane - linked -conjugated compounds such as the clar goblet ( chart 1chart 3 ) constitute very interesting but also computationally challenging cases for describing the polyradical character of these compounds . here , we use the aforementioned mr - aqcc method and the related mr configuration interaction with singles and doubles ( mr - cisd ) to discuss the properties of these types of pahs in their ground state as well as in their lowest lying excited states . one major effort is dedicated to the clarification of which electronic configuration , either the closed - shell quinoid kekul or the open - shell biradical form , better describe the ground state of structures 47 . the related question of the multiplicity of the ground state and of singlet triplet splitting will be addressed also by these high - level calculations . the complicated nature of the multireference wave function will be transformed to simple descriptors such as natural orbital ( no ) occupation numbers and the unpaired density distribution which allow a concise chemical assessment of the polyradical nature of a compound and the location of chemically reactive unpaired density regions in a molecule . by means of comparison with our multireference ab initio results , dft is also considered in the present context in order to obtain better insight into its applicability to these difficult questions concerning the correct description of biradical systems . multiconfiguration self - consistent field ( mcscf ) , mostly in the form of the complete active space self - consistent field ( casscf ) method , mr - cisd , and mr - aqcc calculations have been performed for the structures shown in charts 13 . for most of the calculations , two different sets of complete active spaces ( cas ) have been chosen : ( a ) a cas(7,7 ) and ( b ) a cas(4,4 ) . in active space ( a ) , seven electrons have been distributed in seven orbitals and for active space ( b ) four electrons have been distributed over four orbitals . the choice of these active spaces is based on the no occupation numbers computed from the unrestricted hartree fock ( uhf ) wave function as suggested by pulay et al . the contribution of the single- and double - substituted configurations has been monitored and in the case of a weight larger than 1% extensions of the active space have been made in order to account for intruder states in the mr - aqcc calculations or to ensure degeneracies in the case of the triangular symmetry of structures 13 , since for practical reasons only c2v symmetry was employed . the active spaces for the mcscf , mr - cisd , and mr - aqcc calculations were usually kept the same . size extensivity corrections are computed for the mr - cisd approach by means of an extended davidson correction , denoted as + q ( mr - cisd+q ) . more details on the construction of the mr wave functions and the orientation of the molecules in the cartesian coordinate system can be found in the supporting information ( si ) . three different orbital freezing schemes have been used throughout the calculations : ( i ) core freezing where only the 1s core orbitals of all the carbon atoms are frozen at the mr - cisd level ; ( ii ) -partial freezing ( designated as ( )+-space ) where 1s core orbitals of the carbon atoms ( n ) are frozen at the mcscf level and depending on the degeneracy of the orbitals , additional n or ( n 1 ) orbitals , respectively , are frozen at the mr - cisd level ; and ( iii ) -space where all occupied and virtual orbitals were frozen by transforming the one- and two - electron integrals from the atomic orbital ( ao ) basis into the basis of scf orbitals keeping only the orbitals active . the 6 - 31 g , 6 - 31 g * , and 6 - 311g(2d ) basis sets have been used throughout the calculations . for the analysis of the radical character , we have used ( a ) no occupations as computed from the aqcc density and ( b ) the unpaired density and the number of effectively unpaired electrons ( nu ) as originally introduced by takatsuka et al . as the distribution of odd electrons . this method was further developed by staroverov and davidson . in order to emphasize the contributions from orbitals with occupation near one and suppressing contributions that are nearly doubly occupied or nearly unoccupied , the nonlinear formula of head - gordon1is used in which ni is the occupation of the ith no and m is the total number of nos . all the structures have been optimized using dft with the b3lyp functional and the 6 - 31 g * basis set . harmonic vibrational frequencies have been computed , and none of the structures except the 1ag state of structure 5 shows imaginary frequencies in c2h symmetry , which demonstrates that all other structures are minima . structure 5 possesses two out - of - plane imaginary frequencies ( bg and au ) in c2h symmetry because of the steric congestion between the hydrogen atom of the terminal benzene ring of the anthracene unit and the hydrogen atom of central benzene ring . the final optimized structure 5 has c1 symmetry , and the corresponding frequency calculation confirmed that it is a minimum . it is important to note that the experimental structure with the heptazethrene core also deviates from planarity with a torsional angle of 33.9. the mr - aqcc calculation in c1 symmetry would have been too costly . since the difference between the planar c2h and nonplanar c1 structures in terms of singlet triplet splitting and biradical character computed at dft level is relatively small ( which will discussed below ) and also for comparison purpose with the other isomer ( structure 6 ) , we have decided to focus on the conjugation and keep structure 5 planar by using c2h symmetry . it was found that all the structures have a triplet instability and appropriate geometry reoptimizations have been performed in reduced planar cs symmetry . the real nature ( except planar structure 5 has two imaginary frequencies ) of the vibrational frequencies also confirmed that these structures corresponded to minima . the triplet states ( 1bu for structures 4 - 6 and 1b2u for structures 7 and 8) have also been optimized separately . in the spin - unrestricted kohn sham formalism used , especially the low - spin bs solution suffers from the problem of spin contamination . a high spin calculation with two unpaired electrons with parallel spins is applied to represent the triplet state , and the bs solution with antiparallel spins is used for the singlet state . to correct for spin contamination , the spin corrected formula as proposed by yamaguchi is employed . in this approach the vertical singlet i is given by the expression2where et , es , and ebs represent the energy of the triplet , singlet , and the bs solutions , respectively . st2 and sbs2 are the expectation value of the square of the total spin operator for the triplet and the bs solutions . for quantitative analysis of the open - shell character , along with nu , we have also computed the multiple diradical characteryi ( i = 0 , 1 , 2 ) where 0 yi 1 and yi yi+1 from the spin - projected uhf ( p - uhf ) theory as3where ti is the orbital overlap between the corresponding orbital pairs which can be expressed by where nhono i and nluno+i are the occupation numbers of the ith highest occupied no ( hono ) and the ith lowest unoccupied no ( luno ) computed from uhf nos . y0 = 0 indicates pure closed - shell and y0 = 1 indicates a pure diradical character . comparable y0 and y1 values indicate that in addition to the hono / luno pair , non - hono / luno pairs are also important . in the case of y0 = 1 and a large y1 value the mr calculations have been performed with the parallel version of the columbus program system . population analysis of the unpaired densities has been carried out with the theodore program . for the dft calculations along with the stability analysis and the uhf no calculations , the turbomole program has been used . figure 1 shows the mo occupation schemes for the ground state of the phenalenyl - based neutral radicals . the symmetry is given both in c2v and d3h ( in parentheses ) notations . the figure shows that the triangular pahs exhibit a high - spin ground state and that the spin multiplicity increases with increasing molecular size . n ) as predicted by ovchinnikov s rule and also supports the experimental findings that the ground state of phenalenyl derivatives have a high - spin state . molecular orbital ( mo ) occupation schemes for ( a ) the 1a2 ( 1a1 ) state of phenalenyl ( 1 ) , ( b ) the 1b2 ( 1a2 ) state of triangulene ( 2 ) , and ( c ) the 1b1 ( 1a2 ) state of the -extended triangulene system ( 3 ) . the symmetry is shown as c2v ( d3h ) notation . in table 1 a comparison of the energy differences between the ground and the degenerate first excited states of phenalenyl - based neutral radicals computed at mr - cisd and mr - cisd+q levels is shown . because of the severe occurrence of intruder states in the excited states , mr - aqcc calculations could not be performed in this case . because of the use of the lower c2v symmetry instead of the actual d3h symmetry in the calculations , in some of the cases the degeneracy is slightly lifted . for this reason , we always considered the average energy between the two degenerate states . for phenalenyl ( 1 ) , the degeneracy is well reproduced within 0.001 ev , and in most of the cases for the systems 2 and 3 it remains within 0.02 ev . the first excited doublet state is degenerate ( e symmetry ) . the excitation energies amount to 3.165 ev at -cas(7,7 ) and 2.777 ev at -mr - cisd levels , respectively , using the 6 - 311g(2d ) basis and freezing all orbitals . reducing the basis set to 6 - 31 inclusion of orbitals into the calculation increases the excitation energy at the mr - cisd level by 0.3 ev ; the davidson correction again decreases this value by 0.2 ev . at -mr - cisd level , the symmetry is given as d3h notation , d3h ( c2v ) 1 : a1 ( a2 ) e ( b1,a2 ) ; 2 : a2 ( b2 ) e ( a1 , b2 ) ; 3 : a2 ( b1 ) e ( a2 , b1 ) . for 1 and 3 a -cas(7,7)c and for 2 a -cas(4,7)c reference space are used , respectively . contains intruder states . the next higher non - kekul - type triangular pah investigated is triangulene ( 2 ) . though it has an even number of carbon atoms , no kekul - type formula can be given . 2,6,10-tri - tert - butyltriangulene is the first example of an experimentally synthesized genuine non - kekul pah . the experimentally observed linear dependence of the triplet signal intensity on 1/t for the 2,6,10-tri - tert - butyltriangulene combined with the observed g value of 2.0034 indicates that the high - spin triplet is the ground state . the mr - cisd calculations collected in table 1 for triangulene ( 2 ) confirm this finding . to characterize the manifold of lowest excited states for this system , the lowest triplet excitation energy and the triplet / singlet splitting are given in table 1 . the 1e state is 2.7 ev higher than the 1a2 ground state ; the triplet / singlet splitting is only 0.8 ev . comparison of the excitation energies obtained with 6 - 31 g * and 6 - 311g(2d ) basis sets shows an agreement within a few hundredths of an ev . the same is true for the comparison with the 6 - 31 g basis set ( see table s2 ) . bearpark el al . found for the trioxytriangulene trianion at the casscf level the triplet state is more stable than the singlet state by 0.867 ev , which is in good agreement with our casscf results on triangulene ( 2 ) . for the -extended triangulene system ( 3 ) the ground state is a quartet , in agreement with ovchinnikov s rule . at the -mr - cisd level using the 6 - 311g(2d ) basis , this state is 3.217 ev more stable than the first excited e state , and the quartet / doublet splitting is 0.633 ev . analysis of the progression of the excitation energies along the increase of the triangular system from structures 1 to 3 shows relatively small effects . the excitation energy within the same spin multiplicity even increases from 2 to 3 by 0.3 ev , whereas the corresponding high spin / low spin excitation energies decreased by 0.15 ev . the comparison of the energy differences for the open - shell triangular pahs for the three different orbital schemes ( core freezing , -partial freezing and -space ) using the 6 - 31 g basis set is shown in table s2 . the differences in the results obtained with the polarized basis sets ( table 1 ) are mostly less than 0.1 ev . from these data , one can conclude that ( i ) -space calculations are quite sufficient to describe the energy difference between the ground and the lowest excited states , and ( ii ) the basis set dependence on the addition of polarization functions is quite modest . the no occupations displayed in figure 2 show that phenalenyl ( 1 ) has one ( 1a1 ) , triangulene ( 2 ) has two ( degenerate 4e ) , and -extended triangulene ( 3 ) has three ( one in 2a1 and two in 6e ) singly occupied no(s ) ( sonos ) in agreement with the mo occupation scheme given in figure 1 . it is important to note that the appearance of radical character with high spin multiplicities for the open - shell -conjugated hydrocarbons arises because of the fused -topology and not because of orbital degeneracy derived from the 3-fold symmetry . consequently , extending the -conjugation according to the topology of electrons can lead to an unlimited numbers of electron spins aligned parallel to each other in singly occupied mos ( somos ) . natural orbital occupation of the 1a1 state of phenalenyl ( 1 ) , the 1a2 state of triangulene ( 2 ) , and the 1a2 state of the -extended triangulene system ( 3 ) using a -mr - aqcc / cas(7,7)/6 - 311g(2d ) for 1 and 3 and a -mr - aqcc / cas(4,7)/6 - 311g(2d ) for 2 . in figure 3 it is delocalized over the entire molecule but resides mainly on the edges of the molecule . moreover , for all three cases , the distribution at the edges is alternant and the unpaired density resides only on the starred atoms as indicated in chart 1 . density of unpaired electrons for the ( a ) 1a1 state of phenalenyl ( 1 ) ( nu = 1.33 e ) ; ( b ) 1a2 state of triangulene ( 2 ) ( nu = 2.50 e ) , and ( c ) 1a2 state of -extended triangulene system ( 3 ) ( nu = 3.66 e ) using a -mr - aqcc / cas(7,7)/6 - 311g(2d ) for 1 and 3 and a -mr - aqcc / cas(4,7)/6 - 311g(2d ) calculation for ( 2 ) ( isovalue 0.005 e bohr ) with individual atomic population computed from mulliken analysis . for phenalenyl , the central carbon atom does not carry any unpaired density . this depletion of unpaired density in the central region of the triangle has important consequences on the shape of stacked biradical dimers . for the phenalenyl dimer , it has been shown that the dimer has a convex shape with the edge region being stronger bound due to the enhanced unpaired density in that region . additionally , the possibilities for - and -dimerization were discussed for a series of substituted phenalenyls based on a set of spectroscopic methods and on dft calculations . it is noteworthy that the total unpaired density values , nu presented in figure 3 , are larger as compared to the formal open - shell occupation of the high - spin state ( 1.33 vs 1 for phenalenyl , 2.50 vs 2.0 for triangulene , and 3.66 vs 3.0 for -extended triangulene system ) . this excess unpaired density of these pahs ( 13 ) is derived from non - negligible contributions generated from nos other than the sono(s ) ( figure 2 ) . figures s1 and s2 show selected bond distances of the singlet ground state of structures 46 and 78 as computed at the udft / b3lyp/6 - 31 g * level . the pronounced bond length alternation of the central p - quinodimethane subunit of the structures 4 and 5 indicates that the quinoid resonance form ( chart 2 ) has a dominant contribution to the singlet ground state . the difference in single to double bond lengths in the quinoid ring is between 0.06 and 0.08 for structure 4 . a slightly lower range of 0.040.07 is found for both the c2h- and c1-optimized structures of 1,2:9,10-dibenzoheptazethrene ( 5 ) . this difference is significantly reduced for the alternative isomer , 5,6:13,14-dibenzoheptazethrene ( 6 ) , to a range of 0.030.05 . this trend toward equidistant aromatic bond distances indicates the increasing importance of the biradical vb structure which will find its counterpart in the increase of the values for the total number of unpaired density ( nu ) discussed below . the central benzene ring in structure 7 ( figure s2 ) has almost aromatic character , indicating that this structure has a substantial biradical character . in the case of the structure of clar s goblet ( figure s2 ) , all cc bonds are almost equivalent having a maximum difference of 0.046 with respect to the aromatic cc distance ( 1.39 ) . the optimized bond distances a , b , and c ( chart 2 ) as shown in figure s1 for structures 4 and 5 are very close to the experimentally observed ones with a maximum deviation of 0.01 . the optimized structure of 7 ( figure s2 ) shows that the bond length labeled d ( 1.456 ) , which connects the two phenalenyl rings , and the bond lengths b ( 1.446 ) and c ( 1.396 ) of the central benzene ring are very close to the experimentally observed bond lengths of 1.450 , 1.437 , and 1.394 , respectively . triplet splitting is investigated next in order to continue the discussion of polyradical character of structures 48 . in table 2 , computed vertical and adiabatic ( in parentheses ) singlet triplet splitting energies e(s t ) are listed and compared with available experimental data . in the mr calculations , -cas(4,4)b reference space and the 6 - 31 g * reference space was used where ras refers to a restricted active space and aux to an auxiliary space ( see the si ) . t ) values for structures 48 are positive at all computational levels used , showing that these structures maintain a singlet ground state . t ) for structure 5 ( 0.527 ev at -mr - aqcc , table 2 ) agrees well with the experimentally observed sharp nmr peak and esr silence . this means that the excited triplet state is located relatively high in energy . for structure 6 , the adiabatic e(s t ) is 0.324 ev ( -mr - aqcc ) , somewhat higher than that of the experimentally observed value of 0.165 ev . experimentally , a broadening of the nmr spectrum at room temperature and a broad esr signal were observed for structure 6 and the signal intensity is decreasing with decreasing temperature . these features are consistent with the theoretically observed small e(st ) of structure 6 . t ) computed at the same level is 0.197 ev , very close to the experimentally observed value of 0.211 ev . for structure 8 , the computed e(st ) is very small ( mostly < 0.1 ev , table 2 ) showing that the singlet and the triplet states are almost degenerate . the evolution of the weights of the major configurations computed at -mr - aqcc level ( table s4 ) goes in line with the just - described changes of the singlet triplet splitting has been computed also at the udft level and is given in table 2 together with the expectation values of s for the bs state . two kinds of e(s t ) values have been computed at udft level : ( i ) using the spin - contaminated value and ( ii ) using the spin - corrected form ( eq 3 , eproj ) . triplet splitting , bringing it closer to the mr - aqcc values . for structure 5 , eproj of the nonplanar unrestricted c1 structure is 0.568 ev , very close that of the value of 0.540 ev calculated for the planar c2h structure . a systematic comparison between singlet triplet energies computed at several levels of orbital freezing schemes and basis sets including the unpolarized 6 - 31 g basis ( table s5 ) finds in all cases a good agreement within a few tenths of an ev . these findings apply also to the computation of no occupations ( table s6 ) and unpaired density ( table s7 ) discussed below . the observed relative insensitivity in terms of energies and character of the wave function facilitates the discussion of the polyradical character by means of multireference methods considerably . on the other hand , it is clear that this assessment has to be continuously re - evaluated when different kinds of -conjugated systems are to be investigated . no occupations for the singlet state of structures 48 are presented in figure 4 . several of the no occupation numbers for the singlet state deviate strongly from the closed - shell limiting values of two / zero , indicating that they have substantial polyradical character . natural orbital ( no ) occupation of the 1ag ground state of heptazethrene ( 4 ) , 1,2:9,10-dibenzoheptazethrene ( 5 ) , 5,6:13,14-dibenzoheptazethrene ( 6 ) , p - quinodimethane - linked bisphenalenyl ( 7 ) , and clar goblet ( 8) obtained from -mr - aqcc / cas(4,4)/6 - 311g(2d ) calculations for structures 47 and -mr - aqcc / ras(1)/cas(4,4)/aux(1)/6 - 311g(2d ) calculation for structure 8 . comparison of the hono / luno occupation numbers ( table 3 ) indicates that deviation from the limiting values of two / zero is the smallest for structure 4 and the largest for structure 8 . at the uhf level , these deviations from the closed - shell reference values are considerably larger than the respective mr - aqcc results . in the former case , hono / luno occupations are almost constant along the series 48 , whereas at the mr - aqcc method there is a strong variation of the occupation number , indicating a significant change in radical character . the picture of an almost uniform biradical character throughout the series 48 given by the uhf method is , however , not consistent with the graded evolution of the geometries and the singlet / triplet splitting discussed above . -mr - aqcc / ras(1)/cas(4,4)/aux(1)/6 - 311g(2d ) and uhf/6 - 31 g * calculations . even though most of the open - shell contributions computed at mr - aqcc level are coming from the hono / luno occupation , for all structures , irrespective of singlet or triplet states , there are additional nos , whose occupation numbers deviate significantly from the limiting value of two and zero ( figure 4 ) . this implies that in addition to the hono / luno pair , other nos also provide significant contributions to the radical character which can not be neglected . the densities of unpaired electrons for the singlet state are presented in figure 5 and figure 6 for structures 46 and 78 , respectively . unlike the situation found for the phenalenyl derivatives ( 13 ) where unpaired density is delocalized over the entire molecule , for structures 4 , 5 , and 6 , the radical character is mostly distributed over a few positions . for structure 4 , most of the unpaired density is located at c4/12 ( see chart 2 for numbering ) . for structure 5 the unpaired density is extended also to the atom pair c5/13 . for both of these structures , the unpaired electron density within the benzene ring connecting the two phenalenyl segments is significant . in the case of structure 6 , the unpaired density the main contributions are located equally at c4/12 and c7/15 indicating equal contributions from both the biradical resonance forms ( 6b and 6c ) as shown in chart 2 . however , the unpaired density situated on the other centers can not be neglected . this indicates the existence of several additional vb structures in comparison to which are given in charts 2 and 3 . density of unpaired electrons for the 1ag state of ( a ) heptazethrene ( 4 ) ( nu = 1.03 e ) , ( b ) 1,2:9,10-dibenzoheptazethrene ( 5 ) ( nu = 1.49 e ) , and ( c ) 5,6:13,14-dibenzoheptazethrene ( 6 ) ( nu = 2.24 e ) using the -mr - aqcc / cas(4,4)/6 - 311g(2d ) approach ( isovalue 0.003 e bohr ) with individual atomic population computed from mulliken analysis . the unpaired density for structure 7 ( figure 6 ) shows a pattern which is more delocalized than the one indicated by the two mesomeric forms ( 7a and 7b ) given in chart 3 . thus , in this case , the unpaired density can be better represented by the resonance form 7c , where the structure 7 can be considered as a combination of two phenalenyl systems linked by a benzene ring . density of unpaired electrons for the 1ag state of ( a ) p - quinodimethane - linked bisphenalenyl ( 7 ) ( nu = 1.86 e ) using the -mr - aqcc / cas(4,4)/6 - 311g(2d ) method and ( b ) clar goblet ( 8) ( nu = 2.88 e ) using the -mr - aqcc / ras(1)/cas(4,4)/aux(1)/6 - 311g(2d ) method ( isovalue 0.003 e bohr ) with individual atomic population computed from mulliken analysis . in structure 8 ( figure 6 ) , the unpaired character is mostly located at the zigzag edges with the largest contribution at their centers ( position at c9/18 , see chart 3 ) . it is also noted that for the zethrenes and the structures 5 and 6 , the linking benzene ring seems to play a more important role ( i.e. , there is a significant amount of unpaired density located on this connecting ring relative to the total number of unpaired density ) than in case of the vertical connections between subunits in structure 8 . table 4 compares the nu values computed at -mr - aqcc level with the multiple diradical character indices , yi ( i = 0,1,2 .. ) , obtained from p - uhf theory for structures 48 . to obtain a common basis for comparison with the nu s , it is observed that for the singlet ground states of structures 4 and 5 the 2y0 values , which are computed from the hono / luno uhf occupations , are almost twice of the nu values computed from the mr - aqcc hono / luno occupations . once the structures acquire more biradical character ( for structures 68 ) , the two values approach each other . this behavior is derived from the discrepancies in the no occupation numbers computed with the two different methods ( table 3 ) . the y0 values reported in ref ( 32 ) for the structures 46 are somewhat smaller as compared to our values . however , , the trend of increasing y0 value as one moves from structure 4 to 6 is the same . for structure 5 , the y0 value as computed for c1 optimized structure is 0.649 , very close to that of the c2h planar structure of 0.664 . comparing the nu values derived from different no selections , it is noted that the total nu value is significantly larger than the one computed only from the hono / luno part . these additional contributions come partly from the hono-1/luno+1 set ( table 4 ) , but also from the large number of nos whose occupation numbers deviate from the 0/2 e occupations to a lesser extent . this is in contrast to the tetracyanoethylene anion dimer ( tcne2 ) and neutral k2tcne2 system where the effect of the non - hono / luno pairs is practically negligible . in the present case the contribution of the non - hono / luno pairs to the total nu value is almost 50% for the singlet biradicaloid structures of 4 and 5 , but for the biradical structures 6 - 8 , it decreases from 36% to 31% . comparison of the nu values computed from the non - hono / luno pairs for the singlet and the triplet states of structures 4 - 8 shows that they are almost identical . this indicates that the main difference is coming from the different occupations of the hono and luno pair and the remaining contributions are quite the same . the 6 - 311g(2d ) basis and a -cas(4,4 ) reference space for 47 and ras(1)/-cas(4,4)/aux(1 ) for 8 were used . table 4 shows that for all structures the triplet state maintains practically a constant nu value ( from 2.589 e to 2.816 e ) , whereas for the singlet state of structures 48 a large change in the nu value ( from 1.026 e to 2.880 e ) is observed . as discussed just before , these differences come primarily from the hono / luno pair ( 0.506 e to 1.974 e ) . for structures 4 and 5 , this increase is only moderate from 1.026 e to 1.492 e , but from structures 5 to 6 it is relatively large ( 1.492 e to 2.241 e ) . this clearly indicates that strong variations in the polyradical character within the zethrenes can be achieved by means of relative modest changes in the conjugation . for the singlet state of structure 7 , the nu value is also very large ( 1.865 e ) indicating significant singlet biradical character as well . among the singlet state of all the structures , the clar goblet ( 8) has the largest polyradical character . density of unpaired electrons for the 1bu state of ( a ) heptazethrene ( 4 ) ( nu = 2.59 e ) ; ( b ) 1,2:9,10-dibenzoheptazethrene ( 5 ) ( nu = 2.80 e ) , and ( c ) 5,6:13,14-dibenzoheptazethrene ( 6 ) ( nu = 2.75 e ) using the -mr - aqcc / cas(4,4)/6 - 311g(2d ) approach ( isovalue 0.003 e bohr ) with individual atomic population computed from mulliken analysis . comparison of the distribution of unpaired densities between singlet and triplet states for structures 46 shows characteristic differences ( figures 5 and 7 ) . these differences are naturally larger for the cases with smaller nu values in the singlet state ( especially 4 ) since for the triplet state single occupation of the hono / luno pair is enforced . for structure 4 , the location of maximum density in both the singlet and the triplet states are same ( c4/12 , see chart 2 for numbering ) . however , additionally , for the triplet state the unpaired density extends with significant populations on the c5/13 and c7/15 positions , respectively . enhancement of similar atom position is also observed for the triplet state for structures 5 and 6 . even though the nu values between singlet and triplet start to come closer to each other , the weights on individual atoms still differ . for e.g. in the singlet state of structure 6 , the unpaired density is equally distributed between c4/12 and c7/15 positions , respectively ; for the triplet state the maximum of unpaired density is located at c7/15 . this detailed insight into the unpaired density distribution should provide improved approaches to tune the singlet triplet gap for these compounds . on the other hand , for the singlet and triplet states of both the structures 7 and 8 ( figure 6 and figure s3 ) the distribution of unpaired density is very similar in nature . in this work two different types of pahs have been studied ( a ) non - kekul triangular structures with a high - spin ground state and ( b ) pahs with singlet polyradical character . for the first case , phenalenyl ( 1 ) , triangulene ( 2 ) , and a -extended triangulene system ( 3 ) have been chosen . in the second case , a series of three zethrenes , heptazethrene ( 4 ) , 1,2:9,10-dibenzoheptazethrene ( 5 ) , 5,6:13,14-dibenzoheptazethrene ( 6 ) , and the p - quinodimethane - linked bisphenalenyl ( 7 ) have been investigated . additionally , the non - kekul clar goblet ( 8) has been studied . the motivation in choosing these two types of pahs is that structures 13 already possess open - shell character because of their high - spin ground state , whereas for structures 47 the competition between a closed - shell quinoid kekul valence bond structure and an open - shell singlet biradical resonance form determines the actual electronic structure and the chemical reactivity . for structure 8 , the topology of the -electron arrangement of the non - kekul form is the characteristic feature . to get a reliable quantitative description of these interesting systems , high - level abinitio multireference approaches have been used . unrestricted density functional theory and hartree fock calculations have been performed for structures 48 also in order to assess their applicability to these molecular systems possessing a complicated electronic structure . the triangular structures 13 always have a nondegenerate high - spin state as ground state . the spin state increases with increasing molecular size as predicted by the ovchinnikov s rule and is also in agreement with esr measurements of tri - tert - butyl - substituted phenalenyl , triangulene , and triangulene derivative . although the unpaired density of the ground state of structures 13 is delocalized over the entire molecule , it mainly resides on one of the carbon sublattices , i.e. , the starred atoms as defined above , and is for the most part located on the edges , independent of the size of the triangle . this localization of the chemical reactivity has important consequences on the lengths of the intermolecular cc bonds and the in general convex shape of stacked phenalenyl dimers as has been discussed in detail in ref ( 81 ) . for the second class of systems ( structures 48 ) , the ground state is always singlet with a varying amount of biradicaloid ( structures 4 , 5 ) , biradical ( structure 7 ) , or polyradical ( structures 6 and 8) character . triplet splitting , no occupations , and unpaired densities clearly demonstrate that within the zethrene structure family the singlet state of structure 6 possesses a much larger polyradical character as compared to structures 4 and 5 . structure 7 also has open - shell singlet biradical character in its ground state . interpretation of these results within the valence bond picture confirms that clar s aromatic sextet rule can be successfully applied for the ground state of these types of systems , but for a concrete characterization of the chemical reactivity high level quantum chemical calculations are needed . among structures 48 , the clar goblet ( 8) has the maximum polyradical character having nearly degenerate singlet and triplet states . the low - spin broken symmetry state computed at the udft / b3lyp level is highly spin contaminated . triplet gap and brings the dft and mr - aqcc results into good agreement . however , no occupations derived from uhf calculations in the spirit of the uno - cas method show a strong overshooting of the deviations from closed - shell character for most of the singlet systems investigated and , as a consequence , also an overestimation of the polyradical character as measured by the y0 and y1 indices as compared to total numbers of unpaired electrons computed by the mr - aqcc method . analysis of our mr - aqcc results and also those of previous ones preformed on the singlet triplet splitting in polyacenes shows only a minor influence of basis set effects and of the amount of correlating orbitals . though it is possible to perform large mr calculations by considering both and electrons , this is an attempt to provide a guide for managing even larger systems by performing multireference correlation calculations for the system only where such calculations including both and electrons are too expensive . in spite of the complicated structure of the multireference wave functions , the chemical analysis of the polyradical character is straightforward on the basis of the unpaired densities . such an analysis is very helpful in locating the chemically reactive centers and indicating those regions on which to focus in order to stabilize the highly reactive polyradicals . it will also be possible to accurately assess the effects of different types of substituent attached to systems carrying polyradical character and provide pictorial information on concomitant changes in the chemical reactivity .

in this work , two different classes of polyaromatic hydrocarbon ( pah ) systems have been investigated in order to characterize the amount of polyradical character and to localize the specific regions of chemical reactivity : ( a ) the non - kekul triangular structures phenalenyl , triangulene and a -extended triangulene system with high - spin ground state and ( b ) pahs based on zethrenes , p - quinodimethane - linked bisphenalenyl , and the clar goblet containing varying polyradical character in their singlet ground state . the first class of structures already have open - shell character because of their high - spin ground state , which follows from the bonding pattern , whereas for the second class the open - shell character is generated either because of the competition between the closed - shell quinoid kekul and the open - shell singlet biradical resonance structures or the topology of the -electron arrangement of the non - kekul form . high - level ab initio calculations based on multireference theory have been carried out to compute singlet triplet splitting for the above - listed compounds and to provide insight into their chemical reactivity based on the polyradical character by means of unpaired densities . unrestricted density functional theory and hartree fock calculations have been performed for comparison also in order to obtain better insight into their applicability to these types of complicated radical systems .

Introduction Computational Details Results and Discussion Conclusions

many of the pahs have a closed - shell electronic configuration in their ground state . however , there are types of pahs which possess a high - spin , open - shell radical character in their ground state . these molecules are categorized as open - shell non - kekul polynuclear benzenoid molecules where some electrons are unpaired due to the topology of the electron arrangement . as shown in chart 1 , in the case of phenalenyl ( 1 ) the open - shell character of this system can be understood qualitatively by considering no matter what resonance structure is chosen one starred atom will be left without a bonding partner . on the other hand , zethrenes starting with heptazethrene ( 4 ) ( chart 2 ) , n - acenes longer than pentacene , and some zigzag - edged graphene nanoribbons ( gnrs ) have a singlet ground state but nevertheless significant biradical or even polyradical character . as an alternative to zethrenes , kubo and co - workers designed a p - quinodimethane - linked bisphenalenyl ( 7 ) that contains p - quinodimethane and two phenalenyl rings ( chart 3 ) . a characteristic feature of the zethrenes and structure 7 is the competition between a closed - shell quinoid kekul form and an open - shell biradical resonance form ( charts 2 and 3 ) . because of the presence of these two resonance structures , two interesting questions arise : which resonance form is dominating in the ground state and , consequently , which isomer contains a larger biradical character ? thus , if in different valence bond ( vb ) structures for a given pah the biradical form contains more aromatic sextet rings than the closed - shell quinoid structure , then , as discussed in ref ( 32 ) and shown in charts 2 and 3 , its enhanced stability should be the source of a greater singlet biradical character . for example , in the case of structures 4 and 5 three aromatic sextets occur in the biradical form , whereas in the closed - shell kekul form only two aromatic sextets are present ; for structure 6 , a total of five sextets can be drawn ( 6c ) when the two radical centers are located at the terminal units . structure 8 in chart 3 represents the non - kekul biradical form of the clar goblet . however , due to the partly open - shell character of these compounds , an unrestricted approach has to be used which suffers from the problem of spin - contamination , especially in the case of broken symmetry ( bs ) dft for singlet biradical species or low - spin cases in general . though ovchinnikov s rule and clar s aromatic sextet rule give a general idea about the multiplicity , stability , and electronic character of the ground state , it is nevertheless crucial to obtain a reliable quantitative description of these properties as well . phenalenyl , its extended versions , and the combination to form zethrenes or p - quinodimethane - linked -conjugated compounds such as the clar goblet ( chart 1chart 3 ) constitute very interesting but also computationally challenging cases for describing the polyradical character of these compounds . here , we use the aforementioned mr - aqcc method and the related mr configuration interaction with singles and doubles ( mr - cisd ) to discuss the properties of these types of pahs in their ground state as well as in their lowest lying excited states . one major effort is dedicated to the clarification of which electronic configuration , either the closed - shell quinoid kekul or the open - shell biradical form , better describe the ground state of structures 47 . the related question of the multiplicity of the ground state and of singlet triplet splitting will be addressed also by these high - level calculations . by means of comparison with our multireference ab initio results , dft is also considered in the present context in order to obtain better insight into its applicability to these difficult questions concerning the correct description of biradical systems . multiconfiguration self - consistent field ( mcscf ) , mostly in the form of the complete active space self - consistent field ( casscf ) method , mr - cisd , and mr - aqcc calculations have been performed for the structures shown in charts 13 . for most of the calculations , two different sets of complete active spaces ( cas ) have been chosen : ( a ) a cas(7,7 ) and ( b ) a cas(4,4 ) . in active space ( a ) , seven electrons have been distributed in seven orbitals and for active space ( b ) four electrons have been distributed over four orbitals . the contribution of the single- and double - substituted configurations has been monitored and in the case of a weight larger than 1% extensions of the active space have been made in order to account for intruder states in the mr - aqcc calculations or to ensure degeneracies in the case of the triangular symmetry of structures 13 , since for practical reasons only c2v symmetry was employed . three different orbital freezing schemes have been used throughout the calculations : ( i ) core freezing where only the 1s core orbitals of all the carbon atoms are frozen at the mr - cisd level ; ( ii ) -partial freezing ( designated as ( )+-space ) where 1s core orbitals of the carbon atoms ( n ) are frozen at the mcscf level and depending on the degeneracy of the orbitals , additional n or ( n 1 ) orbitals , respectively , are frozen at the mr - cisd level ; and ( iii ) -space where all occupied and virtual orbitals were frozen by transforming the one- and two - electron integrals from the atomic orbital ( ao ) basis into the basis of scf orbitals keeping only the orbitals active . for the analysis of the radical character , we have used ( a ) no occupations as computed from the aqcc density and ( b ) the unpaired density and the number of effectively unpaired electrons ( nu ) as originally introduced by takatsuka et al . since the difference between the planar c2h and nonplanar c1 structures in terms of singlet triplet splitting and biradical character computed at dft level is relatively small ( which will discussed below ) and also for comparison purpose with the other isomer ( structure 6 ) , we have decided to focus on the conjugation and keep structure 5 planar by using c2h symmetry . for quantitative analysis of the open - shell character , along with nu , we have also computed the multiple diradical characteryi ( i = 0 , 1 , 2 ) where 0 yi 1 and yi yi+1 from the spin - projected uhf ( p - uhf ) theory as3where ti is the orbital overlap between the corresponding orbital pairs which can be expressed by where nhono i and nluno+i are the occupation numbers of the ith highest occupied no ( hono ) and the ith lowest unoccupied no ( luno ) computed from uhf nos . in the case of y0 = 1 and a large y1 value the mr calculations have been performed with the parallel version of the columbus program system . the figure shows that the triangular pahs exhibit a high - spin ground state and that the spin multiplicity increases with increasing molecular size . molecular orbital ( mo ) occupation schemes for ( a ) the 1a2 ( 1a1 ) state of phenalenyl ( 1 ) , ( b ) the 1b2 ( 1a2 ) state of triangulene ( 2 ) , and ( c ) the 1b1 ( 1a2 ) state of the -extended triangulene system ( 3 ) . the experimentally observed linear dependence of the triplet signal intensity on 1/t for the 2,6,10-tri - tert - butyltriangulene combined with the observed g value of 2.0034 indicates that the high - spin triplet is the ground state . for the -extended triangulene system ( 3 ) the ground state is a quartet , in agreement with ovchinnikov s rule . the comparison of the energy differences for the open - shell triangular pahs for the three different orbital schemes ( core freezing , -partial freezing and -space ) using the 6 - 31 g basis set is shown in table s2 . from these data , one can conclude that ( i ) -space calculations are quite sufficient to describe the energy difference between the ground and the lowest excited states , and ( ii ) the basis set dependence on the addition of polarization functions is quite modest . it is important to note that the appearance of radical character with high spin multiplicities for the open - shell -conjugated hydrocarbons arises because of the fused -topology and not because of orbital degeneracy derived from the 3-fold symmetry . natural orbital occupation of the 1a1 state of phenalenyl ( 1 ) , the 1a2 state of triangulene ( 2 ) , and the 1a2 state of the -extended triangulene system ( 3 ) using a -mr - aqcc / cas(7,7)/6 - 311g(2d ) for 1 and 3 and a -mr - aqcc / cas(4,7)/6 - 311g(2d ) for 2 . density of unpaired electrons for the ( a ) 1a1 state of phenalenyl ( 1 ) ( nu = 1.33 e ) ; ( b ) 1a2 state of triangulene ( 2 ) ( nu = 2.50 e ) , and ( c ) 1a2 state of -extended triangulene system ( 3 ) ( nu = 3.66 e ) using a -mr - aqcc / cas(7,7)/6 - 311g(2d ) for 1 and 3 and a -mr - aqcc / cas(4,7)/6 - 311g(2d ) calculation for ( 2 ) ( isovalue 0.005 e bohr ) with individual atomic population computed from mulliken analysis . it is noteworthy that the total unpaired density values , nu presented in figure 3 , are larger as compared to the formal open - shell occupation of the high - spin state ( 1.33 vs 1 for phenalenyl , 2.50 vs 2.0 for triangulene , and 3.66 vs 3.0 for -extended triangulene system ) . the pronounced bond length alternation of the central p - quinodimethane subunit of the structures 4 and 5 indicates that the quinoid resonance form ( chart 2 ) has a dominant contribution to the singlet ground state . triplet splitting is investigated next in order to continue the discussion of polyradical character of structures 48 . the observed relative insensitivity in terms of energies and character of the wave function facilitates the discussion of the polyradical character by means of multireference methods considerably . several of the no occupation numbers for the singlet state deviate strongly from the closed - shell limiting values of two / zero , indicating that they have substantial polyradical character . natural orbital ( no ) occupation of the 1ag ground state of heptazethrene ( 4 ) , 1,2:9,10-dibenzoheptazethrene ( 5 ) , 5,6:13,14-dibenzoheptazethrene ( 6 ) , p - quinodimethane - linked bisphenalenyl ( 7 ) , and clar goblet ( 8) obtained from -mr - aqcc / cas(4,4)/6 - 311g(2d ) calculations for structures 47 and -mr - aqcc / ras(1)/cas(4,4)/aux(1)/6 - 311g(2d ) calculation for structure 8 . even though most of the open - shell contributions computed at mr - aqcc level are coming from the hono / luno occupation , for all structures , irrespective of singlet or triplet states , there are additional nos , whose occupation numbers deviate significantly from the limiting value of two and zero ( figure 4 ) . density of unpaired electrons for the 1ag state of ( a ) heptazethrene ( 4 ) ( nu = 1.03 e ) , ( b ) 1,2:9,10-dibenzoheptazethrene ( 5 ) ( nu = 1.49 e ) , and ( c ) 5,6:13,14-dibenzoheptazethrene ( 6 ) ( nu = 2.24 e ) using the -mr - aqcc / cas(4,4)/6 - 311g(2d ) approach ( isovalue 0.003 e bohr ) with individual atomic population computed from mulliken analysis . density of unpaired electrons for the 1ag state of ( a ) p - quinodimethane - linked bisphenalenyl ( 7 ) ( nu = 1.86 e ) using the -mr - aqcc / cas(4,4)/6 - 311g(2d ) method and ( b ) clar goblet ( 8) ( nu = 2.88 e ) using the -mr - aqcc / ras(1)/cas(4,4)/aux(1)/6 - 311g(2d ) method ( isovalue 0.003 e bohr ) with individual atomic population computed from mulliken analysis . to obtain a common basis for comparison with the nu s , it is observed that for the singlet ground states of structures 4 and 5 the 2y0 values , which are computed from the hono / luno uhf occupations , are almost twice of the nu values computed from the mr - aqcc hono / luno occupations . comparison of the nu values computed from the non - hono / luno pairs for the singlet and the triplet states of structures 4 - 8 shows that they are almost identical . density of unpaired electrons for the 1bu state of ( a ) heptazethrene ( 4 ) ( nu = 2.59 e ) ; ( b ) 1,2:9,10-dibenzoheptazethrene ( 5 ) ( nu = 2.80 e ) , and ( c ) 5,6:13,14-dibenzoheptazethrene ( 6 ) ( nu = 2.75 e ) using the -mr - aqcc / cas(4,4)/6 - 311g(2d ) approach ( isovalue 0.003 e bohr ) with individual atomic population computed from mulliken analysis . in this work two different types of pahs have been studied ( a ) non - kekul triangular structures with a high - spin ground state and ( b ) pahs with singlet polyradical character . for the first case , phenalenyl ( 1 ) , triangulene ( 2 ) , and a -extended triangulene system ( 3 ) have been chosen . in the second case , a series of three zethrenes , heptazethrene ( 4 ) , 1,2:9,10-dibenzoheptazethrene ( 5 ) , 5,6:13,14-dibenzoheptazethrene ( 6 ) , and the p - quinodimethane - linked bisphenalenyl ( 7 ) have been investigated . the motivation in choosing these two types of pahs is that structures 13 already possess open - shell character because of their high - spin ground state , whereas for structures 47 the competition between a closed - shell quinoid kekul valence bond structure and an open - shell singlet biradical resonance form determines the actual electronic structure and the chemical reactivity . for structure 8 , the topology of the -electron arrangement of the non - kekul form is the characteristic feature . unrestricted density functional theory and hartree fock calculations have been performed for structures 48 also in order to assess their applicability to these molecular systems possessing a complicated electronic structure . for the second class of systems ( structures 48 ) , the ground state is always singlet with a varying amount of biradicaloid ( structures 4 , 5 ) , biradical ( structure 7 ) , or polyradical ( structures 6 and 8) character . structure 7 also has open - shell singlet biradical character in its ground state . interpretation of these results within the valence bond picture confirms that clar s aromatic sextet rule can be successfully applied for the ground state of these types of systems , but for a concrete characterization of the chemical reactivity high level quantum chemical calculations are needed . however , no occupations derived from uhf calculations in the spirit of the uno - cas method show a strong overshooting of the deviations from closed - shell character for most of the singlet systems investigated and , as a consequence , also an overestimation of the polyradical character as measured by the y0 and y1 indices as compared to total numbers of unpaired electrons computed by the mr - aqcc method . analysis of our mr - aqcc results and also those of previous ones preformed on the singlet triplet splitting in polyacenes shows only a minor influence of basis set effects and of the amount of correlating orbitals . in spite of the complicated structure of the multireference wave functions , the chemical analysis of the polyradical character is straightforward on the basis of the unpaired densities .

a group of 14 university hospitals participated in this study and 72 patients were recruited between 1992 and 2006 . individuals with confirmed hnf1a - mutated hca who underwent curative resection of the tumor(s ) were eligible for inclusion in this study . the mean age of the patients was 37.5 years ( ranging from 14 to 66 years ) , and there were only seven male patients . among the women studied , 71% ( 34 of 48 ) had a history of oral contraception use , and 17 patients were missing this data . there were 89 hnf1a - mutated hca samples from 72 patients , although 44 samples had been reported previously ( 8) . in 10 patients with multiple and sporadic tumors , different somatic hnf1a mutations were observed in the genotyped nodules . for all patients , a representative part of the hca nodule , as well as of the nontumor liver when it was available , was immediately frozen in liquid nitrogen and stored at 80c until used for molecular studies . the ethical committee of the saint - louis hospital ( paris , france ) approved the overall design of the study . in all 89 tumors , we sequenced the hnf1a gene using direct sequencing of the exons on genomic dna or rt - pcr to identify mutation , as previously described ( 5 ) . all identified mutations ( tables 1 and 2 ) were confirmed by two independent pcr amplifications of genomic dna from tumor , and corresponding nontumor tissues were systematically sequenced to search for germline mutations . in samples with mutations affecting splicing sites , we characterized the transcripts by sequence analysis of the mrna transcripts . in 37 h - hca , the coding exons of myh ( exons 116 ) and ogg1 ( exons 17 ) were screened for mutations by direct sequencing of pcr products . oligonucleotides used for all pcr reactions and experimental conditions are listed in supplementary tables 1 and 2 available in an online appendix at http://care.diabetesjournals.org/cgi/content/full/db09-1819/dc1 . allelic frequency of an ogg1 polymorphism observed in h - hca was compared with those obtained in a control group of non - hnf1a - mutated hepatocellular tumors ( 58 samples ) as previously described ( 13 ) and presented in supplementary table 3 ( available in an online appendix ) . biallelic hnf1 mutations identified in hca cases of hca were previously described in * bluteau et al . mody3 mutations were previously described in ellard and colclough ( 25 ) or bellann - chantelot et al . del , deletion ; fs , frameshift ; ivs , intervening sequence ; loh , loss of heterozygosity ; ins , insertion ; nm , nonmutated . germline hnf1 mutations in monoallelic mutated hca cases of hca were previously described in * bluteau et al . mody3 mutations were previously described in ellard and colclough ( 25 ) or bellann - chantelot et al . total rna was extracted using the rneasy kit ( qiagen , valencia , ca ) from all frozen samples of available hca and nontumor liver tissues . rna isolated from 16 h - hca and 24 nontumor tissue samples was deemed to be of acceptable quality for quantitative rt - pcr experiments ( 23 ) . specific assays for myh ( hs01014866_g1 ) , ogg1 ( hs00213454m1 ) , ape1 ( hs00172396_m1 ) , pol ( hs00160263_m1 ) , fabp1 ( hs00155026_m1 ) , ugt2b7 ( hs00426592_m1 ) , and r18s ( rrna , hs99999901_s1 ) were obtained from applied biosystems ( foster city , ca ) . the relative amount of target gene mrnas was determined using the 2 method ( 24 ) . the values obtained for fabp1 and ugt2b7 were expressed as the n - fold ratio of the gene expression in a tested sample as compared with the mean of 11 nontumor tissues . only six nontumor tissues from patients with a h - hca were used with the myh , ogg1 , ape1 , and pol assays . the values obtained were compared with the mean of seven nontumor tissues from patients with a non - hnf1a mutated hca . western blot analyses were performed as described ( 9 ) using two primary goat polyclonal anti - hnf1 antibodies ( santa cruz biotechnology ) , one detecting the amino terminus , and the other detecting the carboxy terminus of the protein ( sc-6548 and sc-6547 , respectively ) , used at the dilution 1:500 . after centrifugation at 8,000 g for 10 min , the supernatant was assessed for reduced and total glutathione content with an apogsh glutathione colorimetric assay kit ( biovision , mountain view , ca ) , following the manufacturer 's protocol . formalin - fixed , paraffin - embedded sections ( 5 m ) were mounted on glass slides . sections were deparaffinized in xylene , rehydrated in a series of graded alcohol concentrations , and placed in pbs with 0.1% tween 20 . immunostaining was performed using a dako envision system horseradish peroxidase ( dako cytomation , carpinteria , ca ) kit using primary antibody ( 1:2,000 dilution in pbs containing 1% bsa , incubated overnight at 4c ) against 4-hydroxynonenal protein adducts ( alpha diagnostics , san antonio , tx ) . statistical analysis was performed using graphpad prism software ( version 4 , graphpad software , san diego , ca ) . allelic frequencies of the ogg1 polymorphism in h - hca and the group control were compared using contingency tables with a fisher exact test . in all 89 tumors , we sequenced the hnf1a gene using direct sequencing of the exons on genomic dna or rt - pcr to identify mutation , as previously described ( 5 ) . all identified mutations ( tables 1 and 2 ) were confirmed by two independent pcr amplifications of genomic dna from tumor , and corresponding nontumor tissues were systematically sequenced to search for germline mutations . in samples with mutations affecting splicing sites , we characterized the transcripts by sequence analysis of the mrna transcripts . in 37 h - hca , the coding exons of myh ( exons 116 ) and ogg1 ( exons 17 ) were screened for mutations by direct sequencing of pcr products . oligonucleotides used for all pcr reactions and experimental conditions are listed in supplementary tables 1 and 2 available in an online appendix at http://care.diabetesjournals.org/cgi/content/full/db09-1819/dc1 . allelic frequency of an ogg1 polymorphism observed in h - hca was compared with those obtained in a control group of non - hnf1a - mutated hepatocellular tumors ( 58 samples ) as previously described ( 13 ) and presented in supplementary table 3 ( available in an online appendix ) . biallelic hnf1 mutations identified in hca cases of hca were previously described in * bluteau et al . mody3 mutations were previously described in ellard and colclough ( 25 ) or bellann - chantelot et al . del , deletion ; fs , frameshift ; ivs , intervening sequence ; loh , loss of heterozygosity ; ins , insertion ; nm , nonmutated . germline hnf1 mutations in monoallelic mutated hca cases of hca were previously described in * bluteau et al . mody3 mutations were previously described in ellard and colclough ( 25 ) or bellann - chantelot et al . total rna was extracted using the rneasy kit ( qiagen , valencia , ca ) from all frozen samples of available hca and nontumor liver tissues . rna isolated from 16 h - hca and 24 nontumor tissue samples was deemed to be of acceptable quality for quantitative rt - pcr experiments ( 23 ) . specific assays for myh ( hs01014866_g1 ) , ogg1 ( hs00213454m1 ) , ape1 ( hs00172396_m1 ) , pol ( hs00160263_m1 ) , fabp1 ( hs00155026_m1 ) , ugt2b7 ( hs00426592_m1 ) , and r18s ( rrna , hs99999901_s1 ) were obtained from applied biosystems ( foster city , ca ) . the relative amount of target gene mrnas was determined using the 2 method ( 24 ) . the values obtained for fabp1 and ugt2b7 were expressed as the n - fold ratio of the gene expression in a tested sample as compared with the mean of 11 nontumor tissues . only six nontumor tissues from patients with a h - hca were used with the myh , ogg1 , ape1 , and pol assays . the values obtained were compared with the mean of seven nontumor tissues from patients with a non - hnf1a mutated hca . western blot analyses were performed as described ( 9 ) using two primary goat polyclonal anti - hnf1 antibodies ( santa cruz biotechnology ) , one detecting the amino terminus , and the other detecting the carboxy terminus of the protein ( sc-6548 and sc-6547 , respectively ) , used at the dilution 1:500 . after centrifugation at 8,000 g for 10 min , the supernatant was assessed for reduced and total glutathione content with an apogsh glutathione colorimetric assay kit ( biovision , mountain view , ca ) , following the manufacturer 's protocol . formalin - fixed , paraffin - embedded sections ( 5 m ) were mounted on glass slides . sections were deparaffinized in xylene , rehydrated in a series of graded alcohol concentrations , and placed in pbs with 0.1% tween 20 . immunostaining was performed using a dako envision system horseradish peroxidase ( dako cytomation , carpinteria , ca ) kit using primary antibody ( 1:2,000 dilution in pbs containing 1% bsa , incubated overnight at 4c ) against 4-hydroxynonenal protein adducts ( alpha diagnostics , san antonio , tx ) . statistical analysis was performed using graphpad prism software ( version 4 , graphpad software , san diego , ca ) . allelic frequencies of the ogg1 polymorphism in h - hca and the group control were compared using contingency tables with a fisher exact test . among 89 hca samples with hnf1a alteration , mutations and deletion were biallelic in 84 ( 94% ) samples . to further analyze the spectrum of hnf1a mutations , we included 151 hnf1a alterations with a firm somatic origin identified in 75 different hcas ; in contrast , all samples with a proven or highly suspected germline mutation were excluded from this first analysis ( fig . . a : spectrum of 136 somatic hnf1a mutations observed in 75 hca samples ( top ) and in 364 mody3 individuals ( bottom ) ( data from ellard and colclough ) . , in - frame deletion ; , in - frame duplication ; , mutation in splicing site . hca ( ) and mody3 ( ) histograms represent the percentage of total mutations observed in each domain ; substitutions and stops are represented in the upper part and the lower part , respectively . significant differences between hca and mody3 individuals are indicated : * 0.05 > p > 0.01 ; * * 0.01 > p > 0.001 ; * * * p < 0.001 . c : comparison of the number of stops in the transactivation domain ( 281631 ) vs. the rest of the protein observed in individuals with hca ( ) and mody3 ( ) . * * * significant difference ( p < 0.001 ) between the two populations . d : spectrum of eight germline hnf1a mutations identified in h - hca ( top ) and five germline hnf1a mutations identified in monoallelic mutated hca ( bottom ) . somatic alterations , 90% were point mutations , and the remaining 10% ( 15 of 151 ) were gene deletions as evidenced by the loss of heterozygosity at a hnf1a single nucleotide polymorphism(s ) compared with the corresponding nontumor tissues . among the mutations , 57 were missense , 62 were frameshift and nonsense , and 12 were mutations affecting a splicing site . next , we compared the spectrum of somatic hnf1a mutations identified in hca with 364 germline mutations previously described in individuals with mody3 without hca ( fig . 1a , lower panel , supplementary table 4 , available in an online appendix ) and by ellard and colclough ( 25 ) . in both hca and mody3 , a hotspot mutation at codon 291 was present , whereas a hotspot mutation at codon 206 was specifically identified in hca ( fig . as previously reported , codon 291 mutation is located in a polyc-8 tract , whereas codon 206 mutation is not located in a repeated sequence motif . because codon 291 mutations represented 20% of the mutations in both spectra , we did not include them in the following analysis . there was no significant difference ( p = 0.16 ) in the proportion of missense and truncating ( frameshift , nonsense , and mutations affecting a splicing site ) mutations in mody3 and hca spectra . thus , there were 63 and 55% of missense mutations in mody3 and hca , respectively , and 37 and 45% of truncating mutations in mody3 and hca , respectively . in contrast , the distribution of the mutations was significantly different in hca compared with the mody3 spectrum . although almost all of the missense mutations were restricted to the pou - h domain ( p < 0.001 ) in hca , they were distributed in all hnf1 domains in individuals with mody3 ( fig . also , in hca , truncating mutations were localized predominantly in the first 280 amino acids ( p < 0.001 ) , whereas they were distributed equally between the first 280 amino acids and the transactivation domain in the individuals with mody3 ( fig . 1c ) . in a second step , we analyzed the distribution of the 13 germline hnf1a mutations identified in patients with adenomas . in eight patients , we identified a mutation of the second hnf1a allele in the corresponding tumor leading to a biallelic inactivation of hnf1 ( table 1 ) . the corresponding germline mutations fit the distribution of the somatic hnf1a mutations : six of them were nonsense or frameshift occurring before codon 292 , and the remaining two mutations were missense affecting codon 272 , an amino acid found to be mutated by a somatic event ( fig . in contrast , in four hca samples [ 372 ( r583q ) , 769 ( t525s ) , 966 ( l389v ) , and 998 ( r278q ) ] , we observed a germline hnf1a mutation without alteration of the second hnf1a allele . all of these germline mutations were missense ; three of them localized in the transactivation domain , and the last one was at the end of the pou - h domain . none of these mutations were described in the spectrum of somatic hnf1a mutations ; however , three of them ( r583q , t525s , and l389v ) have been previously reported in individuals with mody3 ( 21,25 ) . thus , these four germline mutations did not fit the characteristic distribution of the somatic one . moreover , the corresponding adenomas were classified in different molecular subgroups of hca : one of them was -catenin activated ( case 372 ) , two were inflammatory adenomas with or without a gp130 activating mutation ( cases 769 and 966 ) , and the last one remained unclassified ( case 998 ) . most of the somatic mutations found in hca are predicted to inactivate the protein because they are almost all biallelic and frequently nonsense or missense affecting the pou - h domain that is essential for dna binding . all of these mutations resulted in aberrant splicing , the outcome of which is either an aberrant protein with an early stop ( fig . 2a , b , and d ) , or a protein with an amino acid replacement and a deletion of five amino acids in the dna - binding domain ( fig . then we assessed the protein level of mutant hnf1 in hca and compared it with that in nontumor tissue . samples with mutations leading to an early stop contained little detectable hnf1 protein ( supplementary fig . 1 , available in an online appendix ; samples identified with arrows and data on gradient migration , not shown ) . additionally , some samples harboring missense mutations exhibited a dramatic increase in the expression of hnf1 compared with normal liver ( supplementary fig . consequences of the mutations at different splice sites in hnf1a . a : ivs12 a > t or ivs11 g > t mutations . b : ivs2 + 1 g > a or ivs2 + 1 del 13 bp mutations . the hnf1 transcription factor regulates expression of several key liver genes , including ugt2b7 and fabp1 ( 16,18 ) . indeed , a shutdown of mrna expression for these target genes was observed in all patients with hcas tested with biallelic hnf1a mutations , as previously reported ( 4 ) . an effect of monoallelic hnf1 mutants on expression of ugt2b7 and fabp1 has not been previously examined in liver tissues . our data show that monoallelic mutations did not affect expression of fabp1 and ugt2b7 , which is not in favor of a dominant - negative effect ( fig . however , these results can not completely exclude a subtle dosage effect or a low dominant effect . in contrast to other solid tumors , point mutations were much more frequent in hca than were chromosome deletions . the latter were found in only 10% of the patients with hcas . among the point mutations in hca , we analyzed the proportion of different hnf1a nucleotide changes and found that g - to - t transversions , accounting for 36% of the cases , were the most common type ( fig . 4a ) . moreover , the distribution of nucleotide substitution subtypes in h - hca was strikingly different from that in mody3 ( fig . 4b ) but very similar to a spectrum of mutations known to be induced by genotoxic events , that is , in tp53-mutated lung cancers in smokers ( fig . 4c ) ( 26 ) . in addition , when the hnf1a nucleotide substitutions were partitioned between the two dna strands , we observed that g - to - t transversions were significantly more frequent on a nontranscribed strand ( p = 0.01 ) ( fig . 4d ) . taking these results together , we suggest that hnf1a somatic mutations showed a typical spectrum of mutations induced by the genotoxic exposure targeting a specific nucleotide sequence . comparison of the mutation profiles in h - hca ( 67 transversions and transitions , a ) , mody3 ( 229 transversions and transitions , b ) , and tp53-mutated lung cancers ( 311 transversions and transitions , c ) and smokers ( 26 ) ( d ) . repartitioning of transition and transversion mutations between the transcribed and the nontranscribed strands in h - hca . black and white histograms indicate the number of nucleotide substitutions on the nontranscribed and the transcribed strand , respectively . * significant ( p = 0.01 ) elevation in g - to - t transversions on the nontranscribed strand . because both direct dna damage by an environmental agent and oxidative stress are known to result in g - to - t transversion mutations ( 27,28 ) , we also assessed oxidative stress markers in nontumor liver from six patients with h - hca and seven patients with non - hnf1a mutated hca ( supplementary fig . we observed no difference in total or reduced glutathione content , expression of four base excision dna repair genes , markers of oxidative dna damage ( 29 ) , or 4-hydroxynonenal protein adducts , a marker of lipid peroxidation ( supplementary fig . these data showed a lack of evidence for elevated oxidative stress in livers of patients with h - hca as a potential source of dna mutations . in addition , it is possible that a large number of g - to - t transversions could be caused by inefficient repair of dna damage due to mutations or polymorphisms in myh and ogg1 , genes involved in the repair of 8-hydroxyguanine ( 3032 ) . thus , we sequenced myh and ogg1 dna in 37 subjects with an hnf1a - mutated hca , and no mutations were detected in either gene . a known functional polymorphism in ogg1 ( s326c ) was detected in hnf1a - mutated hca subjects , albeit there was no difference in the frequency of this polymorphism compared with that in control subjects ( supplementary table 3 , available in an online appendix ) . among 89 hca samples with hnf1a alteration , mutations and deletion were biallelic in 84 ( 94% ) samples . to further analyze the spectrum of hnf1a mutations , we included 151 hnf1a alterations with a firm somatic origin identified in 75 different hcas ; in contrast , all samples with a proven or highly suspected germline mutation were excluded from this first analysis ( fig . . a : spectrum of 136 somatic hnf1a mutations observed in 75 hca samples ( top ) and in 364 mody3 individuals ( bottom ) ( data from ellard and colclough ) . , in - frame deletion ; , in - frame duplication ; , mutation in splicing site . hca ( ) and mody3 ( ) histograms represent the percentage of total mutations observed in each domain ; substitutions and stops are represented in the upper part and the lower part , respectively . significant differences between hca and mody3 individuals are indicated : * 0.05 > p > 0.01 ; * * 0.01 > p > 0.001 ; * * * p < 0.001 . c : comparison of the number of stops in the transactivation domain ( 281631 ) vs. the rest of the protein observed in individuals with hca ( ) and mody3 ( ) . * * * significant difference ( p < 0.001 ) between the two populations . d : spectrum of eight germline hnf1a mutations identified in h - hca ( top ) and five germline hnf1a mutations identified in monoallelic mutated hca ( bottom ) . somatic alterations , 90% were point mutations , and the remaining 10% ( 15 of 151 ) were gene deletions as evidenced by the loss of heterozygosity at a hnf1a single nucleotide polymorphism(s ) compared with the corresponding nontumor tissues . among the mutations , 57 were missense , 62 were frameshift and nonsense , and 12 were mutations affecting a splicing site . next , we compared the spectrum of somatic hnf1a mutations identified in hca with 364 germline mutations previously described in individuals with mody3 without hca ( fig . 1a , lower panel , supplementary table 4 , available in an online appendix ) and by ellard and colclough ( 25 ) . in both hca and mody3 , a hotspot mutation at codon 291 was present , whereas a hotspot mutation at codon 206 was specifically identified in hca ( fig . as previously reported , codon 291 mutation is located in a polyc-8 tract , whereas codon 206 mutation is not located in a repeated sequence motif . because codon 291 mutations represented 20% of the mutations in both spectra , we did not include them in the following analysis . there was no significant difference ( p = 0.16 ) in the proportion of missense and truncating ( frameshift , nonsense , and mutations affecting a splicing site ) mutations in mody3 and hca spectra . thus , there were 63 and 55% of missense mutations in mody3 and hca , respectively , and 37 and 45% of truncating mutations in mody3 and hca , respectively . in contrast , the distribution of the mutations was significantly different in hca compared with the mody3 spectrum . although almost all of the missense mutations were restricted to the pou - h domain ( p < 0.001 ) in hca , they were distributed in all hnf1 domains in individuals with mody3 ( fig . also , in hca , truncating mutations were localized predominantly in the first 280 amino acids ( p < 0.001 ) , whereas they were distributed equally between the first 280 amino acids and the transactivation domain in the individuals with mody3 ( fig . in a second step , we analyzed the distribution of the 13 germline hnf1a mutations identified in patients with adenomas . in eight patients , we identified a mutation of the second hnf1a allele in the corresponding tumor leading to a biallelic inactivation of hnf1 ( table 1 ) . the corresponding germline mutations fit the distribution of the somatic hnf1a mutations : six of them were nonsense or frameshift occurring before codon 292 , and the remaining two mutations were missense affecting codon 272 , an amino acid found to be mutated by a somatic event ( fig . in contrast , in four hca samples [ 372 ( r583q ) , 769 ( t525s ) , 966 ( l389v ) , and 998 ( r278q ) ] , we observed a germline hnf1a mutation without alteration of the second hnf1a allele . all of these germline mutations were missense ; three of them localized in the transactivation domain , and the last one was at the end of the pou - h domain . none of these mutations were described in the spectrum of somatic hnf1a mutations ; however , three of them ( r583q , t525s , and l389v ) have been previously reported in individuals with mody3 ( 21,25 ) . thus , these four germline mutations did not fit the characteristic distribution of the somatic one . moreover , the corresponding adenomas were classified in different molecular subgroups of hca : one of them was -catenin activated ( case 372 ) , two were inflammatory adenomas with or without a gp130 activating mutation ( cases 769 and 966 ) , and the last one remained unclassified ( case 998 ) . most of the somatic mutations found in hca are predicted to inactivate the protein because they are almost all biallelic and frequently nonsense or missense affecting the pou - h domain that is essential for dna binding . all of these mutations resulted in aberrant splicing , the outcome of which is either an aberrant protein with an early stop ( fig . 2a , b , and d ) , or a protein with an amino acid replacement and a deletion of five amino acids in the dna - binding domain ( fig . then we assessed the protein level of mutant hnf1 in hca and compared it with that in nontumor tissue . samples with mutations leading to an early stop contained little detectable hnf1 protein ( supplementary fig . 1 , available in an online appendix ; samples identified with arrows and data on gradient migration , not shown ) . additionally , some samples harboring missense mutations exhibited a dramatic increase in the expression of hnf1 compared with normal liver ( supplementary fig . consequences of the mutations at different splice sites in hnf1a . a : ivs12 a > t or ivs11 g > t mutations . b : ivs2 + 1 g > a or ivs2 + 1 del 13 bp mutations . the hnf1 transcription factor regulates expression of several key liver genes , including ugt2b7 and fabp1 ( 16,18 ) . indeed , a shutdown of mrna expression for these target genes was observed in all patients with hcas tested with biallelic hnf1a mutations , as previously reported ( 4 ) . an effect of monoallelic hnf1 mutants on expression of ugt2b7 and fabp1 has not been previously examined in liver tissues . our data show that monoallelic mutations did not affect expression of fabp1 and ugt2b7 , which is not in favor of a dominant - negative effect ( fig . , these results can not completely exclude a subtle dosage effect or a low dominant effect . in contrast to other solid tumors , point mutations were much more frequent in hca than were chromosome deletions . the latter were found in only 10% of the patients with hcas . among the point mutations in hca , we analyzed the proportion of different hnf1a nucleotide changes and found that g - to - t transversions , accounting for 36% of the cases , were the most common type ( fig . 4a ) . moreover , the distribution of nucleotide substitution subtypes in h - hca was strikingly different from that in mody3 ( fig . 4b ) but very similar to a spectrum of mutations known to be induced by genotoxic events , that is , in tp53-mutated lung cancers in smokers ( fig . 4c ) ( 26 ) . in addition , when the hnf1a nucleotide substitutions were partitioned between the two dna strands , we observed that g - to - t transversions were significantly more frequent on a nontranscribed strand ( p = 0.01 ) ( fig . 4d ) . taking these results together , we suggest that hnf1a somatic mutations showed a typical spectrum of mutations induced by the genotoxic exposure targeting a specific nucleotide sequence . comparison of the mutation profiles in h - hca ( 67 transversions and transitions , a ) , mody3 ( 229 transversions and transitions , b ) , and tp53-mutated lung cancers ( 311 transversions and transitions , c ) and smokers ( 26 ) ( d ) . repartitioning of transition and transversion mutations between the transcribed and the nontranscribed strands in h - hca . black and white histograms indicate the number of nucleotide substitutions on the nontranscribed and the transcribed strand , respectively . * significant ( p = 0.01 ) elevation in g - to - t transversions on the nontranscribed strand . because both direct dna damage by an environmental agent and oxidative stress are known to result in g - to - t transversion mutations ( 27,28 ) , we also assessed oxidative stress markers in nontumor liver from six patients with h - hca and seven patients with non - hnf1a mutated hca ( supplementary fig . 2 , available in an online appendix ) . we observed no difference in total or reduced glutathione content , expression of four base excision dna repair genes , markers of oxidative dna damage ( 29 ) , or 4-hydroxynonenal protein adducts , a marker of lipid peroxidation ( supplementary fig . these data showed a lack of evidence for elevated oxidative stress in livers of patients with h - hca as a potential source of dna mutations . in addition , it is possible that a large number of g - to - t transversions could be caused by inefficient repair of dna damage due to mutations or polymorphisms in myh and ogg1 , genes involved in the repair of 8-hydroxyguanine ( 3032 ) . thus , we sequenced myh and ogg1 dna in 37 subjects with an hnf1a - mutated hca , and no mutations were detected in either gene . a known functional polymorphism in ogg1 ( s326c ) was detected in hnf1a - mutated hca subjects , albeit there was no difference in the frequency of this polymorphism compared with that in control subjects ( supplementary table 3 , available in an online appendix ) . in this study we analyzed the spectrum of hnf1a mutations in hca and showed a significant difference in pattern in comparison with individuals with mody3 , both at the nucleotide and amino acid levels . in hca , location of the mutations is very restricted because almost all of the truncating mutations led to the loss of the transactivation domain and the missense mutations altered mainly the pou - h domain . when we take into account the two largest series of hnf1a screening in mody3 ( 21,25 ) , only 48% of the germline mutations ( 117 of 720 are missense mutations in pou - h , and 227 of 720 are truncating mutations localized in the first 291 amino acids ) fit the features of the hnf1a somatic mutations . this observation suggests that only a part of hnf1a mutations that are associated with diabetes could predispose to the development of a h - hca . previous analysis of the mody3 mutations showed that the age at onset of diabetes is modulated according to the position of the mutation relative to the hnf1a isoforms : missense mutations located in exons 7 to 10 that affect only a or b isoforms of hnf1a are associated with the late onset of diabetes ( 21,22 ) . interestingly , these mutations are predicted to have a less severe effect on the hnf1 function and are not found in hca . similarly , we can hypothesize that frequent mody3 missense mutations located outside the pou - h domain and mutations truncating hnf1 after codon 291 are possibly less inactivating than the mutations found in hca . in contrast to mody3 , in hca the lack of mutations leading to amino acid substitution in pou - s suggests that the pou - s and pou - h domains are functionally different . chi et al . ( 20 ) found that the pou - s domain interacts with the pou - h domain in the recognition and binding on promoter sequences ; however , the pou - s domain is not conserved in homeobox transcription factors outside the pou family . thus , based on the profile of the hnf1a mutations that occur in hca , it may be of interest to reanalyze clinical severity of the diabetes and putative associated phenotypes in patients with mody3 , taking into the account only a premature stop before codon 291 , missense restricted at pou - h , and mutations affecting splicing sites from exon 1 to 7 . interestingly , among the five hcas with a monoallelic germline hnf1a mutation , three of them were inflammatory . we previously noted that inflammatory adenomas are associated with obesity ( 4 ) , and the present observation raised the question of an association with mody3 and possibly with other subtypes of adenomas . we can hypothesize that according to the nature of their hnf1a germline mutation , patients with mody3 could be at risk of different subtypes of hca : mutations leading to a severe impaired hnf1 function predispose to the development of h - hca , whereas germline mutation with a mild functional consequence could predispose to inflammatory or - catenin activated hcas without a familial context . in consideration of the high risk of malignant transformation of the -catenin - activated hca , this observation could have important clinical consequences . our analysis of the expression of mutant hnf1 proteins in liver tissue did not reveal a detectable level of truncated proteins , and most of the mutated proteins were not expressed . moreover , mutations of both hnf1a alleles are required to observe a downregulation of the genes physiologically regulated by hnf1. these observations suggest that if a dominant - negative effect exists , as suggested by the in vitro analyses for particular hnf1a mutants ( 15 ) , it is not sufficient to shut down activity of hnf1 in vivo . accordingly , other researchers ( 33,34 ) , including harries et al . ( 35 ) showed that hnf1a transcripts harboring a premature termination codon ( ptc ) were less expressed in lymphoblastoid cells than the normal allele resulting from a nonsense - mediated mrna decay , which detects and degrades the transcripts with a ptc to prevent the synthesis of truncated proteins . in the present study , we observed a decrease of mrna expression for 16 of 21 different mutants leading to ptc ( data not shown ) . altogether , in hepatocytes , a dominant - negative effect of an hnf1 mutant is unlikely in vivo . this study not only confirms , in a large series of adenomas , that the spectrum of hnf1a mutations in hca is different from that in individuals with mody3 , it also supports the hypothesis that hnf1a mutations in hca arise because of a genotoxic mechanism . first , we observed a consistent hotspot mutation at the codon 206 of hnf1a in hca . hotspots of transversion identified in tumors without a repeated nucleotide sequence context were previously suggested to be a hallmark of an exposure to a genotoxic compound , such as an r249s mutation in the tp53 gene in hepatocellular carcinoma ( hcc ) associated with an exposure to the aflatoxin b1 mycotoxin ( 36,37 ) . second , hainaut and pfeifer ( 26 ) showed that there is a significant difference in the pattern of tp53 mutations in lung cancers between smokers and nonsmokers , with a 30% frequency of g - to - t transversions in smoking - related lung cancer vs. 10% in non - smoking related lung cancer . the overabundance of g - to - t transversions in smoking - related lung cancers was consistent with the studies of dna damage and mutations due to benzo[a]pyrene and other cigarette smoke - derived carcinogens and their metabolites ( 38,39 ) . our observation of an uneven distribution among hnf1a nucleotide substitutions in hca and high frequency of g - to - t transversions is in many ways similar to that in lung cancer caused by tobacco ( fig . 4a ) . third , denissenko et al . ( 40 ) showed that the repair of benzo[a]pyrene diol epoxide adducts in the nontranscribed strand was slower than that in the transcribed strand and may explain the strand bias of transversions in cancer . similarly , we observed that g - to - t transversions were significantly localized to the nontranscribed strand in h - hca , an additional argument in favor of a genotoxic effect and , possibly , a presence of bulky dna adducts . we can hypothesize that it could be related to a particular genetic susceptibility and/or to an exposure to a specific toxic . according to the first hypothesis , we searched for mutations in ogg1 and myh , both genes specifically involved in the repair of 8-hydroxyguanine , a product of oxidative dna damage that can also lead to g - to - t transversions ( 3032 ) . in addition , we did not observe a higher level of oxidative stress in the livers of patients with a h - hca . these results strongly suggest that exposure to an environmental agent appears to be the most likely event to explain the elevated rate of g - to - t transversions in h - hca . because hca occurrence is highly associated with oral contraception , we can hypothesize that estrogen exposure could play a role in this mechanism . this last hypothesis is supported by the facts that 1 ) the genotoxic activity of estrogen metabolites has been shown , particularly for catechol estrogens that may be oxidized to a reactive quinone capable of direct formation of dna adducts ( 41,42 ) ; and 2 ) germline heterozygous mutations of cyp1b1 , a key metabolism enzyme responsible for the formation of hydroxylated and genotoxic metabolites of estrogen , contribute to the development of h - hca in women using hormonal contraception ( 13 ) . in conclusion , when analyzing a large series of individuals with hcas , we observed a significant differences between hnf1a somatic mutations identified in those with hcas and the germline mutations identified in individuals with mody3 . somatic mutations in hca predict a more inactivating profile , resulting in a complete loss of function of the protein when biallelic . moreover , we showed that the origin of the mutations might be the result of dna damage caused by a genotoxic agent , possibly resulting from the metabolism of estrogen that could also be associated with a genetic susceptibility that remains to be identified .

objectivematurity onset diabetes of the young type 3 ( mody3 ) is a consequence of heterozygous germline mutation in hnf1a . a subtype of hepatocellular adenoma ( hca ) is also caused by biallelic somatic hnf1a mutations ( h - hca ) , and rare hca may be related to mody3 . to better understand a relationship between the development of mody3 and hca , we compared both germline and somatic spectra of hnf1a mutations.research design and methodswe compared 151 somatic hnf1a mutations in hca with 364 germline mutations described in mody3 . we searched for genotoxic and oxidative stress features in hca and surrounding liver tissue.resultsa spectrum of hnf1a somatic mutations significantly differed from the germline changes in mody3 . in hca , we identified a specific hot spot at codon 206 , nonsense and frameshift mutations mainly in the nh2-terminal part , and almost all amino acid substitutions were restricted to the pou - h domain . the high frequency of g - to - t tranversions , predominantly found on the nontranscribed dna strand , suggested a genotoxic mechanism . however , no features of oxidative stress were observed in the nontumor liver tissue . finally , in a few mody3 patients with hnf1a germline mutation leading to amino acid substitutions outside the pou - h domain , we identified a different subtype of hca either with a gp130 and/or ctnnb1 activating mutation.conclusionsgermline hnf1a mutations could be associated with different molecular subtypes of hca . h - hca showed mutations profoundly inactivating hepatocyte nuclear factor-1 function ; they are associated with a genotoxic signature suggesting a specific toxicant exposure that could be associated with genetic predisposition .

RESEARCH DESIGN AND METHODS Mutation screening. Quantitative RT-PCR. Western blotting. Determination of reduced and total glutathione levels. Immunohistochemistry. Statistical analysis. RESULTS Spectrum of Germline Functional consequences of HNF1A mutations in HCA are likely to arise due to a genotoxic event. HNF1A spectrum of mutation is not related to measurable increase in oxidative stress in liver. DISCUSSION Supplementary Material

in 10 patients with multiple and sporadic tumors , different somatic hnf1a mutations were observed in the genotyped nodules . allelic frequency of an ogg1 polymorphism observed in h - hca was compared with those obtained in a control group of non - hnf1a - mutated hepatocellular tumors ( 58 samples ) as previously described ( 13 ) and presented in supplementary table 3 ( available in an online appendix ) . only six nontumor tissues from patients with a h - hca were used with the myh , ogg1 , ape1 , and pol assays . allelic frequencies of the ogg1 polymorphism in h - hca and the group control were compared using contingency tables with a fisher exact test . allelic frequency of an ogg1 polymorphism observed in h - hca was compared with those obtained in a control group of non - hnf1a - mutated hepatocellular tumors ( 58 samples ) as previously described ( 13 ) and presented in supplementary table 3 ( available in an online appendix ) . only six nontumor tissues from patients with a h - hca were used with the myh , ogg1 , ape1 , and pol assays . allelic frequencies of the ogg1 polymorphism in h - hca and the group control were compared using contingency tables with a fisher exact test . to further analyze the spectrum of hnf1a mutations , we included 151 hnf1a alterations with a firm somatic origin identified in 75 different hcas ; in contrast , all samples with a proven or highly suspected germline mutation were excluded from this first analysis ( fig . a : spectrum of 136 somatic hnf1a mutations observed in 75 hca samples ( top ) and in 364 mody3 individuals ( bottom ) ( data from ellard and colclough ) . d : spectrum of eight germline hnf1a mutations identified in h - hca ( top ) and five germline hnf1a mutations identified in monoallelic mutated hca ( bottom ) . next , we compared the spectrum of somatic hnf1a mutations identified in hca with 364 germline mutations previously described in individuals with mody3 without hca ( fig . because codon 291 mutations represented 20% of the mutations in both spectra , we did not include them in the following analysis . there was no significant difference ( p = 0.16 ) in the proportion of missense and truncating ( frameshift , nonsense , and mutations affecting a splicing site ) mutations in mody3 and hca spectra . thus , there were 63 and 55% of missense mutations in mody3 and hca , respectively , and 37 and 45% of truncating mutations in mody3 and hca , respectively . although almost all of the missense mutations were restricted to the pou - h domain ( p < 0.001 ) in hca , they were distributed in all hnf1 domains in individuals with mody3 ( fig . also , in hca , truncating mutations were localized predominantly in the first 280 amino acids ( p < 0.001 ) , whereas they were distributed equally between the first 280 amino acids and the transactivation domain in the individuals with mody3 ( fig . in a second step , we analyzed the distribution of the 13 germline hnf1a mutations identified in patients with adenomas . in eight patients , we identified a mutation of the second hnf1a allele in the corresponding tumor leading to a biallelic inactivation of hnf1 ( table 1 ) . the corresponding germline mutations fit the distribution of the somatic hnf1a mutations : six of them were nonsense or frameshift occurring before codon 292 , and the remaining two mutations were missense affecting codon 272 , an amino acid found to be mutated by a somatic event ( fig . in contrast , in four hca samples [ 372 ( r583q ) , 769 ( t525s ) , 966 ( l389v ) , and 998 ( r278q ) ] , we observed a germline hnf1a mutation without alteration of the second hnf1a allele . all of these germline mutations were missense ; three of them localized in the transactivation domain , and the last one was at the end of the pou - h domain . none of these mutations were described in the spectrum of somatic hnf1a mutations ; however , three of them ( r583q , t525s , and l389v ) have been previously reported in individuals with mody3 ( 21,25 ) . moreover , the corresponding adenomas were classified in different molecular subgroups of hca : one of them was -catenin activated ( case 372 ) , two were inflammatory adenomas with or without a gp130 activating mutation ( cases 769 and 966 ) , and the last one remained unclassified ( case 998 ) . most of the somatic mutations found in hca are predicted to inactivate the protein because they are almost all biallelic and frequently nonsense or missense affecting the pou - h domain that is essential for dna binding . 2a , b , and d ) , or a protein with an amino acid replacement and a deletion of five amino acids in the dna - binding domain ( fig . among the point mutations in hca , we analyzed the proportion of different hnf1a nucleotide changes and found that g - to - t transversions , accounting for 36% of the cases , were the most common type ( fig . in addition , when the hnf1a nucleotide substitutions were partitioned between the two dna strands , we observed that g - to - t transversions were significantly more frequent on a nontranscribed strand ( p = 0.01 ) ( fig . taking these results together , we suggest that hnf1a somatic mutations showed a typical spectrum of mutations induced by the genotoxic exposure targeting a specific nucleotide sequence . comparison of the mutation profiles in h - hca ( 67 transversions and transitions , a ) , mody3 ( 229 transversions and transitions , b ) , and tp53-mutated lung cancers ( 311 transversions and transitions , c ) and smokers ( 26 ) ( d ) . repartitioning of transition and transversion mutations between the transcribed and the nontranscribed strands in h - hca . * significant ( p = 0.01 ) elevation in g - to - t transversions on the nontranscribed strand . because both direct dna damage by an environmental agent and oxidative stress are known to result in g - to - t transversion mutations ( 27,28 ) , we also assessed oxidative stress markers in nontumor liver from six patients with h - hca and seven patients with non - hnf1a mutated hca ( supplementary fig . in addition , it is possible that a large number of g - to - t transversions could be caused by inefficient repair of dna damage due to mutations or polymorphisms in myh and ogg1 , genes involved in the repair of 8-hydroxyguanine ( 3032 ) . to further analyze the spectrum of hnf1a mutations , we included 151 hnf1a alterations with a firm somatic origin identified in 75 different hcas ; in contrast , all samples with a proven or highly suspected germline mutation were excluded from this first analysis ( fig . a : spectrum of 136 somatic hnf1a mutations observed in 75 hca samples ( top ) and in 364 mody3 individuals ( bottom ) ( data from ellard and colclough ) . d : spectrum of eight germline hnf1a mutations identified in h - hca ( top ) and five germline hnf1a mutations identified in monoallelic mutated hca ( bottom ) . next , we compared the spectrum of somatic hnf1a mutations identified in hca with 364 germline mutations previously described in individuals with mody3 without hca ( fig . in both hca and mody3 , a hotspot mutation at codon 291 was present , whereas a hotspot mutation at codon 206 was specifically identified in hca ( fig . because codon 291 mutations represented 20% of the mutations in both spectra , we did not include them in the following analysis . there was no significant difference ( p = 0.16 ) in the proportion of missense and truncating ( frameshift , nonsense , and mutations affecting a splicing site ) mutations in mody3 and hca spectra . thus , there were 63 and 55% of missense mutations in mody3 and hca , respectively , and 37 and 45% of truncating mutations in mody3 and hca , respectively . although almost all of the missense mutations were restricted to the pou - h domain ( p < 0.001 ) in hca , they were distributed in all hnf1 domains in individuals with mody3 ( fig . also , in hca , truncating mutations were localized predominantly in the first 280 amino acids ( p < 0.001 ) , whereas they were distributed equally between the first 280 amino acids and the transactivation domain in the individuals with mody3 ( fig . in a second step , we analyzed the distribution of the 13 germline hnf1a mutations identified in patients with adenomas . in eight patients , we identified a mutation of the second hnf1a allele in the corresponding tumor leading to a biallelic inactivation of hnf1 ( table 1 ) . the corresponding germline mutations fit the distribution of the somatic hnf1a mutations : six of them were nonsense or frameshift occurring before codon 292 , and the remaining two mutations were missense affecting codon 272 , an amino acid found to be mutated by a somatic event ( fig . in contrast , in four hca samples [ 372 ( r583q ) , 769 ( t525s ) , 966 ( l389v ) , and 998 ( r278q ) ] , we observed a germline hnf1a mutation without alteration of the second hnf1a allele . all of these germline mutations were missense ; three of them localized in the transactivation domain , and the last one was at the end of the pou - h domain . none of these mutations were described in the spectrum of somatic hnf1a mutations ; however , three of them ( r583q , t525s , and l389v ) have been previously reported in individuals with mody3 ( 21,25 ) . moreover , the corresponding adenomas were classified in different molecular subgroups of hca : one of them was -catenin activated ( case 372 ) , two were inflammatory adenomas with or without a gp130 activating mutation ( cases 769 and 966 ) , and the last one remained unclassified ( case 998 ) . most of the somatic mutations found in hca are predicted to inactivate the protein because they are almost all biallelic and frequently nonsense or missense affecting the pou - h domain that is essential for dna binding . 2a , b , and d ) , or a protein with an amino acid replacement and a deletion of five amino acids in the dna - binding domain ( fig . among the point mutations in hca , we analyzed the proportion of different hnf1a nucleotide changes and found that g - to - t transversions , accounting for 36% of the cases , were the most common type ( fig . in addition , when the hnf1a nucleotide substitutions were partitioned between the two dna strands , we observed that g - to - t transversions were significantly more frequent on a nontranscribed strand ( p = 0.01 ) ( fig . taking these results together , we suggest that hnf1a somatic mutations showed a typical spectrum of mutations induced by the genotoxic exposure targeting a specific nucleotide sequence . comparison of the mutation profiles in h - hca ( 67 transversions and transitions , a ) , mody3 ( 229 transversions and transitions , b ) , and tp53-mutated lung cancers ( 311 transversions and transitions , c ) and smokers ( 26 ) ( d ) . repartitioning of transition and transversion mutations between the transcribed and the nontranscribed strands in h - hca . * significant ( p = 0.01 ) elevation in g - to - t transversions on the nontranscribed strand . because both direct dna damage by an environmental agent and oxidative stress are known to result in g - to - t transversion mutations ( 27,28 ) , we also assessed oxidative stress markers in nontumor liver from six patients with h - hca and seven patients with non - hnf1a mutated hca ( supplementary fig . in addition , it is possible that a large number of g - to - t transversions could be caused by inefficient repair of dna damage due to mutations or polymorphisms in myh and ogg1 , genes involved in the repair of 8-hydroxyguanine ( 3032 ) . in this study we analyzed the spectrum of hnf1a mutations in hca and showed a significant difference in pattern in comparison with individuals with mody3 , both at the nucleotide and amino acid levels . in hca , location of the mutations is very restricted because almost all of the truncating mutations led to the loss of the transactivation domain and the missense mutations altered mainly the pou - h domain . when we take into account the two largest series of hnf1a screening in mody3 ( 21,25 ) , only 48% of the germline mutations ( 117 of 720 are missense mutations in pou - h , and 227 of 720 are truncating mutations localized in the first 291 amino acids ) fit the features of the hnf1a somatic mutations . this observation suggests that only a part of hnf1a mutations that are associated with diabetes could predispose to the development of a h - hca . previous analysis of the mody3 mutations showed that the age at onset of diabetes is modulated according to the position of the mutation relative to the hnf1a isoforms : missense mutations located in exons 7 to 10 that affect only a or b isoforms of hnf1a are associated with the late onset of diabetes ( 21,22 ) . similarly , we can hypothesize that frequent mody3 missense mutations located outside the pou - h domain and mutations truncating hnf1 after codon 291 are possibly less inactivating than the mutations found in hca . in contrast to mody3 , in hca the lack of mutations leading to amino acid substitution in pou - s suggests that the pou - s and pou - h domains are functionally different . ( 20 ) found that the pou - s domain interacts with the pou - h domain in the recognition and binding on promoter sequences ; however , the pou - s domain is not conserved in homeobox transcription factors outside the pou family . thus , based on the profile of the hnf1a mutations that occur in hca , it may be of interest to reanalyze clinical severity of the diabetes and putative associated phenotypes in patients with mody3 , taking into the account only a premature stop before codon 291 , missense restricted at pou - h , and mutations affecting splicing sites from exon 1 to 7 . we previously noted that inflammatory adenomas are associated with obesity ( 4 ) , and the present observation raised the question of an association with mody3 and possibly with other subtypes of adenomas . we can hypothesize that according to the nature of their hnf1a germline mutation , patients with mody3 could be at risk of different subtypes of hca : mutations leading to a severe impaired hnf1 function predispose to the development of h - hca , whereas germline mutation with a mild functional consequence could predispose to inflammatory or - catenin activated hcas without a familial context . this study not only confirms , in a large series of adenomas , that the spectrum of hnf1a mutations in hca is different from that in individuals with mody3 , it also supports the hypothesis that hnf1a mutations in hca arise because of a genotoxic mechanism . hotspots of transversion identified in tumors without a repeated nucleotide sequence context were previously suggested to be a hallmark of an exposure to a genotoxic compound , such as an r249s mutation in the tp53 gene in hepatocellular carcinoma ( hcc ) associated with an exposure to the aflatoxin b1 mycotoxin ( 36,37 ) . second , hainaut and pfeifer ( 26 ) showed that there is a significant difference in the pattern of tp53 mutations in lung cancers between smokers and nonsmokers , with a 30% frequency of g - to - t transversions in smoking - related lung cancer vs. 10% in non - smoking related lung cancer . the overabundance of g - to - t transversions in smoking - related lung cancers was consistent with the studies of dna damage and mutations due to benzo[a]pyrene and other cigarette smoke - derived carcinogens and their metabolites ( 38,39 ) . our observation of an uneven distribution among hnf1a nucleotide substitutions in hca and high frequency of g - to - t transversions is in many ways similar to that in lung cancer caused by tobacco ( fig . similarly , we observed that g - to - t transversions were significantly localized to the nontranscribed strand in h - hca , an additional argument in favor of a genotoxic effect and , possibly , a presence of bulky dna adducts . we can hypothesize that it could be related to a particular genetic susceptibility and/or to an exposure to a specific toxic . according to the first hypothesis , we searched for mutations in ogg1 and myh , both genes specifically involved in the repair of 8-hydroxyguanine , a product of oxidative dna damage that can also lead to g - to - t transversions ( 3032 ) . in addition , we did not observe a higher level of oxidative stress in the livers of patients with a h - hca . these results strongly suggest that exposure to an environmental agent appears to be the most likely event to explain the elevated rate of g - to - t transversions in h - hca . this last hypothesis is supported by the facts that 1 ) the genotoxic activity of estrogen metabolites has been shown , particularly for catechol estrogens that may be oxidized to a reactive quinone capable of direct formation of dna adducts ( 41,42 ) ; and 2 ) germline heterozygous mutations of cyp1b1 , a key metabolism enzyme responsible for the formation of hydroxylated and genotoxic metabolites of estrogen , contribute to the development of h - hca in women using hormonal contraception ( 13 ) . in conclusion , when analyzing a large series of individuals with hcas , we observed a significant differences between hnf1a somatic mutations identified in those with hcas and the germline mutations identified in individuals with mody3 . somatic mutations in hca predict a more inactivating profile , resulting in a complete loss of function of the protein when biallelic . moreover , we showed that the origin of the mutations might be the result of dna damage caused by a genotoxic agent , possibly resulting from the metabolism of estrogen that could also be associated with a genetic susceptibility that remains to be identified .

the united states faces ongoing questions about how to plan for the mix of long - term services and supports ( ltss ) to meet the needs of its aging population . by 2030 , census bureau projects people aged 65 and above will number nearly 72 million and comprise about 19% of the population in contrast to 40 million ( 13% ) in 2010 ( vincent & velkoff , 2010 ) . because the likelihood of functional and cognitive disabilities increases with age , an increase in the demand for long - term services and supports is expected to accompany this demographic shift . over their remaining years of life , more than two thirds of 65 year olds are likely to need help dealing with decreases in functioning ( kemper , komisar , & alecxih , 2005 ) . ltss may be provided in the community by informal or formal care givers or in institutional settings . although support for home and community care programs has increased , the elderly who are unable to live independently must rely on institutional care ( hagen , 2013 ) . thus , with the aging population , such institutions will continue to be an important way to provide needed care for the elderly . for many years , nursing homes ( nhs ) were the primary source of around - the - clock institutional long - term care for the elderly in the united states . however , assisted living facilities ( alfs ) , which provide care at a lower level in a more home - like setting than nhs , have emerged and grown over the past few decades . estimates of assisted living capacity from different sources during a given year vary , but the data are consistent with rapid growth from 1990 to the early 2000s and slower growth after 2000 ( bishop , 1999 ; harrington , chapman , miller , miller , & newcomer , 2005 ; mollica , houser , & ujvari , 2012 ; mollica , sims - kastelein , & okeeffe , 2007 ; park - lee et al . , 2011 ; stevenson & grabowski , 2010 ) . the number of nhs in the united states decreased by approximately 4% and the number of nh beds decreased by about 3% ; occupancy went from 84.5% to 82% ( national center for health statistics , 2013 ) . data collected by the long - term care focus ( ltcf ) project at brown university also show increases in average nh case - mix from 2000 through 2010 ( ltcf ) . the change in nursing home capacity and use may be related to several factors including greater availability of home and community care ( bishop , 1999 ) , improvements in health among the elderly ( martin , schoeni , & andreski , 2010 ) , or declining disability rates ( freedman , schoeni , martin , & cornman , 2007 ; kaye , 2013 ) . the role of assisted living capacity , if any , in changes in nh case mix is not well understood . however , it is important for policy makers and communities planning for ltss to understand how alfs and nhs do or do not interact in providing services to the elderly . one possible interaction is substitution of assisted living for nh care especially for the elderly whose care needs fall toward the lower end of the spectrum . they may select alf care because they may prefer the more home - like setting and alfs are less costly than nhs ( brodie & blendon , 2001 ; genworth financial , 2010 ) . according to the genworth 2010 cost of care survey , the median annual cost of nh care in the united states for a semi - private room in 2010 was $ 67,525 but for assisted living care , it was $ 38,200 ( genworth financial , 2010 ) . assisted living care could also be a temporary substitute for nh care , delaying nh entry until a resident s care needs grow . if there is such a substitution , nh residents would have more intensive care needs where more alfs are present in the market , resulting in a higher case mix in nhs . alternatively , alfs may be unrelated to nh case mix if they address a previously unmet need for services , that is , a need that was not being met by nhs anyway . without the option of alf care , some elderly , especially those with lower care needs , would not choose to enter a nh . instead , their care needs would be addressed by family care giving or purchasing care at home or from other community sources . in this case , alfs would not be a substitute for nhs , and higher alf market capacity would not be associated with a higher nh case mix . in this study there has been little direct examination of how assisted living market capacity may be related to nh case mix in the same market . this is , in part , because of the difficulty in obtaining data on alfs and beds , which must be gathered separately for each state from a state agency . the time period for which data may be available is dependent on state data retention policies . a study by newcomer et al . ( 2001 ) of the relationship between assisted living market capacity and nh resident case mix found little evidence of substitution of assisted living for nh care . to avoid the inability to control for state policy differences , the authors evaluated the relationship between nh case mix ( facility average case mix and case mix for new admissions ) and assisted living capacity separately for five states in 1995 . low ( but not high ) alf capacity was significantly related to higher nh case mix in one state . in the other four states , there were no statistically significant relationships between assisted living capacity and nh case mix . grabowski , stevenson , and cornell ( 2012 ) report a small statistically significant positive relationship between growth in assisted living units per 1,000 residents aged 65 or older and in nh facility average case mix . the lengths of the panels for the states were not uniform , ranging from 5 to 12 years , due to limited assisted living data availability . the times covered by the panels also differed by pace of growth in assisted living capacity . finally , a more focused study of texas nhs in 2004 found that the presence of assisted living beds in a county was associated with a higher acuity for residents in nh dementia special care units ( gruneir , lapane , miller , & mor , 2007 ) . however , the authors also concluded that nh administrators may not view alfs as competitors because their findings show that nhs invest in dementia special care units when other nhs do but not in response to competition from alfs . thus , the findings are mixed , with some evidence indicating a small increase in nh case mix with higher alf market capacity and other evidence supporting no such relationship . however , these studies examined time periods when alfs were new entrants to the market or periods characterized by growth sometimes rapid in assisted living capacity . during such times even if there was substitution in earlier time periods , it is possible that after initial entry of alfs or a period of growth in these alternative providers in their markets , nhs adapted to alfs and made changes to attract potential residents including those with lower care needs , and there was less substitution . alternatively , it is also possible that communities became more knowledgeable about alfs and substitution became more common . consequently , the current relationship between alf capacity and nh resident case mix may be different from what has been previously reported . we examine this possibility by studying the relationship between assisted living bed capacity and nh case mix using more recent data in a state where assisted living capacity has stabilized . there has been little direct examination of how assisted living market capacity may be related to nh case mix in the same market . this is , in part , because of the difficulty in obtaining data on alfs and beds , which must be gathered separately for each state from a state agency . the time period for which data may be available is dependent on state data retention policies . a study by newcomer et al . ( 2001 ) of the relationship between assisted living market capacity and nh resident case mix found little evidence of substitution of assisted living for nh care . to avoid the inability to control for state policy differences , the authors evaluated the relationship between nh case mix ( facility average case mix and case mix for new admissions ) and assisted living capacity separately for five states in 1995 . low ( but not high ) alf capacity was significantly related to higher nh case mix in one state . in the other four states , grabowski , stevenson , and cornell ( 2012 ) report a small statistically significant positive relationship between growth in assisted living units per 1,000 residents aged 65 or older and in nh facility average case mix . the lengths of the panels for the states were not uniform , ranging from 5 to 12 years , due to limited assisted living data availability . the times covered by the panels also differed by pace of growth in assisted living capacity . finally , a more focused study of texas nhs in 2004 found that the presence of assisted living beds in a county was associated with a higher acuity for residents in nh dementia special care units ( gruneir , lapane , miller , & mor , 2007 ) . however , the authors also concluded that nh administrators may not view alfs as competitors because their findings show that nhs invest in dementia special care units when other nhs do but not in response to competition from alfs . thus , the findings are mixed , with some evidence indicating a small increase in nh case mix with higher alf market capacity and other evidence supporting no such relationship . however , these studies examined time periods when alfs were new entrants to the market or periods characterized by growth sometimes rapid in assisted living capacity . during such times even if there was substitution in earlier time periods , it is possible that after initial entry of alfs or a period of growth in these alternative providers in their markets , nhs adapted to alfs and made changes to attract potential residents including those with lower care needs , and there was less substitution . alternatively , it is also possible that communities became more knowledgeable about alfs and substitution became more common . consequently , the current relationship between alf capacity and nh resident case mix may be different from what has been previously reported . we examine this possibility by studying the relationship between assisted living bed capacity and nh case mix using more recent data in a state where assisted living capacity has stabilized . given the limited availability of data on assisted living capacity and our study question , we focus on a single state , virginia , during a recent time period , 2010 . virginia and this time period are interesting because the capacity of both alfs and nhs was relatively stable for the 4 years prior to and 3 years after our study period . in contrast to the national trend , published reports show that the total number of licensed alfs and beds in virginia peaked in 2001 and declined from 679 to 579 facilities ( 14.7% ) and from 34,696 to 31,824 beds ( 8.3% ) from 2001 through 2007 . however , the assisted living bed decline abated from 2007 through 2010 averaging only 0.6% per year . in addition , in the 3 years following the study period , bed capacity was stable , increasing slightly , by an average of less than 1% per year , from 2010 through 2013 ( mollica et al . , 2012 ; virginia department of social services , 2014 ) . from 2007 through 2010 , the total number of nhs increased by 8 ( 3% ) , whereas the total number of beds increased by 1.5% and occupancy held steady at 89.4% and 89.5% , respectively ( national center for health statistics , 2013 ) . thus , the relationship between the two types of care providers had the opportunity to stabilize and mature . virginia falls in the mid - range of assisted living capacity nationwide ( mollica et al . , 2012 ) . the percentage of elderly population is only somewhat lower than the national average . in 2000 , 11.2% of virginia s population was above 65 years of age , whereas in 2010 , it was 12.4% . the national average was 12.4% in 2000 and 13% in 2010 ( vincent & velkoff , 2010 ) . similar to other states , for many years , certificate of public need ( copn ) review has been required for nhs but not for alfs . in 2010 , 36 states in the united states required copn for nhs , but only 5 required certificate of public need review for alfs ( american health planning association , 2011 ) . as in many states , in virginia , public funding for alf care is limited ( mollica , 2009 ) . there is a supplement to income for recipients of federal supplemental security income program ( joint legislative audit and review committee [ jlarc ] , 2012 ) , but medicaid does not pay for alf care except for a waiver program paying $ 50 per day for up to 200 residents statewide with alzheimer s disease ( virginia department of medical assistance services , 2013 ) . finally , virginia has been close to the national average in percent of medicaid long - term care funds spent on home - based care ( 36% vs. 39% nationally ) ( ng , harrington , & kitchener , 2010 ) . thus , virginia has enough similarities to other states that findings from study of assisted living and nh case mix can provide some insights for planning of ltss in a number of other states . in addition to the market stability from 2008 to 2013 , we chose 2010 as our study year for several reasons . first , although statewide totals of alfs and beds in virginia are available from written reports for a number of years , we were unable to obtain lists of facilities and their bed totals for years prior to 2008 from virginia state sources . finally , using 2010 allows sufficient time for alfs to adapt to regulations passed in 2005 requiring licensing of alf administrators and more training for medication administration aides among other regulations that increased costs to alfs in virginia ( jlarc , 2005 ) . although an increase in costs to alfs could decrease capacity , the decline in alf capacity preceded the 2005 regulations and abated within 2 years of the regulation . first , data for nh case mix and other nh characteristics are obtained from the ltcf project at brown university . available on the ltcfocus.org website , through the year 2010 , the ltcf data are compiled using a variety of primary and secondary sources , including the minimum data set ( mds ) and online survey certification and reporting system ( oscar ) from the centers for medicare and medicaid services , as well as other sources that describe market forces relevant to nhs . the mds is a tool to assess the physical , psychological , and psychosocial functioning status for all residents in medicare and/or medicaid - certified long - term care facilities . the oscar data show regulatory compliance , operational characteristics , and aggregate patient characteristics for each facility ; they are collected during state surveys occurring at least every 15 months . second , the virginia department of social services supplied data on the number and bed capacity of alfs in nh market areas . other nh market demographic data are drawn from the u.s . department of commerce , bureau of economic analysis ( bea ) ( third data source ) and the u.s . data from the latter three data sets are merged with the nh data by market area . because all nh resident data are aggregated to the nh and are from public sources , and all market data are also publicly available , the study is exempt from institutional review board review . the outcome of interest , nh case mix , is measured as the average activities of daily living ( adl ) score and the average resource utilization group nursing case mix index ( ncmi ) during the calendar year for the nh . the adl score is the nh s average of residents total scores on seven adl categories bed mobility , transfer , locomotion , dressing , eating , toilet use , and personal hygiene . thus , the total ranges from 0 , which indicates complete independence to 28 , indicating complete dependence . the average facility resource utilization group ncmi reflects the relative staff time to care for different residents . a higher number means a higher average acuity of residents in the nh . to normalize their distributions adl = activities of daily living ; rug = resource utilization group ; ncmi = nursing case mix index ; alf = assisted living facility ; bea = bureau of economic analysis ; oscar = online survey certification and reporting system . online survey certification and reporting system . explanatory variables reflect market as well as nh organizational characteristics likely to be related to case mix ( arling & daneman , 2002 ; feng , grabowski , intrator , & mor , 2006 ; grabowski et al . , 2012 ; newcomer et al . , 2001 similar to most research concerning nh performance , we use the county as the market area ( banaszak - holl , zinn , & mor , 1996 ) . however , in virginia , because cities are not included in counties , no matter how small they are , we follow the u.s . the key explanatory variable is the density of assisted living bed capacity in a market , which is measured with the number of licensed assisted living beds per 1,000 population above age 65 ( grabowski et al . , 2012 ) . the remainder of markets , those in which nhs and alfs were present , were divided into 3 equal groups using the number of licensed assisted living beds per 1,000 population above age 65 to create categories of low , medium , and high assisted living market capacity . drawing on previous research , the other market variables chosen indicate competition among nhs and other nh alternatives in the market as well as sociodemographic characteristics . nh competition is measured with a nh herfindahl index , which indicates nh market share concentration of beds ( banaszak - holl et al . , 1996 ) . it is defined as the sum of the squares of the market share of nh beds . a value of one indicates that there is no market competition , that is , there is one nh in the market . availability of home health care , which may be an alternative to nh care for some elderly , is indicated with the number of home health agencies ( hha ) per 1,000 persons aged 65 and older . sociodemographic characteristics related to demand for nh care and health status are per capita income , percent population above age 65 , and percent black population ( scanlon , 1980 ) . it is important to control for per capita income in part because of the difference in sources of payment for alf and nh services . payment for assisted living services is largely from private sources , mostly out of pocket . although some states provide some support for assisted living care , the large majority of alf residents receive no assistance from state programs , and for those who do receive assistance , the dollar amounts provided are often low ( mollica , 2009 ) . in contrast , the largest payer for long - term nh care is the medicaid program ( american health care association , 2013 ) . nh organizational variables reflect total bed size , ownership , chain membership , and service mix ( an alzheimer s specialty unit ; arling & daneman , 2002 ) . the empirical model is cm = a+b1alf+b2mkt+b3org+e , where cm refers to nh case mix , alf refers to alf bed capacity in the market , mkt refers to a vector of market variables , org refers to a vector of organizational variables , and e is the error term . we tested for endogeneity of nh competition using lagged nh market structure as an instrumental variable as suggested by bowblis ( 2012 ) using the durbin we also tested for heteroskedasticity and adjusted standard errors for clustering of nhs within markets when it was present . given the limited availability of data on assisted living capacity and our study question , we focus on a single state , virginia , during a recent time period , 2010 . virginia and this time period are interesting because the capacity of both alfs and nhs was relatively stable for the 4 years prior to and 3 years after our study period . in contrast to the national trend , published reports show that the total number of licensed alfs and beds in virginia peaked in 2001 and declined from 679 to 579 facilities ( 14.7% ) and from 34,696 to 31,824 beds ( 8.3% ) from 2001 through 2007 . however , the assisted living bed decline abated from 2007 through 2010 averaging only 0.6% per year . in addition , in the 3 years following the study period , bed capacity was stable , increasing slightly , by an average of less than 1% per year , from 2010 through 2013 ( mollica et al . , 2012 ; virginia department of social services , 2014 ) . from 2007 through 2010 , the total number of nhs increased by 8 ( 3% ) , whereas the total number of beds increased by 1.5% and occupancy held steady at 89.4% and 89.5% , respectively ( national center for health statistics , 2013 ) . thus , the relationship between the two types of care providers had the opportunity to stabilize and mature . virginia falls in the mid - range of assisted living capacity nationwide ( mollica et al . , 2012 ) . the percentage of elderly population is only somewhat lower than the national average . in 2000 , 11.2% of virginia s population was above 65 years of age , whereas in 2010 , it was 12.4% . the national average was 12.4% in 2000 and 13% in 2010 ( vincent & velkoff , 2010 ) . similar to other states , for many years , certificate of public need ( copn ) review has been required for nhs but not for alfs . in 2010 , 36 states in the united states required copn for nhs , but only 5 required certificate of public need review for alfs ( american health planning association , 2011 ) . as in many states , in virginia , public funding for alf care is limited ( mollica , 2009 ) . there is a supplement to income for recipients of federal supplemental security income program ( joint legislative audit and review committee [ jlarc ] , 2012 ) , but medicaid does not pay for alf care except for a waiver program paying $ 50 per day for up to 200 residents statewide with alzheimer s disease ( virginia department of medical assistance services , 2013 ) . finally , virginia has been close to the national average in percent of medicaid long - term care funds spent on home - based care ( 36% vs. 39% nationally ) ( ng , harrington , & kitchener , 2010 ) . thus , virginia has enough similarities to other states that findings from study of assisted living and nh case mix can provide some insights for planning of ltss in a number of other states . in addition to the market stability from 2008 to 2013 , we chose 2010 as our study year for several reasons . first , although statewide totals of alfs and beds in virginia are available from written reports for a number of years , we were unable to obtain lists of facilities and their bed totals for years prior to 2008 from virginia state sources . finally , using 2010 allows sufficient time for alfs to adapt to regulations passed in 2005 requiring licensing of alf administrators and more training for medication administration aides among other regulations that increased costs to alfs in virginia ( jlarc , 2005 ) . although an increase in costs to alfs could decrease capacity , the decline in alf capacity preceded the 2005 regulations and abated within 2 years of the regulation . first , data for nh case mix and other nh characteristics are obtained from the ltcf project at brown university . available on the ltcfocus.org website , through the year 2010 , the ltcf data are compiled using a variety of primary and secondary sources , including the minimum data set ( mds ) and online survey certification and reporting system ( oscar ) from the centers for medicare and medicaid services , as well as other sources that describe market forces relevant to nhs . the mds is a tool to assess the physical , psychological , and psychosocial functioning status for all residents in medicare and/or medicaid - certified long - term care facilities . the oscar data show regulatory compliance , operational characteristics , and aggregate patient characteristics for each facility ; they are collected during state surveys occurring at least every 15 months . second , the virginia department of social services supplied data on the number and bed capacity of alfs in nh market areas . other nh market demographic data are drawn from the u.s . department of commerce , bureau of economic analysis ( bea ) ( third data source ) and the u.s . data from the latter three data sets are merged with the nh data by market area . because all nh resident data are aggregated to the nh and are from public sources , and all market data are also publicly available , the study is exempt from institutional review board review . the outcome of interest , nh case mix , is measured as the average activities of daily living ( adl ) score and the average resource utilization group nursing case mix index ( ncmi ) during the calendar year for the nh . the adl score is the nh s average of residents total scores on seven adl categories bed mobility , transfer , locomotion , dressing , eating , toilet use , and personal hygiene . thus , the total ranges from 0 , which indicates complete independence to 28 , indicating complete dependence . the average facility resource utilization group ncmi reflects the relative staff time to care for different residents . a higher number means a higher average acuity of residents in the nh . to normalize their distributions adl = activities of daily living ; rug = resource utilization group ; ncmi = nursing case mix index ; alf = assisted living facility ; bea = bureau of economic analysis ; oscar = online survey certification and reporting system . online survey certification and reporting system . explanatory variables reflect market as well as nh organizational characteristics likely to be related to case mix ( arling & daneman , 2002 ; feng , grabowski , intrator , & mor , 2006 ; grabowski et al . , 2012 ; newcomer et al . , 2001 similar to most research concerning nh performance , we use the county as the market area ( banaszak - holl , zinn , & mor , 1996 ) . however , in virginia , because cities are not included in counties , no matter how small they are , we follow the u.s . the key explanatory variable is the density of assisted living bed capacity in a market , which is measured with the number of licensed assisted living beds per 1,000 population above age 65 ( grabowski et al . , 2012 ) . the remainder of markets , those in which nhs and alfs were present , were divided into 3 equal groups using the number of licensed assisted living beds per 1,000 population above age 65 to create categories of low , medium , and high assisted living market capacity . drawing on previous research , the other market variables chosen indicate competition among nhs and other nh alternatives in the market as well as sociodemographic characteristics . nh competition is measured with a nh herfindahl index , which indicates nh market share concentration of beds ( banaszak - holl et al . , 1996 ) . it is defined as the sum of the squares of the market share of nh beds . a value of one indicates that there is no market competition , that is , there is one nh in the market . availability of home health care , which may be an alternative to nh care for some elderly , is indicated with the number of home health agencies ( hha ) per 1,000 persons aged 65 and older . sociodemographic characteristics related to demand for nh care and health status are per capita income , percent population above age 65 , and percent black population ( scanlon , 1980 ) . it is important to control for per capita income in part because of the difference in sources of payment for alf and nh services . payment for assisted living services is largely from private sources , mostly out of pocket . although some states provide some support for assisted living care , the large majority of alf residents receive no assistance from state programs , and for those who do receive assistance , the dollar amounts provided are often low ( mollica , 2009 ) . in contrast , the largest payer for long - term nh care is the medicaid program ( american health care association , 2013 ) . nh organizational variables reflect total bed size , ownership , chain membership , and service mix ( an alzheimer s specialty unit ; arling & daneman , 2002 ) . the empirical model is cm = a+b1alf+b2mkt+b3org+e , where cm refers to nh case mix , alf refers to alf bed capacity in the market , mkt refers to a vector of market variables , org refers to a vector of organizational variables , and e is the error term . we tested for endogeneity of nh competition using lagged nh market structure as an instrumental variable as suggested by bowblis ( 2012 ) using the durbin we also tested for heteroskedasticity and adjusted standard errors for clustering of nhs within markets when it was present . first , data for nh case mix and other nh characteristics are obtained from the ltcf project at brown university . available on the ltcfocus.org website , through the year 2010 , the ltcf data are compiled using a variety of primary and secondary sources , including the minimum data set ( mds ) and online survey certification and reporting system ( oscar ) from the centers for medicare and medicaid services , as well as other sources that describe market forces relevant to nhs . the mds is a tool to assess the physical , psychological , and psychosocial functioning status for all residents in medicare and/or medicaid - certified long - term care facilities . the oscar data show regulatory compliance , operational characteristics , and aggregate patient characteristics for each facility ; they are collected during state surveys occurring at least every 15 months . second , the virginia department of social services supplied data on the number and bed capacity of alfs in nh market areas . other nh market demographic data are drawn from the u.s . department of commerce , bureau of economic analysis ( bea ) ( third data source ) and the u.s . data from the latter three data sets are merged with the nh data by market area . because all nh resident data are aggregated to the nh and are from public sources , and all market data are also publicly available , the study is exempt from institutional review board review . the outcome of interest , nh case mix , is measured as the average activities of daily living ( adl ) score and the average resource utilization group nursing case mix index ( ncmi ) during the calendar year for the nh . the adl score is the nh s average of residents total scores on seven adl categories bed mobility , transfer , locomotion , dressing , eating , toilet use , and personal hygiene . thus , the total ranges from 0 , which indicates complete independence to 28 , indicating complete dependence . the average facility resource utilization group ncmi reflects the relative staff time to care for different residents . a higher number means a higher average acuity of residents in the nh . to normalize their distributions adl = activities of daily living ; rug = resource utilization group ; ncmi = nursing case mix index ; alf = assisted living facility ; bea = bureau of economic analysis ; oscar = online survey certification and reporting system . long - term care focus . online survey certification and reporting system . explanatory variables reflect market as well as nh organizational characteristics likely to be related to case mix ( arling & daneman , 2002 ; feng , grabowski , intrator , & mor , 2006 ; grabowski et al . similar to most research concerning nh performance , we use the county as the market area ( banaszak - holl , zinn , & mor , 1996 ) . however , in virginia , because cities are not included in counties , no matter how small they are , we follow the u.s . the key explanatory variable is the density of assisted living bed capacity in a market , which is measured with the number of licensed assisted living beds per 1,000 population above age 65 ( grabowski et al . , 2012 ) . the remainder of markets , those in which nhs and alfs were present , were divided into 3 equal groups using the number of licensed assisted living beds per 1,000 population above age 65 to create categories of low , medium , and high assisted living market capacity . drawing on previous research , the other market variables chosen indicate competition among nhs and other nh alternatives in the market as well as sociodemographic characteristics . nh competition is measured with a nh herfindahl index , which indicates nh market share concentration of beds ( banaszak - holl et al . , it is defined as the sum of the squares of the market share of nh beds . a value of one indicates that there is no market competition , that is , there is one nh in the market . availability of home health care , which may be an alternative to nh care for some elderly , is indicated with the number of home health agencies ( hha ) per 1,000 persons aged 65 and older . sociodemographic characteristics related to demand for nh care and health status are per capita income , percent population above age 65 , and percent black population ( scanlon , 1980 ) . it is important to control for per capita income in part because of the difference in sources of payment for alf and nh services . payment for assisted living services is largely from private sources , mostly out of pocket . although some states provide some support for assisted living care , the large majority of alf residents receive no assistance from state programs , and for those who do receive assistance , the dollar amounts provided are often low ( mollica , 2009 ) . in contrast , the largest payer for long - term nh care is the medicaid program ( american health care association , 2013 ) . nh organizational variables reflect total bed size , ownership , chain membership , and service mix ( an alzheimer s specialty unit ; arling & daneman , 2002 ) . the empirical model is cm = a+b1alf+b2mkt+b3org+e , where cm refers to nh case mix , alf refers to alf bed capacity in the market , mkt refers to a vector of market variables , org refers to a vector of organizational variables , and e is the error term . we tested for endogeneity of nh competition using lagged nh market structure as an instrumental variable as suggested by bowblis ( 2012 ) using the durbin we also tested for heteroskedasticity and adjusted standard errors for clustering of nhs within markets when it was present . however , because hospital - based facilities are fundamentally different from other nhs ( banaszak - holl et al . , 1996 ) , similar to most studies of nhs , we excluded 19 such facilities from this study . we also excluded specialty facilities such as training centers and acute long - term care facilities as well as nhs in operation for a year or less . six facilities were excluded due to missing data for the dependent variables . these six were smaller facilities that were part of a continuing care retirement community . the mean herfindahl index of 0.38 shows that most nhs were located in markets with competition from other nhs ; the range was from 0.08 to 1.0 . average bed size was 117 and nhs were largely for - profit and members of chains . both of our potential instruments for the 2010 herfindahl index were strongly related to the index . however , results of tests of our models with the lagged ( 2009 ) herfindahl index as an instrumental variable for nh competition indicated endogeneity for the ncmi but not for the adl equations , and tests of the models total 2009 nursing home market beds indicated no endogeneity . thus , in table 2 , we present the ordinary least squares models for average nh adl with standard errors adjusting for clustering by nh markets . we present the instrumental variable models for the average nh ncmi models ; these results were very similar to the results for the models without the instrumental variable . note.adl = activities of daily living ; ncmi = nursing case mix index ; alf = assisted living facility ; hha = home health agencies . estimated with ordinary least squares with standard errors adjusted for clustering by nursing home market area . estimated with instrumental variable for nursing home competition and unadjusted standard errors . * p < .05 . * * p < .01 . * * * p < .001 . initial models using the continuous measure of assisted living capacity showed no significant linear relationship between alf capacity and either average facility adl or ncmi . however , as shown in table 2 , when we examined the series of binary indicators of alf capacity or included a squared term , we found some support for a nonlinear relationship . nhs in markets with low and mid - range of alf capacity had a higher average all resident adl case mix than those in market with no alf beds ( p = .04 and .09 , respectively ) . however , nhs in markets with the highest alf ratios did not have case - mix indices that were significantly different from nhs with no alf capacity in their markets . an examination of the average adl for nhs by alf market capacity shows 17.04 for nhs in markets with no alf beds , but 17.93 and 17.92 for nhs in the low and mid - range alf markets , respectively . instead of continuing the increase with alf capacity , the average adl for nhs in the markets with the highest capacity turns down to 17.51 . a regression model with a squared alf capacity term shows a nonlinear relationship with facility resident adl case mix , confirming the downturn in case mix at higher alf capacity levels ( table 2 ) . the only other variable significantly related ( positive ) to average facility adl is per capita income ( p < .10 ) . there is no statistically significant relationship between alf capacity and this measure of nh resident case mix . however , both the percentage of medicare and medicaid residents are significantly positively associated with ncmi , whereas having an alzheimer s unit is associated with a lower ncmi . first , although there was little change statewide in assisted living capacity in the 3 years preceding the study , in some markets in the state , there was greater change . we reestimated the models after eliminating markets with the highest and lowest deciles of change in alf beds per 1,000 population above 65 from 2008 through 2010 . in these markets , the alf capacity ratio increased by more than 3.66 or decreased by more than 5.83 . second , we eliminated nhs that are part of continuing care retirement communities ( ccrcs ) , which include assisted living care , because they may not compete with alfs for nh residents . this study examined the relationship between assisted living market capacity and nh case mix in virginia after the growth in alfs subsided and bed capacity stabilized and , seemingly , adjusted to demand . although it is interesting to study markets in flux , for planning purposes , it is also important to examine what happens after periods of turbulence and adaptation . our findings provide some support for substitution of assisted living for nh care , but the relationship does not appear to be linear . that is , in markets with low- to mid - level alf bed capacity , the facility average adl score is higher than where there are no alf beds . however , this is not the case in areas with the highest alf bed capacity where there was no statistically significant relationship . these results suggest that nhs in high alf markets , perhaps because of their more frequent interaction with them , may have found a way to compete with alfs for residents with care needs at the lower end of the case - mix spectrum . in contrast , we did not find any significant relationship to alf bed capacity for the ncmi case - mix measure . it is likely that the ncmi is more reflective of characteristics of nh residents likely to have a short stay for rehabilitation or medical care than the characteristics of residents who must choose a long - term stay in assisted living or nh facility ( feng et al . , ( 2001 ) for one state ; however , our findings suggest a substitution in the mid - range of alf capacity as well as the low range , although the results agree that there is no substitution where alf capacity is high . they report a linear relationship between the change in alf capacity and the change in both the average facility adl and the ncmi measure . in their study , the coefficients for alf capacity in both models are small , but especially in the ncmi equation . it is possible that the smaller size of our sample was not sufficient to detect a relationship of this small magnitude . still , we differ by not finding a linear relationship between alf capacity and nh case mix . our results suggest that policies that make more assisted living care accessible to the elderly may result in better matching of care needs to the appropriate type of facility . that is , the care needs of some of the residents in nhs where there are no alfs may be able to be addressed by assisted living rather than nh care . because state medicaid programs pay for much care , states , and virginia , in particular , may be able to lower their long - term care costs with such policies . however , families , care givers and policy makers must be careful to match individual elderly persons to the best care setting for each . they must be careful in assuming that equivalent outcomes are obtained in different settings . for example , recent research shows that placement in a home care setting rather than a nh may lead to a higher likelihood of potentially preventable hospitalizations ( wysocki et al . , 2014 ) . other research concludes that the assumption that people always prefer less institutional care is not accurate ( guo , et al . the current study involves nhs in one state chosen for the stability of alf and nh capacity . although the strengths of studying a single state include identifying an interesting market situation of stability and eliminating potentially confounding state policy differences , limitations include smaller sample size and , potentially , lack of generalizability of the results to other states . however , as noted previously , virginia has many demographic and policy similarities to other states that are likely to make the findings relevant to other states . although the results of this study increase our understanding of the market interplay between alf capacity and the case mix of residents in nhs , state and federal policy changes could affect the relationship between alfs and nhs . for example , as hospitals focus on avoiding rehospitalization payment penalties from medicare , alfs may find opportunities to assist hospitals by increasing their service capabilities and forming partnerships with them ( stone , 2014 ) . planners will need to consider the evolution of alfs as they address ltss needs of the elderly in the united states . consequently , nhs and policy makers must continue to monitor the alf nh market interplay to ensure the availability and best fit of long - term care services and supports for the aging u.s . population .

assisted living facilities ( alfs ) have grown over the past few decades . if they attract residents with lower care needs away from nursing homes ( nhs ) , nhs may be left with higher case mix residents . we study the relationship between alf bed market capacity and nh case mix in a state ( virginia ) where alf bed capacity stabilized after a period of growth . similarly , nh capacity and use had been stable . while it is interesting to study markets in flux , for planning purposes , it is also important to examine what happens after periods of turbulence and adaptation . our findings show some substitution of alf for nh care , but the relationship is not linear with alf market capacity . communities need to consider the interplay of alfs and nhs in planning for long - term care services and supports . policies supporting alfs may enable care needs to be met in a lower cost setting than the nh .

Introduction Previous Research Method Sample Data Variables and analysis Results Discussion

the united states faces ongoing questions about how to plan for the mix of long - term services and supports ( ltss ) to meet the needs of its aging population . because the likelihood of functional and cognitive disabilities increases with age , an increase in the demand for long - term services and supports is expected to accompany this demographic shift . for many years , nursing homes ( nhs ) were the primary source of around - the - clock institutional long - term care for the elderly in the united states . however , assisted living facilities ( alfs ) , which provide care at a lower level in a more home - like setting than nhs , have emerged and grown over the past few decades . estimates of assisted living capacity from different sources during a given year vary , but the data are consistent with rapid growth from 1990 to the early 2000s and slower growth after 2000 ( bishop , 1999 ; harrington , chapman , miller , miller , & newcomer , 2005 ; mollica , houser , & ujvari , 2012 ; mollica , sims - kastelein , & okeeffe , 2007 ; park - lee et al . data collected by the long - term care focus ( ltcf ) project at brown university also show increases in average nh case - mix from 2000 through 2010 ( ltcf ) . the change in nursing home capacity and use may be related to several factors including greater availability of home and community care ( bishop , 1999 ) , improvements in health among the elderly ( martin , schoeni , & andreski , 2010 ) , or declining disability rates ( freedman , schoeni , martin , & cornman , 2007 ; kaye , 2013 ) . the role of assisted living capacity , if any , in changes in nh case mix is not well understood . however , it is important for policy makers and communities planning for ltss to understand how alfs and nhs do or do not interact in providing services to the elderly . one possible interaction is substitution of assisted living for nh care especially for the elderly whose care needs fall toward the lower end of the spectrum . according to the genworth 2010 cost of care survey , the median annual cost of nh care in the united states for a semi - private room in 2010 was $ 67,525 but for assisted living care , it was $ 38,200 ( genworth financial , 2010 ) . assisted living care could also be a temporary substitute for nh care , delaying nh entry until a resident s care needs grow . if there is such a substitution , nh residents would have more intensive care needs where more alfs are present in the market , resulting in a higher case mix in nhs . alternatively , alfs may be unrelated to nh case mix if they address a previously unmet need for services , that is , a need that was not being met by nhs anyway . without the option of alf care , some elderly , especially those with lower care needs , would not choose to enter a nh . in this case , alfs would not be a substitute for nhs , and higher alf market capacity would not be associated with a higher nh case mix . in this study there has been little direct examination of how assisted living market capacity may be related to nh case mix in the same market . ( 2001 ) of the relationship between assisted living market capacity and nh resident case mix found little evidence of substitution of assisted living for nh care . to avoid the inability to control for state policy differences , the authors evaluated the relationship between nh case mix ( facility average case mix and case mix for new admissions ) and assisted living capacity separately for five states in 1995 . low ( but not high ) alf capacity was significantly related to higher nh case mix in one state . in the other four states , there were no statistically significant relationships between assisted living capacity and nh case mix . grabowski , stevenson , and cornell ( 2012 ) report a small statistically significant positive relationship between growth in assisted living units per 1,000 residents aged 65 or older and in nh facility average case mix . finally , a more focused study of texas nhs in 2004 found that the presence of assisted living beds in a county was associated with a higher acuity for residents in nh dementia special care units ( gruneir , lapane , miller , & mor , 2007 ) . thus , the findings are mixed , with some evidence indicating a small increase in nh case mix with higher alf market capacity and other evidence supporting no such relationship . during such times even if there was substitution in earlier time periods , it is possible that after initial entry of alfs or a period of growth in these alternative providers in their markets , nhs adapted to alfs and made changes to attract potential residents including those with lower care needs , and there was less substitution . alternatively , it is also possible that communities became more knowledgeable about alfs and substitution became more common . consequently , the current relationship between alf capacity and nh resident case mix may be different from what has been previously reported . we examine this possibility by studying the relationship between assisted living bed capacity and nh case mix using more recent data in a state where assisted living capacity has stabilized . there has been little direct examination of how assisted living market capacity may be related to nh case mix in the same market . ( 2001 ) of the relationship between assisted living market capacity and nh resident case mix found little evidence of substitution of assisted living for nh care . to avoid the inability to control for state policy differences , the authors evaluated the relationship between nh case mix ( facility average case mix and case mix for new admissions ) and assisted living capacity separately for five states in 1995 . low ( but not high ) alf capacity was significantly related to higher nh case mix in one state . in the other four states , grabowski , stevenson , and cornell ( 2012 ) report a small statistically significant positive relationship between growth in assisted living units per 1,000 residents aged 65 or older and in nh facility average case mix . finally , a more focused study of texas nhs in 2004 found that the presence of assisted living beds in a county was associated with a higher acuity for residents in nh dementia special care units ( gruneir , lapane , miller , & mor , 2007 ) . thus , the findings are mixed , with some evidence indicating a small increase in nh case mix with higher alf market capacity and other evidence supporting no such relationship . during such times even if there was substitution in earlier time periods , it is possible that after initial entry of alfs or a period of growth in these alternative providers in their markets , nhs adapted to alfs and made changes to attract potential residents including those with lower care needs , and there was less substitution . alternatively , it is also possible that communities became more knowledgeable about alfs and substitution became more common . consequently , the current relationship between alf capacity and nh resident case mix may be different from what has been previously reported . we examine this possibility by studying the relationship between assisted living bed capacity and nh case mix using more recent data in a state where assisted living capacity has stabilized . given the limited availability of data on assisted living capacity and our study question , we focus on a single state , virginia , during a recent time period , 2010 . thus , the relationship between the two types of care providers had the opportunity to stabilize and mature . there is a supplement to income for recipients of federal supplemental security income program ( joint legislative audit and review committee [ jlarc ] , 2012 ) , but medicaid does not pay for alf care except for a waiver program paying $ 50 per day for up to 200 residents statewide with alzheimer s disease ( virginia department of medical assistance services , 2013 ) . finally , virginia has been close to the national average in percent of medicaid long - term care funds spent on home - based care ( 36% vs. 39% nationally ) ( ng , harrington , & kitchener , 2010 ) . thus , virginia has enough similarities to other states that findings from study of assisted living and nh case mix can provide some insights for planning of ltss in a number of other states . first , data for nh case mix and other nh characteristics are obtained from the ltcf project at brown university . the mds is a tool to assess the physical , psychological , and psychosocial functioning status for all residents in medicare and/or medicaid - certified long - term care facilities . second , the virginia department of social services supplied data on the number and bed capacity of alfs in nh market areas . the outcome of interest , nh case mix , is measured as the average activities of daily living ( adl ) score and the average resource utilization group nursing case mix index ( ncmi ) during the calendar year for the nh . the key explanatory variable is the density of assisted living bed capacity in a market , which is measured with the number of licensed assisted living beds per 1,000 population above age 65 ( grabowski et al . the remainder of markets , those in which nhs and alfs were present , were divided into 3 equal groups using the number of licensed assisted living beds per 1,000 population above age 65 to create categories of low , medium , and high assisted living market capacity . availability of home health care , which may be an alternative to nh care for some elderly , is indicated with the number of home health agencies ( hha ) per 1,000 persons aged 65 and older . it is important to control for per capita income in part because of the difference in sources of payment for alf and nh services . although some states provide some support for assisted living care , the large majority of alf residents receive no assistance from state programs , and for those who do receive assistance , the dollar amounts provided are often low ( mollica , 2009 ) . in contrast , the largest payer for long - term nh care is the medicaid program ( american health care association , 2013 ) . the empirical model is cm = a+b1alf+b2mkt+b3org+e , where cm refers to nh case mix , alf refers to alf bed capacity in the market , mkt refers to a vector of market variables , org refers to a vector of organizational variables , and e is the error term . virginia and this time period are interesting because the capacity of both alfs and nhs was relatively stable for the 4 years prior to and 3 years after our study period . thus , the relationship between the two types of care providers had the opportunity to stabilize and mature . there is a supplement to income for recipients of federal supplemental security income program ( joint legislative audit and review committee [ jlarc ] , 2012 ) , but medicaid does not pay for alf care except for a waiver program paying $ 50 per day for up to 200 residents statewide with alzheimer s disease ( virginia department of medical assistance services , 2013 ) . finally , virginia has been close to the national average in percent of medicaid long - term care funds spent on home - based care ( 36% vs. 39% nationally ) ( ng , harrington , & kitchener , 2010 ) . thus , virginia has enough similarities to other states that findings from study of assisted living and nh case mix can provide some insights for planning of ltss in a number of other states . first , data for nh case mix and other nh characteristics are obtained from the ltcf project at brown university . the mds is a tool to assess the physical , psychological , and psychosocial functioning status for all residents in medicare and/or medicaid - certified long - term care facilities . the outcome of interest , nh case mix , is measured as the average activities of daily living ( adl ) score and the average resource utilization group nursing case mix index ( ncmi ) during the calendar year for the nh . explanatory variables reflect market as well as nh organizational characteristics likely to be related to case mix ( arling & daneman , 2002 ; feng , grabowski , intrator , & mor , 2006 ; grabowski et al . the key explanatory variable is the density of assisted living bed capacity in a market , which is measured with the number of licensed assisted living beds per 1,000 population above age 65 ( grabowski et al . the remainder of markets , those in which nhs and alfs were present , were divided into 3 equal groups using the number of licensed assisted living beds per 1,000 population above age 65 to create categories of low , medium , and high assisted living market capacity . availability of home health care , which may be an alternative to nh care for some elderly , is indicated with the number of home health agencies ( hha ) per 1,000 persons aged 65 and older . it is important to control for per capita income in part because of the difference in sources of payment for alf and nh services . although some states provide some support for assisted living care , the large majority of alf residents receive no assistance from state programs , and for those who do receive assistance , the dollar amounts provided are often low ( mollica , 2009 ) . in contrast , the largest payer for long - term nh care is the medicaid program ( american health care association , 2013 ) . the empirical model is cm = a+b1alf+b2mkt+b3org+e , where cm refers to nh case mix , alf refers to alf bed capacity in the market , mkt refers to a vector of market variables , org refers to a vector of organizational variables , and e is the error term . first , data for nh case mix and other nh characteristics are obtained from the ltcf project at brown university . the mds is a tool to assess the physical , psychological , and psychosocial functioning status for all residents in medicare and/or medicaid - certified long - term care facilities . second , the virginia department of social services supplied data on the number and bed capacity of alfs in nh market areas . the outcome of interest , nh case mix , is measured as the average activities of daily living ( adl ) score and the average resource utilization group nursing case mix index ( ncmi ) during the calendar year for the nh . to normalize their distributions adl = activities of daily living ; rug = resource utilization group ; ncmi = nursing case mix index ; alf = assisted living facility ; bea = bureau of economic analysis ; oscar = online survey certification and reporting system . long - term care focus . explanatory variables reflect market as well as nh organizational characteristics likely to be related to case mix ( arling & daneman , 2002 ; feng , grabowski , intrator , & mor , 2006 ; grabowski et al . the key explanatory variable is the density of assisted living bed capacity in a market , which is measured with the number of licensed assisted living beds per 1,000 population above age 65 ( grabowski et al . availability of home health care , which may be an alternative to nh care for some elderly , is indicated with the number of home health agencies ( hha ) per 1,000 persons aged 65 and older . it is important to control for per capita income in part because of the difference in sources of payment for alf and nh services . although some states provide some support for assisted living care , the large majority of alf residents receive no assistance from state programs , and for those who do receive assistance , the dollar amounts provided are often low ( mollica , 2009 ) . in contrast , the largest payer for long - term nh care is the medicaid program ( american health care association , 2013 ) . the empirical model is cm = a+b1alf+b2mkt+b3org+e , where cm refers to nh case mix , alf refers to alf bed capacity in the market , mkt refers to a vector of market variables , org refers to a vector of organizational variables , and e is the error term . we also excluded specialty facilities such as training centers and acute long - term care facilities as well as nhs in operation for a year or less . initial models using the continuous measure of assisted living capacity showed no significant linear relationship between alf capacity and either average facility adl or ncmi . nhs in markets with low and mid - range of alf capacity had a higher average all resident adl case mix than those in market with no alf beds ( p = .04 and .09 , respectively ) . an examination of the average adl for nhs by alf market capacity shows 17.04 for nhs in markets with no alf beds , but 17.93 and 17.92 for nhs in the low and mid - range alf markets , respectively . there is no statistically significant relationship between alf capacity and this measure of nh resident case mix . first , although there was little change statewide in assisted living capacity in the 3 years preceding the study , in some markets in the state , there was greater change . second , we eliminated nhs that are part of continuing care retirement communities ( ccrcs ) , which include assisted living care , because they may not compete with alfs for nh residents . this study examined the relationship between assisted living market capacity and nh case mix in virginia after the growth in alfs subsided and bed capacity stabilized and , seemingly , adjusted to demand . although it is interesting to study markets in flux , for planning purposes , it is also important to examine what happens after periods of turbulence and adaptation . our findings provide some support for substitution of assisted living for nh care , but the relationship does not appear to be linear . however , this is not the case in areas with the highest alf bed capacity where there was no statistically significant relationship . these results suggest that nhs in high alf markets , perhaps because of their more frequent interaction with them , may have found a way to compete with alfs for residents with care needs at the lower end of the case - mix spectrum . in contrast , we did not find any significant relationship to alf bed capacity for the ncmi case - mix measure . it is likely that the ncmi is more reflective of characteristics of nh residents likely to have a short stay for rehabilitation or medical care than the characteristics of residents who must choose a long - term stay in assisted living or nh facility ( feng et al . , ( 2001 ) for one state ; however , our findings suggest a substitution in the mid - range of alf capacity as well as the low range , although the results agree that there is no substitution where alf capacity is high . still , we differ by not finding a linear relationship between alf capacity and nh case mix . our results suggest that policies that make more assisted living care accessible to the elderly may result in better matching of care needs to the appropriate type of facility . that is , the care needs of some of the residents in nhs where there are no alfs may be able to be addressed by assisted living rather than nh care . because state medicaid programs pay for much care , states , and virginia , in particular , may be able to lower their long - term care costs with such policies . the current study involves nhs in one state chosen for the stability of alf and nh capacity . although the results of this study increase our understanding of the market interplay between alf capacity and the case mix of residents in nhs , state and federal policy changes could affect the relationship between alfs and nhs . planners will need to consider the evolution of alfs as they address ltss needs of the elderly in the united states . consequently , nhs and policy makers must continue to monitor the alf nh market interplay to ensure the availability and best fit of long - term care services and supports for the aging u.s .

gene therapy is defined as induction or inhibition of genes by means of introducing various forms of nucleic acids into cells . since the entry of nucleic acids into cells is a very inefficient process , successful gene therapy requires an efficient drug delivery system . viral vectors and nonviral delivery systems are used for delivery of nucleic acids into cells . viral vectors are very efficient in introducing genes into cells but have limitations regarding the size of the genes that can be delivered and the safety of such formulations . alternatively , nonviral delivery systems are much safer and not limited in their delivery of large pieces of dna but are often not sufficiently efficient.13 nonviral delivery systems include various physical modes of delivery ( eg , gene gun , electroporation , hydrodynamic delivery , and ultrasound ) whereas chemical systems include various cationic polymers and cationic liposomes.4,5 the most frequently used nonviral systems are liposomes as can be seen from the proportions of vectors used in clinical trials.6 cationic liposomes interact with negatively charged nucleic acids and these complexes enter the cell by endocytosis , then fuse with endosomal membranes and release nucleic acids into the cytoplasm.711 liposomes have been investigated for over 20 years as delivery systems for nucleic acids , but the process is not fully understood and depends on various physicochemical characteristics of the liposome / dna , such as size,1215 lamellarity,16 structure,17 fusogenicity,18 charge ratio,13,14,19 and charge density.20 furthermore , these properties often influence each other , further complicating the picture.21,22 also , various studies have been conducted with the aim of synthesizing new cationic lipids with improved properties and studying structure - activity relationships.23,24 however , since various factors influence the transfection efficiency , it is difficult to draw a definite conclusion about the influence of structural characteristics of lipids and physicochemical properties of liposome / dna complexes on transfection efficiency . finding the aim of this study was to systemically investigate liposome / dna formulations differing in lipid composition , size , and charge ratio and to define the most effective liposome / dna characteristics promoting transfection efficiency in vitro . such a study was undertaken as a preliminary screening for the best formulation to be studied in a future design of a liposome / dna vaccine and investigation of its efficacy in vivo . to monitor transfection efficiency we used plasmid dna encoding - enhanced green fluorescent protein ( pegfp ) and the percentage of green fluorescent protein ( gfp)-expressing cells was determined by flow cytometry . it was incorporated into three differently composed liposomal formulations : ( 1 ) phosphatidylcholine ( pc ) , 1,2-dioleoyl - sn - glycero-3 phosphatidylethanolamine ( dope ) and n-(1-(2,3-dioleoyloxy)propyl)n , n , n trimethylammonium chloride ( dotap ) ; ( 2 ) dope and dotap ; ( 3 ) pc , cholesterol ( chol ) , and dotap . in addition , the charge ratio was varied from 1 to 50 by changing the quantities of positive lipid ( dotap ) in the process of liposome preparation . charge ratios well above those usually studied ( 110 ) were investigated here since it has already been shown that liposome / dna complexes have better transfection efficiency with higher charge ratios ( + / 15),25 presumably due to the existence of free liposomes . 26 interestingly , as far as we are aware , the higher ratios have not been studied before , so this is the first such study . the obtained charge and size of the formulated liposome complexes were subsequently monitored by dynamic light scattering . in addition , lipoplexes were prepared by hydrating lipid films directly with dna solution and not by mixing of the preformed liposomes and dna as is usually the method employed in preparing complexes . our results show that after extrusion of liposome complexes , their size is decreased , but is mainly governed by the charge ratio . also , increase in charge ratio and decrease in size of complexes improved transfection efficiency . cytotoxicity of liposomal preparations was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium ( mtt ) cell viability assay and it was found that with increasing amount of dotap cytotoxicity also increased . the dope , pc , and dotap were from avanti polar lipids ( alabaster , al ) . plasmid dna ( pdna ) encoding pegfp was from clontech ( mountain view , ca ) . the pdna was purified on a deae cim disk from bia separation ( ljubljana , slovenia ) as previously described.27 the concentration and purity of the pdna was determined by the ratio a260/280 and by agarose gel electrophoresis . thin lipid films were obtained by rotary evaporation of chloroform lipid solution in round - bottom flasks . rehydration was performed with 1.35 ml pegfp solution ( 30 g / ml ) in phosphate - buffered saline ( pbs ) ( 10 mm phosphate buffer ph = 7.4 with 0.15 m nacl ) . in three component systems the amount of pc was twice that of the other helper lipid and the amount of dotap was directed by the charge ratio . this setup resulted in the following lipid molar ratios ( charge ratios are denoted in square brackets ) : pc - dope - dotap 2 - 1 - 0.04 , pc - dope - dotap 2 - 1 - 0.5 , pc - dope - dotap 2 - 1 - 1.3 , pc - dope - dotap 2 - 1 - 4.3 , pc - dope 1 - 1.4 , pc - chol - dotap 1 - 1 - 2.25 . to reduce the size of the lipoplexes , some of the lipoplex preparations were extruded ( 29 ) through polycarbonate membranes of specific pore size using a lipofast extruder from avestin ( ottawa , canada ) . the following cell lines were used : 293 t cells ( sv40 t - antigen expressing human kidney cells ) , cos7 ( sv40 transformed african green monkey kidney cells ) , and vero cells ( normal african green monkey kidney cells ) . the cells were cultivated in dulbecco s modified eagle s medium ( dmem ) supplemented with 10% fetal calf serum ( moregate biotech , australia ) and neomycin 50 g / ml ( gibco invitrogen corporation , ca ) and incubated at 37c in a humidified atmosphere of 5% co2 . the cells were trypsinized ( 0.25% ) every 34 days and further subcultivated by splitting them in a ratio of 1:6 . cells were seeded in six well plates ( 2.5 10 cells / well ) in complete medium at 37c in a humidified atmosphere containing 5% co2 . after 24 hours cells were washed with dmem once and liposome / dna complexes ( 500 l ) plus 500 l dmem were added . after 3 hours at 37c in a humidified atmosphere containing 5% co2 , the transfection solution was replaced with complete medium and cells were further cultured for 48 hours . as a positive control , 5 10 cells were electroporated at ( 700 v , 450 s , 1 ) in a 4 mm gap cuvette ( multiporator , eppendorf , germany ) with pegfp and cultivated for 48 hours . electroporated or lipofected cells were trypsinized , washed once with complete medium , and then once with pbs . cells were resuspended in 500 l of pbs supplemented with 0.1% sodium azide and 2% fetal calf serum and analyzed on a facscalibure flow cytometer ( becton dickinson , franklin lakes , nj ) . 293 t cells were seeded in a 96-well plate ( 1 10 cells / well ) and cultured in complete medium at 37c in a humidified atmosphere containing 5% co2 . after 24 hours , liposome / dna complexes ( 50 l ) plus 50 l dmem were added . cells were cultivated for a further 48 hours at 37c in a humidified atmosphere containing 5% co2 . medium was removed and subsequently 50 l of mtt ( 0.5 mg / ml ) was added and cells were cultivated for another 4 hours . formed formazan crystals were dissolved by addition of 200 l dimethyl sulfoxide . the plates were read on a thermoscientific microplate spectrophotometer ( thermo fisher scientific , ma ) at 580 nm and at 680 nm as a reference wavelength . liposome size and zeta potential were measured by dynamic light scattering using zetasizer nano zs ( malvern instruments , malvern , uk ) equipped with 532 nm liposomal formulations for zeta and size measurements were diluted with pbs to a final concentration 0.125 mg / ml . size of lipoplexes is expressed as an average diameter ( z - average ) that is obtained from the zetasizer nano software using intensity derived size distribution . for comparison of results , the diameter of the major population obtained from number - derived size distribution was taken into account . the dope , pc , and dotap were from avanti polar lipids ( alabaster , al ) . plasmid dna ( pdna ) encoding pegfp was from clontech ( mountain view , ca ) . the pdna was purified on a deae cim disk from bia separation ( ljubljana , slovenia ) as previously described.27 the concentration and purity of the pdna was determined by the ratio a260/280 and by agarose gel electrophoresis . thin lipid films were obtained by rotary evaporation of chloroform lipid solution in round - bottom flasks . rehydration was performed with 1.35 ml pegfp solution ( 30 g / ml ) in phosphate - buffered saline ( pbs ) ( 10 mm phosphate buffer ph = 7.4 with 0.15 m nacl ) . in three component systems the amount of pc was twice that of the other helper lipid and the amount of dotap was directed by the charge ratio . this setup resulted in the following lipid molar ratios ( charge ratios are denoted in square brackets ) : pc - dope - dotap 2 - 1 - 0.04 , pc - dope - dotap 2 - 1 - 0.5 , pc - dope - dotap 2 - 1 - 1.3 , pc - dope - dotap 2 - 1 - 4.3 , pc - dope 1 - 1.4 , pc - chol - dotap 1 - 1 - 2.25 . to reduce the size of the lipoplexes , some of the lipoplex preparations were extruded ( 29 ) through polycarbonate membranes of specific pore size using a lipofast extruder from avestin ( ottawa , canada ) . the following cell lines were used : 293 t cells ( sv40 t - antigen expressing human kidney cells ) , cos7 ( sv40 transformed african green monkey kidney cells ) , and vero cells ( normal african green monkey kidney cells ) . the cells were cultivated in dulbecco s modified eagle s medium ( dmem ) supplemented with 10% fetal calf serum ( moregate biotech , australia ) and neomycin 50 g / ml ( gibco invitrogen corporation , ca ) and incubated at 37c in a humidified atmosphere of 5% co2 . the cells were trypsinized ( 0.25% ) every 34 days and further subcultivated by splitting them in a ratio of 1:6 . cells were seeded in six well plates ( 2.5 10 cells / well ) in complete medium at 37c in a humidified atmosphere containing 5% co2 . after 24 hours cells were washed with dmem once and liposome / dna complexes ( 500 l ) plus 500 l dmem were added . after 3 hours at 37c in a humidified atmosphere containing 5% co2 , the transfection solution was replaced with complete medium and cells were further cultured for 48 hours . as a positive control , 5 10 cells were electroporated at ( 700 v , 450 s , 1 ) in a 4 mm gap cuvette ( multiporator , eppendorf , germany ) with pegfp and cultivated for 48 hours . electroporated or lipofected cells were trypsinized , washed once with complete medium , and then once with pbs . cells were resuspended in 500 l of pbs supplemented with 0.1% sodium azide and 2% fetal calf serum and analyzed on a facscalibure flow cytometer ( becton dickinson , franklin lakes , nj ) . 293 t cells were seeded in a 96-well plate ( 1 10 cells / well ) and cultured in complete medium at 37c in a humidified atmosphere containing 5% co2 . after 24 hours , liposome / dna complexes ( 50 l ) plus 50 l dmem were added . cells were cultivated for a further 48 hours at 37c in a humidified atmosphere containing 5% co2 . medium was removed and subsequently 50 l of mtt ( 0.5 mg / ml ) was added and cells were cultivated for another 4 hours . formed formazan crystals were dissolved by addition of 200 l dimethyl sulfoxide . the plates were read on a thermoscientific microplate spectrophotometer ( thermo fisher scientific , ma ) at 580 nm and at 680 nm as a reference wavelength . liposome size and zeta potential were measured by dynamic light scattering using zetasizer nano zs ( malvern instruments , malvern , uk ) equipped with 532 nm green laser . liposomal formulations for zeta and size measurements were diluted with pbs to a final concentration 0.125 mg / ml . size of lipoplexes is expressed as an average diameter ( z - average ) that is obtained from the zetasizer nano software using intensity derived size distribution . for comparison of results , the diameter of the major population obtained from number - derived size distribution was taken into account . size and charge ratio ( ratio of number of positive charges originating from cationic lipid and number of negative charges from dna s phosphates ) of liposome / dna complexes are considered very important physicochemical characteristics for transfection efficiency of formulation.1215,19 we prepared lipoplexes ( pc - dope - dotap ) with charge ratios 1 , 10 , 25 , and 50 by varying the amount of dotap . to investigate the influence of the size of liposome / dna complexes on the transfection efficiency we extruded lipoplexes through membranes with various pore sizes : 100 , 200 , and 400 nm . size measured by dynamic light scattering can be given as a number distribution , volume distribution , or intensity distribution and mean diameter ( z - average ) which is calculated from intensity distribution using a non - negatively least squares algorithm ( software provided by the manufacturer ) and these results can differ from each other . dynamic light scattering results revealed that tested liposomal formulations were not 100% homogenous in size but rather contained one major vesicle population ( generally comprising more than 90% of all vesicles ) . therefore , we processed results from number distributions ( we took into account the largest size population which was generally above 90% ) and compared them to z - average . it should be noted that three measurements of the same sample expressed as z - average had lower standard deviations then the mean size of the major population from number distributions . the trend of liposome size in respect to charge ratio was the same for both , mean diameter and number distribution , but the values of mean diameters were always higher ( figure 1 ) . liposome size of extruded formulations was reduced in comparison to unextruded , however it was not determined by the size of membrane pores but by the charge ratio . the mean diameters of all preparations ( pc - dope - dotap ) , regardless of membrane pore size , with charge ratio 50 , 25 , 10 , and 1 were 561 29.7 , 727 74.0 , 1477 100.3 , and 7121 increase in charge ratio resulted in decrease in the size of liposome / dna complex . sizes of complexes that were not extruded ( figure 1b ) did not show any trend but this might be because they were very large and on the upper limit of the instrument measurement ability ( in some runs the instrument could not even measure the sizes of these large complexes ) so precision of obtained results is quite low . interestingly , liposome / dna complexes with charge ratio 50 and 25 ( regardless of composition or extrusion ) after ultracentrifugation ( 300 , 000 g ) and also after a period of no disturbance in a container floated at the surface whereas lipoplexes with charge ratio 10 and 1 settled at the bottom of the cuvette . it can be seen that the zeta potential was governed by the charge ratio and was not changed by extrusion as could be expected ( table 1 ) . since it is well known that lipid composition and structure of lipids can influence transfection efficiency we investigated three formulations ; pc - dope - dotap , dope - dotap , and pc - chol - dotap . we used pegfp to asses transfection efficiency of liposomal formulations which was measured by flow cytometry . we have tested liposome / pegfp formulations in three different types of cells ( vero , cos7 , and 293 t ) . the best transfection results were obtained in 293 t cells ( figure 2 ) so they were used throughout this work . we used dotap as cationic lipid as it is the most widely used lipid for lipofection.28 as neutral ( helper ) lipids we used pc , dope , and chol . dope is known to be fusogenic and promotes transfection efficiency in vitro29,30 whereas chol decreases fluidity of the bilayer , increases stability , and is considered to be more efficient in vivo;25,31 and in the presence of serum.32 also , we used three - component systems since it has been shown that multicomponent systems may also promote transfection efficiency of liposome / dna complexes . 33 pc - dope - dotap formulation was prepared with charge ratios 1 , 10 , 25 , and 50 while dope - dotap and pc - chol - dotap were prepared only at ratio 50 . liposome / pegfp complexes were prepared by hydration of thin lipid film which yields large vesicles and these preparations were extruded to reduce the size . zeta potential and size of prepared formulations are shown in figure 1 and tables 1 and 2 . pc - dope - dotap / pegfp complexes showed increase of transfection efficiency upon increase of charge ratio and reduction of the size of liposome / dna complexes ( figure 3 ) . lipoplexes composed of dope - dotap were investigated regarding size but only at charge ratio 50 and showed the best efficiency of all formulations tested ( figure 3 ) . we did not investigate dope - dotap formulations at other charge ratios in more detail because preliminary experiments showed that these formulations strongly adhered to flasks and were very hard to hydrate and resuspend with dna solution , although these experiments also showed a trend of increase in transfection efficiency with increase in charge ratio ( data not shown ) . results from transfection efficiency experiments with dope - dotap formulations showed that size reduction of complexes resulted in enhancement of transfection efficiency ( figure 3 ) . in the case of formulations consisting of pc - chol - dotap it was shown that decrease in size resulted in just a slight increase in transfection efficiency ( figure 3 ) . to conclude , for different liposome compositions , size reduction ( from micrometer to nanometer sizes , figure 1 and table 2 ) improved transfection efficiency which was most evident for pc - dope - dotap complexes while slightly lower for dope - dotap and the lowest for pc - chol - dotap . increasing charge ratio of liposomal formulations ie , higher amount of dotap ( in pc - dope - dotap ) increased cytotoxicity of liposome / dna complexes and caused cell viability decrease from 90% for charge ratio 1 to 50% for charge ratio 50 ( figure 4 ) . also , in two other formulations , dope- dotap and pc - chol - dotap with charge ratio 50 , toxicity was 40% and 43% , respectively . furthermore , we observed that liposome / dna complexes exhibit greater cytotoxicity than liposomes alone ( data not shown ) as previously published by nguyen et al.34 size and charge ratio ( ratio of number of positive charges originating from cationic lipid and number of negative charges from dna s phosphates ) of liposome / dna complexes are considered very important physicochemical characteristics for transfection efficiency of formulation.1215,19 we prepared lipoplexes ( pc - dope - dotap ) with charge ratios 1 , 10 , 25 , and 50 by varying the amount of dotap . to investigate the influence of the size of liposome / dna complexes on the transfection efficiency we extruded lipoplexes through membranes with various pore sizes : 100 , 200 , and 400 nm . size measured by dynamic light scattering can be given as a number distribution , volume distribution , or intensity distribution and mean diameter ( z - average ) which is calculated from intensity distribution using a non - negatively least squares algorithm ( software provided by the manufacturer ) and these results can differ from each other . dynamic light scattering results revealed that tested liposomal formulations were not 100% homogenous in size but rather contained one major vesicle population ( generally comprising more than 90% of all vesicles ) . therefore , we processed results from number distributions ( we took into account the largest size population which was generally above 90% ) and compared them to z - average . it should be noted that three measurements of the same sample expressed as z - average had lower standard deviations then the mean size of the major population from number distributions . the trend of liposome size in respect to charge ratio was the same for both , mean diameter and number distribution , but the values of mean diameters were always higher ( figure 1 ) . liposome size of extruded formulations was reduced in comparison to unextruded , however it was not determined by the size of membrane pores but by the charge ratio . the mean diameters of all preparations ( pc - dope - dotap ) , regardless of membrane pore size , with charge ratio 50 , 25 , 10 , and 1 were 561 29.7 , 727 74.0 , 1477 100.3 , and 7121 increase in charge ratio resulted in decrease in the size of liposome / dna complex . sizes of complexes that were not extruded ( figure 1b ) did not show any trend but this might be because they were very large and on the upper limit of the instrument measurement ability ( in some runs the instrument could not even measure the sizes of these large complexes ) so precision of obtained results is quite low . interestingly , liposome / dna complexes with charge ratio 50 and 25 ( regardless of composition or extrusion ) after ultracentrifugation ( 300 , 000 g ) and also after a period of no disturbance in a container floated at the surface whereas lipoplexes with charge ratio 10 and 1 settled at the bottom of the cuvette . it can be seen that the zeta potential was governed by the charge ratio and was not changed by extrusion as could be expected ( table 1 ) . since it is well known that lipid composition and structure of lipids can influence transfection efficiency we investigated three formulations ; pc - dope - dotap , dope - dotap , and pc - chol - dotap . we used pegfp to asses transfection efficiency of liposomal formulations which was measured by flow cytometry . results were expressed as a percentage of cells expressing gfp . we have tested liposome / pegfp formulations in three different types of cells ( vero , cos7 , and 293 t ) . the best transfection results were obtained in 293 t cells ( figure 2 ) so they were used throughout this work . we used dotap as cationic lipid as it is the most widely used lipid for lipofection.28 as neutral ( helper ) lipids we used pc , dope , and chol . dope is known to be fusogenic and promotes transfection efficiency in vitro29,30 whereas chol decreases fluidity of the bilayer , increases stability , and is considered to be more efficient in vivo;25,31 and in the presence of serum.32 also , we used three - component systems since it has been shown that multicomponent systems may also promote transfection efficiency of liposome / dna complexes . 33 pc - dope - dotap formulation was prepared with charge ratios 1 , 10 , 25 , and 50 while dope - dotap and pc - chol - dotap were prepared only at ratio 50 . liposome / pegfp complexes were prepared by hydration of thin lipid film which yields large vesicles and these preparations were extruded to reduce the size . zeta potential and size of prepared formulations are shown in figure 1 and tables 1 and 2 . pc - dope - dotap / pegfp complexes showed increase of transfection efficiency upon increase of charge ratio and reduction of the size of liposome / dna complexes ( figure 3 ) . lipoplexes composed of dope - dotap were investigated regarding size but only at charge ratio 50 and showed the best efficiency of all formulations tested ( figure 3 ) . we did not investigate dope - dotap formulations at other charge ratios in more detail because preliminary experiments showed that these formulations strongly adhered to flasks and were very hard to hydrate and resuspend with dna solution , although these experiments also showed a trend of increase in transfection efficiency with increase in charge ratio ( data not shown ) . results from transfection efficiency experiments with dope - dotap formulations showed that size reduction of complexes resulted in enhancement of transfection efficiency ( figure 3 ) . in the case of formulations consisting of pc - chol - dotap it was shown that decrease in size resulted in just a slight increase in transfection efficiency ( figure 3 ) . to conclude , for different liposome compositions , size reduction ( from micrometer to nanometer sizes , figure 1 and table 2 ) improved transfection efficiency which was most evident for pc - dope - dotap complexes while slightly lower for dope - dotap and the lowest for pc - chol - dotap . increasing charge ratio of liposomal formulations ie , higher amount of dotap ( in pc - dope - dotap ) increased cytotoxicity of liposome / dna complexes and caused cell viability decrease from 90% for charge ratio 1 to 50% for charge ratio 50 ( figure 4 ) . also , in two other formulations , dope- dotap and pc - chol - dotap with charge ratio 50 , toxicity was 40% and 43% , respectively . furthermore , we observed that liposome / dna complexes exhibit greater cytotoxicity than liposomes alone ( data not shown ) as previously published by nguyen et al.34 a large amount of research has been conducted with the aim of defining physicochemical characteristics of liposome / dna complexes leading to better transfection efficiency . overall , it can be only concluded that characteristics of liposome / dna complexes influence the efficiency of liposomes as delivery systems and additionally influence each other so it is very difficult to unambiguously define superior characteristics leading to superior efficiency . in the presented work we wanted to explore the efficiency of liposome / dna complexes with higher charge ratios up to 50 since usually only charge ratios in the range from 1 to 10 furthermore , we wanted to characterize and define liposomal formulations with respect to charge and size . regardless of the method used for mammalian cell transfection , it is important to optimize transfection conditions . this includes the optimization of the liposome / dna formulation and the ratio of the formulation to medium for every cell type used . in order to avoid a protracted optimization process we tested three robust cell lines ( vero , cos7 , and 293 t ) , which are well known for their susceptibility to transfection and are therefore often used experimentally for generation of either transient or stable transformants . as shown in figure 2 these cell lines display different susceptibilities for transfection when electroporation and lipofection were conducted under conditions described herein . 293 t cells were transfected to an almost identical level by both methods so for further experiments we decided to use this cell line . results shown in figure 1 demonstrate that the size of liposome / dna complexes was reduced after extrusion but is very dependent on the charge ratio and not so much on the pore size of extrusion membrane since regardless of the pore size ( 400 , 200 , or 100 nm ) liposome size was about the same for certain charge ratios . increase in the size of complexes approaching equivalence point and decrease at higher and lower charge ratios ( 80.5 ) have already been observed by xu et al , except that only charge ratios up to 8 were used.35 furthermore , we have observed that lipoplexes with higher charge ratios floated after ultracentrifugation and those with smaller charge ratios sedimented . xu et al found that on sucrose gradient position of the band corresponding to lipoplexes was determined by charge ratio.35 increase in complex size when the zeta potential approached zero was also observed by birchall et al . this group studied charge ratios in the range of 2.80.8 ( + /).36 the same effect was also reported for charge ratios 60.125.13,14 taken together it can be concluded that charge ratio influences the size of complexes and moreover it does so even after extrusion through membranes . some report a peak of the transfection efficiency being above a charge ratio of 2.3 but only in one type of cell whereas in the other type the transfection efficiency seemed not to be influenced by the charge ratio.36 there are some reports that the highest transfection efficiency is at a charge ratio of 1.5 and decreasing at higher and lower charge ratios.13 liu et al reported that the charge ratio of 15.9 ( corresponding to 48 nmol : 1 g ) is best in the range 0.33 to 15.9 when studied in vivo.19 some reports imply that increase in size ( above 1000 nm ) of the complex is the major determinant of in vitro transfection leading to improvement of efficiency.12 others showed that the high transfection activity correlates with the presence of complexes of sizes in the range 6501500 nm.37 also it has been shown that the type of endocytosis ( clathrin or caveolae mediated ) is size dependent and influences the type of processing in the cell . particles about 500 nm in size are endocytosized by a caveolae - mediated process which does not lead to a degradative pathway in contrast to particles smaller than 200 nm that undergo clathrin - dependent endocytosis leading to lysosomal degradation.11 this means that complexes smaller than 500 nm would be able to deliver nucleic acids to cytoplasm only if they were able to exit the endosome , whereas those larger than 500 nm would be more favorable for delivery . our results show that the transfection efficiency in vitro increased with the increasing charge ratio and decreasing size in the case of pc - dope - dotap and dope - dotap ( figure 3 ) , but it is important to say that size was decreased only to about 500 nm . the effect of charge ratio is however evident with unextruded pc - dope - dotap lipoplexes where mean diameter is above 4 m for all charge ratios and the highest transfection efficiency is achieved with the highest charge ratio . in addition , transfection efficiency of extruded lipoplexes is also charge ratio dependent in the same manner ( figure 3 ) , although size differences are more pronounced in this case and should be considered . apparently , both the effect of size reduction and increase in charge ratio influence the transfection efficiency but also each other as shown in figure 1 . taken together , it might be concluded that increase in charge ratio ie , charge density , is contributing to transfection efficiency most probably due to stronger interaction with the cell membrane or easier escape from the endosome ( since charge density was shown to correlate with endosome escape).20 formulations containing chol showed the smallest transfection efficiency in comparison to dope - containing formulations as is usually the case in in vitro experiments . in addition , chol - containing formulations showed only slight enhancement of transfection efficiency following size reduction . it can be concluded that , in the experimental conditions used , increase in charge ratio leads to enhancement of transfection efficiency and together with size reduction ( also influenced by charge ratio ) leads to more efficient formulations for delivery of nucleic acids . finally , we tested cytotoxicity of the formulations used for transfection and found that pc - chol - dotap , pc - dope - dotap , and dope - dotap all at charge ratio 50 are the most toxic probably due to the large amounts of dotap . these formulations had much higher concentrations of dotap ( 5 mm ) than those shown to be nontoxic for cells ( caski ) 40 m but this was required for obtaining high charge ratios.38 our results indicate that the reduction of the size and increase in charge ratio of liposome / dna complexes promotes transfection efficiency but unfortunately also induces higher cytotoxicity due to the higher amounts of cationic lipid dotap . also , we have demonstrated that the size of liposome / dna complexes after extrusion through certain pore sizes is reduced to some extent but is strongly governed by the charge ratio . this emphasizes the complex relationship of liposome properties and activity and the necessity for further experiments studying mechanism of action of liposome- based transfection and liposome characteristics promoting transfection to achieve effective and safe nucleic acids delivery .

backgroundphysicochemical characteristics of liposome / dna complexes influence transfection efficiency and affect each other in a very intricate way . the result of this is discrepancies in conclusions drawn about the individual influence of each one.methodsaiming to elucidate the influence of liposome / dna charge ratio and size on transfection efficiency and on each other , we used liposome / dna complexes with charge ratio ( + / ) in the range of 150 and extruded through membranes of 400 , 200 , and 100 nm . plasmid dna encoding green fluorescent protein was used to measure transfection efficiency by flow cytometry . sizes of liposome / dna complexes were measured by dynamic light scattering.resultsliposome size was reduced after extrusion but this was mainly driven by the charge ratio and not by the size of the membrane pores . reduction of complex size at each charge ratio positively correlated with transfection efficiency . when the size of the complexes was approximately constant , increasing the charge ratio was found to promote transfection efficiency . cationic lipid n-(1-(2,3-dioleoyloxy)propyl)n , n , n trimethylammonium chloride was used for modulation of positive charge and a cytotoxicity test showed that increasing its amount increases cytotoxicity.conclusionit can be concluded that charge ratio dictates the size of the complex whereas overall size reduction and higher charge ratios promote transfection efficiency in vitro .

Introduction Materials and methods Chemicals Liposome/DNA preparation Cell culture Lipofection procedure Measurement of transfection efficiency by flow cytometry Cytotoxicity Size and charge of liposome/DNA complexes Results Influence of the charge ratio on the size of extruded liposome/DNA complexes Influence of liposome composition, charge ratio, and size of the liposome/DNA complex on transfection efficiency Cytotoxicity of liposome/DNA complexes Discussion Conclusion

viral vectors are very efficient in introducing genes into cells but have limitations regarding the size of the genes that can be delivered and the safety of such formulations . alternatively , nonviral delivery systems are much safer and not limited in their delivery of large pieces of dna but are often not sufficiently efficient.13 nonviral delivery systems include various physical modes of delivery ( eg , gene gun , electroporation , hydrodynamic delivery , and ultrasound ) whereas chemical systems include various cationic polymers and cationic liposomes.4,5 the most frequently used nonviral systems are liposomes as can be seen from the proportions of vectors used in clinical trials.6 cationic liposomes interact with negatively charged nucleic acids and these complexes enter the cell by endocytosis , then fuse with endosomal membranes and release nucleic acids into the cytoplasm.711 liposomes have been investigated for over 20 years as delivery systems for nucleic acids , but the process is not fully understood and depends on various physicochemical characteristics of the liposome / dna , such as size,1215 lamellarity,16 structure,17 fusogenicity,18 charge ratio,13,14,19 and charge density.20 furthermore , these properties often influence each other , further complicating the picture.21,22 also , various studies have been conducted with the aim of synthesizing new cationic lipids with improved properties and studying structure - activity relationships.23,24 however , since various factors influence the transfection efficiency , it is difficult to draw a definite conclusion about the influence of structural characteristics of lipids and physicochemical properties of liposome / dna complexes on transfection efficiency . finding the aim of this study was to systemically investigate liposome / dna formulations differing in lipid composition , size , and charge ratio and to define the most effective liposome / dna characteristics promoting transfection efficiency in vitro . to monitor transfection efficiency we used plasmid dna encoding - enhanced green fluorescent protein ( pegfp ) and the percentage of green fluorescent protein ( gfp)-expressing cells was determined by flow cytometry . it was incorporated into three differently composed liposomal formulations : ( 1 ) phosphatidylcholine ( pc ) , 1,2-dioleoyl - sn - glycero-3 phosphatidylethanolamine ( dope ) and n-(1-(2,3-dioleoyloxy)propyl)n , n , n trimethylammonium chloride ( dotap ) ; ( 2 ) dope and dotap ; ( 3 ) pc , cholesterol ( chol ) , and dotap . in addition , the charge ratio was varied from 1 to 50 by changing the quantities of positive lipid ( dotap ) in the process of liposome preparation . charge ratios well above those usually studied ( 110 ) were investigated here since it has already been shown that liposome / dna complexes have better transfection efficiency with higher charge ratios ( + / 15),25 presumably due to the existence of free liposomes . the obtained charge and size of the formulated liposome complexes were subsequently monitored by dynamic light scattering . our results show that after extrusion of liposome complexes , their size is decreased , but is mainly governed by the charge ratio . also , increase in charge ratio and decrease in size of complexes improved transfection efficiency . in three component systems the amount of pc was twice that of the other helper lipid and the amount of dotap was directed by the charge ratio . to reduce the size of the lipoplexes , some of the lipoplex preparations were extruded ( 29 ) through polycarbonate membranes of specific pore size using a lipofast extruder from avestin ( ottawa , canada ) . after 24 hours cells were washed with dmem once and liposome / dna complexes ( 500 l ) plus 500 l dmem were added . liposome size and zeta potential were measured by dynamic light scattering using zetasizer nano zs ( malvern instruments , malvern , uk ) equipped with 532 nm liposomal formulations for zeta and size measurements were diluted with pbs to a final concentration 0.125 mg / ml . in three component systems the amount of pc was twice that of the other helper lipid and the amount of dotap was directed by the charge ratio . to reduce the size of the lipoplexes , some of the lipoplex preparations were extruded ( 29 ) through polycarbonate membranes of specific pore size using a lipofast extruder from avestin ( ottawa , canada ) . after 24 hours cells were washed with dmem once and liposome / dna complexes ( 500 l ) plus 500 l dmem were added . after 24 hours , liposome / dna complexes ( 50 l ) plus 50 l dmem were added . liposome size and zeta potential were measured by dynamic light scattering using zetasizer nano zs ( malvern instruments , malvern , uk ) equipped with 532 nm green laser . size and charge ratio ( ratio of number of positive charges originating from cationic lipid and number of negative charges from dna s phosphates ) of liposome / dna complexes are considered very important physicochemical characteristics for transfection efficiency of formulation.1215,19 we prepared lipoplexes ( pc - dope - dotap ) with charge ratios 1 , 10 , 25 , and 50 by varying the amount of dotap . to investigate the influence of the size of liposome / dna complexes on the transfection efficiency we extruded lipoplexes through membranes with various pore sizes : 100 , 200 , and 400 nm . size measured by dynamic light scattering can be given as a number distribution , volume distribution , or intensity distribution and mean diameter ( z - average ) which is calculated from intensity distribution using a non - negatively least squares algorithm ( software provided by the manufacturer ) and these results can differ from each other . the trend of liposome size in respect to charge ratio was the same for both , mean diameter and number distribution , but the values of mean diameters were always higher ( figure 1 ) . liposome size of extruded formulations was reduced in comparison to unextruded , however it was not determined by the size of membrane pores but by the charge ratio . the mean diameters of all preparations ( pc - dope - dotap ) , regardless of membrane pore size , with charge ratio 50 , 25 , 10 , and 1 were 561 29.7 , 727 74.0 , 1477 100.3 , and 7121 increase in charge ratio resulted in decrease in the size of liposome / dna complex . sizes of complexes that were not extruded ( figure 1b ) did not show any trend but this might be because they were very large and on the upper limit of the instrument measurement ability ( in some runs the instrument could not even measure the sizes of these large complexes ) so precision of obtained results is quite low . interestingly , liposome / dna complexes with charge ratio 50 and 25 ( regardless of composition or extrusion ) after ultracentrifugation ( 300 , 000 g ) and also after a period of no disturbance in a container floated at the surface whereas lipoplexes with charge ratio 10 and 1 settled at the bottom of the cuvette . it can be seen that the zeta potential was governed by the charge ratio and was not changed by extrusion as could be expected ( table 1 ) . since it is well known that lipid composition and structure of lipids can influence transfection efficiency we investigated three formulations ; pc - dope - dotap , dope - dotap , and pc - chol - dotap . we used pegfp to asses transfection efficiency of liposomal formulations which was measured by flow cytometry . we used dotap as cationic lipid as it is the most widely used lipid for lipofection.28 as neutral ( helper ) lipids we used pc , dope , and chol . dope is known to be fusogenic and promotes transfection efficiency in vitro29,30 whereas chol decreases fluidity of the bilayer , increases stability , and is considered to be more efficient in vivo;25,31 and in the presence of serum.32 also , we used three - component systems since it has been shown that multicomponent systems may also promote transfection efficiency of liposome / dna complexes . liposome / pegfp complexes were prepared by hydration of thin lipid film which yields large vesicles and these preparations were extruded to reduce the size . pc - dope - dotap / pegfp complexes showed increase of transfection efficiency upon increase of charge ratio and reduction of the size of liposome / dna complexes ( figure 3 ) . we did not investigate dope - dotap formulations at other charge ratios in more detail because preliminary experiments showed that these formulations strongly adhered to flasks and were very hard to hydrate and resuspend with dna solution , although these experiments also showed a trend of increase in transfection efficiency with increase in charge ratio ( data not shown ) . results from transfection efficiency experiments with dope - dotap formulations showed that size reduction of complexes resulted in enhancement of transfection efficiency ( figure 3 ) . increasing charge ratio of liposomal formulations ie , higher amount of dotap ( in pc - dope - dotap ) increased cytotoxicity of liposome / dna complexes and caused cell viability decrease from 90% for charge ratio 1 to 50% for charge ratio 50 ( figure 4 ) . furthermore , we observed that liposome / dna complexes exhibit greater cytotoxicity than liposomes alone ( data not shown ) as previously published by nguyen et al.34 size and charge ratio ( ratio of number of positive charges originating from cationic lipid and number of negative charges from dna s phosphates ) of liposome / dna complexes are considered very important physicochemical characteristics for transfection efficiency of formulation.1215,19 we prepared lipoplexes ( pc - dope - dotap ) with charge ratios 1 , 10 , 25 , and 50 by varying the amount of dotap . to investigate the influence of the size of liposome / dna complexes on the transfection efficiency we extruded lipoplexes through membranes with various pore sizes : 100 , 200 , and 400 nm . size measured by dynamic light scattering can be given as a number distribution , volume distribution , or intensity distribution and mean diameter ( z - average ) which is calculated from intensity distribution using a non - negatively least squares algorithm ( software provided by the manufacturer ) and these results can differ from each other . the trend of liposome size in respect to charge ratio was the same for both , mean diameter and number distribution , but the values of mean diameters were always higher ( figure 1 ) . liposome size of extruded formulations was reduced in comparison to unextruded , however it was not determined by the size of membrane pores but by the charge ratio . the mean diameters of all preparations ( pc - dope - dotap ) , regardless of membrane pore size , with charge ratio 50 , 25 , 10 , and 1 were 561 29.7 , 727 74.0 , 1477 100.3 , and 7121 increase in charge ratio resulted in decrease in the size of liposome / dna complex . sizes of complexes that were not extruded ( figure 1b ) did not show any trend but this might be because they were very large and on the upper limit of the instrument measurement ability ( in some runs the instrument could not even measure the sizes of these large complexes ) so precision of obtained results is quite low . interestingly , liposome / dna complexes with charge ratio 50 and 25 ( regardless of composition or extrusion ) after ultracentrifugation ( 300 , 000 g ) and also after a period of no disturbance in a container floated at the surface whereas lipoplexes with charge ratio 10 and 1 settled at the bottom of the cuvette . it can be seen that the zeta potential was governed by the charge ratio and was not changed by extrusion as could be expected ( table 1 ) . we used pegfp to asses transfection efficiency of liposomal formulations which was measured by flow cytometry . we used dotap as cationic lipid as it is the most widely used lipid for lipofection.28 as neutral ( helper ) lipids we used pc , dope , and chol . dope is known to be fusogenic and promotes transfection efficiency in vitro29,30 whereas chol decreases fluidity of the bilayer , increases stability , and is considered to be more efficient in vivo;25,31 and in the presence of serum.32 also , we used three - component systems since it has been shown that multicomponent systems may also promote transfection efficiency of liposome / dna complexes . 33 pc - dope - dotap formulation was prepared with charge ratios 1 , 10 , 25 , and 50 while dope - dotap and pc - chol - dotap were prepared only at ratio 50 . liposome / pegfp complexes were prepared by hydration of thin lipid film which yields large vesicles and these preparations were extruded to reduce the size . pc - dope - dotap / pegfp complexes showed increase of transfection efficiency upon increase of charge ratio and reduction of the size of liposome / dna complexes ( figure 3 ) . we did not investigate dope - dotap formulations at other charge ratios in more detail because preliminary experiments showed that these formulations strongly adhered to flasks and were very hard to hydrate and resuspend with dna solution , although these experiments also showed a trend of increase in transfection efficiency with increase in charge ratio ( data not shown ) . results from transfection efficiency experiments with dope - dotap formulations showed that size reduction of complexes resulted in enhancement of transfection efficiency ( figure 3 ) . increasing charge ratio of liposomal formulations ie , higher amount of dotap ( in pc - dope - dotap ) increased cytotoxicity of liposome / dna complexes and caused cell viability decrease from 90% for charge ratio 1 to 50% for charge ratio 50 ( figure 4 ) . furthermore , we observed that liposome / dna complexes exhibit greater cytotoxicity than liposomes alone ( data not shown ) as previously published by nguyen et al.34 a large amount of research has been conducted with the aim of defining physicochemical characteristics of liposome / dna complexes leading to better transfection efficiency . overall , it can be only concluded that characteristics of liposome / dna complexes influence the efficiency of liposomes as delivery systems and additionally influence each other so it is very difficult to unambiguously define superior characteristics leading to superior efficiency . in the presented work we wanted to explore the efficiency of liposome / dna complexes with higher charge ratios up to 50 since usually only charge ratios in the range from 1 to 10 furthermore , we wanted to characterize and define liposomal formulations with respect to charge and size . this includes the optimization of the liposome / dna formulation and the ratio of the formulation to medium for every cell type used . results shown in figure 1 demonstrate that the size of liposome / dna complexes was reduced after extrusion but is very dependent on the charge ratio and not so much on the pore size of extrusion membrane since regardless of the pore size ( 400 , 200 , or 100 nm ) liposome size was about the same for certain charge ratios . increase in the size of complexes approaching equivalence point and decrease at higher and lower charge ratios ( 80.5 ) have already been observed by xu et al , except that only charge ratios up to 8 were used.35 furthermore , we have observed that lipoplexes with higher charge ratios floated after ultracentrifugation and those with smaller charge ratios sedimented . xu et al found that on sucrose gradient position of the band corresponding to lipoplexes was determined by charge ratio.35 increase in complex size when the zeta potential approached zero was also observed by birchall et al . this group studied charge ratios in the range of 2.80.8 ( + /).36 the same effect was also reported for charge ratios 60.125.13,14 taken together it can be concluded that charge ratio influences the size of complexes and moreover it does so even after extrusion through membranes . some report a peak of the transfection efficiency being above a charge ratio of 2.3 but only in one type of cell whereas in the other type the transfection efficiency seemed not to be influenced by the charge ratio.36 there are some reports that the highest transfection efficiency is at a charge ratio of 1.5 and decreasing at higher and lower charge ratios.13 liu et al reported that the charge ratio of 15.9 ( corresponding to 48 nmol : 1 g ) is best in the range 0.33 to 15.9 when studied in vivo.19 some reports imply that increase in size ( above 1000 nm ) of the complex is the major determinant of in vitro transfection leading to improvement of efficiency.12 others showed that the high transfection activity correlates with the presence of complexes of sizes in the range 6501500 nm.37 also it has been shown that the type of endocytosis ( clathrin or caveolae mediated ) is size dependent and influences the type of processing in the cell . our results show that the transfection efficiency in vitro increased with the increasing charge ratio and decreasing size in the case of pc - dope - dotap and dope - dotap ( figure 3 ) , but it is important to say that size was decreased only to about 500 nm . in addition , transfection efficiency of extruded lipoplexes is also charge ratio dependent in the same manner ( figure 3 ) , although size differences are more pronounced in this case and should be considered . apparently , both the effect of size reduction and increase in charge ratio influence the transfection efficiency but also each other as shown in figure 1 . taken together , it might be concluded that increase in charge ratio ie , charge density , is contributing to transfection efficiency most probably due to stronger interaction with the cell membrane or easier escape from the endosome ( since charge density was shown to correlate with endosome escape).20 formulations containing chol showed the smallest transfection efficiency in comparison to dope - containing formulations as is usually the case in in vitro experiments . it can be concluded that , in the experimental conditions used , increase in charge ratio leads to enhancement of transfection efficiency and together with size reduction ( also influenced by charge ratio ) leads to more efficient formulations for delivery of nucleic acids . finally , we tested cytotoxicity of the formulations used for transfection and found that pc - chol - dotap , pc - dope - dotap , and dope - dotap all at charge ratio 50 are the most toxic probably due to the large amounts of dotap . these formulations had much higher concentrations of dotap ( 5 mm ) than those shown to be nontoxic for cells ( caski ) 40 m but this was required for obtaining high charge ratios.38 our results indicate that the reduction of the size and increase in charge ratio of liposome / dna complexes promotes transfection efficiency but unfortunately also induces higher cytotoxicity due to the higher amounts of cationic lipid dotap . also , we have demonstrated that the size of liposome / dna complexes after extrusion through certain pore sizes is reduced to some extent but is strongly governed by the charge ratio .

patients suffering from nasal polyps remain one of the more challenging groups of patients to manage . however , inflammation of the upper and lower airways is well documented , and epidemiologic and pathophysiological links between chronic rhinosinusitis ( crs ) without or with nasal polyps , asthma , and/or eosinophilic inflammation have been established by recent investigation [ 13 ] . the association of nasal polyps , asthma , and hypersensitivity to aspirin was first described by widal et.al in 1922 and thereafter popularized by samter and beers in 1968 thoroughly characterizing the clinical picture . this syndrome has been termed syndrome de widal or samter 's triad . severe cutaneous and systemic adverse reactions upon ingestion of aspirin were first documented in 1902 by hirschberg , shortly after the market launch of aspirin . the diverse terms used in medical literature describing the adverse reactions upon ingestion of nonsteroidal anti - inflammatory drugs ( nsaids ) had been recently reviewed and summarized . this subset of patients with recurrent nasal polyps , asthma , and nsaids remains one of the more challenging groups of patients . the term aspirin - exacerbated respiratory disease ( aerd ) refers to the clinical syndromes of chronic rhinosinusitis ( crs ) , nasal polyps , bronchoconstriction in asthmatics , and/or eosinophil inflammation in the upper and lower airways , urticaria , angioedema , and anaphyalxis following the ingestion of nsaids blocking the cox-1 enzyme . in this concern , nsaids are an exacerbating factor rather than an underlying disease . this classification system was proposed by stevenson et al . in 2001 allowing a better understanding , which type or clinical reactions constitute the subject of the publication . aerd comprises the description of physical reactions , underlying airway - related diseases , and inhibitors of cyclooxygenase ( cox ) . aerd is subdivided , based on physical reactions , to ( 1 ) nsaid - induced rhinitis and asthma , ( 2 ) nsaid - induced urticaria / angioedema , ( 3 ) multiple - drug - induced urticaria / angioedema , ( 4 ) single - drug - induce anaphylaxis , and ( 5 ) single - drug- or nsaid - induced blended reaction ; by definition , there are none underlying diseases concerning the subclassification ( 3 ) to ( 5 ) . the appearance of diseases mentioned above in combination with the intake of nsaids constitutes a fatal combination for some patients . therefore , current epidemiology , clinical features , diagnostic approaches , molecular pathogenesis , and aerd specific therapies will be elaborated and postulates of future attempts to gain new insights into this disease will be presented . aerd has been estimated to affect 0.3 to 2.5% of the general population [ 2 , 8 , 9 ] . the frequency of symptoms associated with aerd published in literature is 510% with rhinitis , 530% with nasal polyps , 10% with bronchial asthma , 2530% with nasal polyps and bronchial asthma , and 510% with urticaria / quincke 's edema [ 1 , 2 , 913 ] . the estimation of prevalence of aerd varies depending on the determination through questionnaire ( 1120% ) , medical record ( ~3% ) , or oral provocation test ( 21% ) . therefore , aerd might be over- as well as underdiagnosed depending on the diagnostic tool used . the onset of aerd is typically during the third decade and is more commonly reported in females ( ~3 : 2 ) [ 14 , 15 ] . ethnic preferences are not described and only rare familial associations were mentioned [ 2 , 912 ] . crs is estimated as the most frequent chronic diseases worldwide with an intense impact on healthcare system and on the quality of life of patients . more than 30 million americans are involved causing over 6 billion us $ burden for the health care system worldwide . the prevalence of crs is difficult to estimate due to different diagnostic criteria , heterogenous group of patients , treatment by different medical professions , and inconsistent definitions but is assumed to reveal 5% ranging from 1 to 19% . up to 70% of patients with crs also suffer from asthma and aspirin sensitivity [ 14 , 1823 ] . the incidence and prevalence of crs with nasal polyps(crswnp ) is estimated with 24% [ 14 , 1824 ] and 3160% [ 13 , 25 , 26 ] , respectively . crswnp was observed in 7% of asthmatics but rises to 15% of patients with crswnp and aspirin sensitivity [ 13 , 27 , 28 ] . these tremendous discrepancies of aspirin - sensitive patients who suffer from nasal polyps have most likely been caused by the diagnostic techniques which have been used . in patients classified by aerd rhinitis appears first during the third decade with concomitant onset of nasal congestion , hyposmia , chronic rhinorrhea , and progress to chronic pansinusitis followed by nasal polyps which frequently relapses after surgery . finally , the disease results in nsaid - triggered hypersensitivity of the lower airways with the symptoms of asthma . about fifty percent of the patients demonstrate chronic , severe , corticoid - dependent asthma , often accompanied by systemic anaphylactoid reactions . based on our recent knowledge , once appeared , aerd remains throughout life , even though sporadic disappearance of intolerance has been reported [ 13 , 2931 ] . although there are typical clinical features , aerd is most likely underdiagnosed as exemplified above . atopy is present in approximately 30% of patients classified by aerd , which was significantly higher in patients with positive rather than negative skin tests . the formation of nasal polyps in patients suffering from aerd follows an aggressive course filling the nasal cavity , often protruding anteriorly in the face or posteriorly into the nasopharynx . facial deformation is common , due to midfacial expansion , which occurs as a consequence of the increased pressure on the bones from nasal polyps [ 32 , 33 ] . a strong positive correlation has also been found between the number of polypectomies and the peripheral blood eosinophil count [ 34 , 35 ] . the diagnosis of aerd is a major challenge not only in patients suffering from crs with / without nasal polyps and/or bronchial asthma , but also in individuals without known underlying airway - related diseases [ 13 ] . this will be supported in preposition by imaging techniques , including computed tomography or endoscopy . some patients have a definitive history of adverse reactions to nsaids . however , many patients also had not experienced aerd , suggesting that aspirin challenge tests are critical for diagnosis . the confirmative diagnosis for aerd can definitely be established by aspirin challenge [ 13 , 12 , 32 , 3639 ] . there are four routes of provocation challenge : ( 1 ) oral , ( 2 ) bronchial inhalation , ( 3 ) nasal inhalation , ( 4 ) and intravenous [ 3641 ] . in europe , inhalation , nasal , or oral challenge are used ; in the united states oral aspirin challenge is performed . the challenge test should be performed when asthma is stable ( i.e. , forced expiratory volume in the first second of expiration ( fev1 ) is > 70% of expected value , with a variability of < 10% ) . increasing challenge doses of aspirin are administered ( oral : ~20 to 500 mg , inhalation : ~0.2 to 182 mg cumulative dose , or lower , depending on the route of administration and severity of reported symptoms ) , spaced by 1.5 to 2 hours ( or shorter ) . nasal obstruction is measured by rhinomanometry , acoustic rhinomanometry , and/or peak nasal inspiratory flow ; bronchial obstruction by fev1 . challenges are interrupted , if a decrease of fev1 20% of baseline is measured [ 28 , 4245 ] . the challenge procedure bears the risk of severe asthma exacerbations and should be done only by trained specialists with availability of equipment and medication for the treatment of acute asthma attacks if they develop . hypersensitivity emerged in general within 1 to 4 hours after intake of nsaids but may occur within minutes or takes up to 24 hours . life - threatening reactions may occur in some patients , especially those with aerd [ 13 , 28 , 3942 , 4447 ] . therefore , nsaid challenge must be performed in a specialized hospital under supervision of the patient by skilled health professionals . challenge tests will fail if aerd is still not thoroughly distinctive or provocation is precluded on ethical grounds ( e.g. , pregnancy , children of young age ) , unstable asthma , asthma nonresponsive to corticosteroids , patients on -blocker , anatomical alterations ( e.g. , massive nasal polyps ) , missing compliance of the patient ( e.g. , asthmatic experiences and therefore fear of life threatening symptoms ) , unavailability of specific technical and/or medical equipment ( e.g. , measurement of respiratory function , appropriate emergency unit ) , or inadequately trained staff [ 1 , 38 ] . furthermore , long - term developments such as crs with and without polyps or gastrointestinal complications can not be adequately followed or if a prognostic goal has to be considered in patients without typical aspirin - exacerbated respiratory symptoms . in addition , oral challenge tests ruled out hypersensitivity in 50% of the patients otherwise characterised by nsaid hypersensitivity . medical history revealed best positive and negative predictive values in comparison to oral challenges , but high false - positive and negative rates were also reported . in such cases in vitro tests might be an useful option . during the last decades several in vitro tests had been developed . the most promising in vitro tests are analysing peripheral blood leukocytes ( pbls ) focusing on genes , receptors , and metabolites of the eicosanoid cascade . the characteristics and relevance for supporting the diagnosis of aerd are very recently reviewed by schfer and maune . the diagnosis of aerd is crucial for the onset of an appropriate therapy , but aspirin sensitivity usually is diagnosed very late during the chronological sequence of the disease . as outlined above , aspirin sensitivity might become obvious in several organs until all symptoms of aerd will have been developed ( for more details , see ) . therefore , a full medical history placing special attention to the existence of respiratory symptoms associated to aerd is essential for an early diagnosis ( see table 1 ) . the theories trying to explain aerd include ( i ) alteration of the arachidonic metabolism and its receptors / enzymes , ( ii ) release of inflammatory mediators and cytokines , and ( iii ) microorganisms such as virus and bacteria . driven by the known inhibitory action of nsaids on a subset of the eicosanoid pathway , that is , the cyclooxygenase pathway , szczeklik and colleagues speculated in an early paper in 1975 that the cyclooxygenases and their metabolic products , mainly prostaglandin e ( pge ) , reflect a central modulating role in patients classified by aerd . a decade later he presented his theory , that a viral respiratory infection may be an inciting event that starts the inflammatory processes that lead to respiratory inflammation and aerd in genetically susceptible individuals . the bronchoprotective effects of pge2 were confirmed by pavord and tattersfield in 1992 , and elevated peptido leukotrienes ( plt ) in nasal polyps were documented in 1996 by baenkler and colleagues . the interdependence of the pathways of cyclooxygenase and 5-lipoxygenase elucidated and exemplified first in 1999 by schfer and colleagues . they demonstrated that a profile of eicosanoids ( i.e. , pge2 and plt ) is specific for aerd and might play a role in the pathogenesis of aspirin exacerbated asthma . furthermore , an altered profile of expression , synthesis , and metabolic activity of receptors and enzymes was hypothesised . subsequent studies confirmed this concept of an aerd - specific eicosanoid pattern investigating the synthesis of eicosanoid mediators as well as the expression of receptors and enzymes in nasal secretion , urinary excretion , polypous , mucosal , and bronchial tissues in respect to the cyclooxygenase and lipoxygenase pathways [ 5457 ] . a concept of pathogenic mechanisms , focusing on the imbalance of eicosanoid pathway was outlined recently . to adumbrate this complex concept in brief , pge as well as the corresponding receptors and enzymes are diminished even before nsaid challenge , whereas the plt and the corresponding receptors and selected enzymes are elevated . this imbalance is potentiated upon intake of nsaids ( for further details , see and references therein ) . in addition , it was shown that t cells , cocultured with parainfluenza , repiratory syncytial virus , or rhinovirus produced proliferation of cd3 + and cdr4 + cells and released cytokines that attract eosinophils , supporting the theory of szczeklik . recent studies also have implicated interleukin-10 and tumor growth factor-1 polymorphism through gene interaction in aerd and rhinosinusitis , and cyclooxygenases are modulated by cytokines . patients suffering from asthma and aspirin sensitivity as well as expressing the hla a1/b8 phenotype have a higher incidence of aerd . elevated release of inflammatory mediators , for example , tryptase , histamine , ecp , il-5 , gm - csf , rantes , and eotaxin predominantly by mast cells and eosinophils , were described in patients suffering from crswnp and aspirin sensitivity [ 6171 ] . the overproduction of il-5 might intensify eosinophilic inflammation in aspirin - sensitive patients and is correlated to increased levels of ige and staphylococcus aureus enterotoxins(sae ) present in nasal polyp tissue but is not specific for patients suffering from crswnp and aspirin sensitivity . in summary , there is a complex network of molecular pathomechanisms and transcellular metabolism of eicosanoids which are implicated in the pathogenesis of crswnp and aspirin sensitivity [ 7 , 66 , 67 , 73 , 74 ] . the treatment of crs depends on the stage and extent of the disease . several reviews and meta - analyses during the last decade point to slight differences in the pathogenesis of crswnp in association with aspirin intolerance compared to crs . therefore , we will review therapeutic approaches , which will specifically address this issue . for more details aspirin desensitization might be beneficial for some patients classified with aerd demonstrating upper and lower airway inflammation . aspirin desensitization is recommended for those aerd individuals with corticoid - dependent asthma or those requiring daily nsaid therapy for other medical reasons , for example , coronary artery diseases or chronic arthritis . oral administration may reveal definitive improvements in both lower and upper airways in most patients with aspirin sensitivity . however , the synthesis of plt by pbls and nasal mucosa was reduced after desensitization [ 42 , 77 ] . another study demonstrated decreased bronchial responsiveness to inhaled leukotriene e4 on the day of desensitization therapy . modulation of further intracellular biochemical parameters might be another molecular mechanism ( for more details on suggested mechanisms , see [ 7 , 42 ] ) . focusing on the clinical symptoms , long - term aspirin desensitization ( 1 to 6 years ) reduced significantly the use of oral corticoid treatment for asthma , the dosage of nasal corticosteroids , the numbers of sinus infections , sinus surgery per year , and hospitalization and also improved olfaction . furthermore , the rate of recurrence of nasal polyp in patients undergoing desensitization after one and six years was 6.9% and 65% , whereas without aspirin desensitization the rate was 51.3% and 93.5% , respectively . intranasal aspirin treatment in patients with bilateral crswnp resulted in delayed polyps recurrence , and 8 of 16 patients remained without symptoms for 15 months . the clinical outcome was significantly better than that from those treated with corticosteroids for recurrence prevention . furthermore , endoscopy and acoustic rhinomanometry indicated a lower polyp size on the aspirin - treated nostril . a double - blind , randomized , placebo - controlled trial revealed no effect on nasal airway using 16 mg of intranasal aspirin every 48 h after 6 months of treatment , but significantly decreased expression of the cys - lt1 receptor in the turbinate mucosa of aspirin treated patients . intranasal lysine - aspirin ( up to 50 mg / d ) in addition to routine therapy reduced polyp size without adverse effects in the lower airways . therefore , currently there is level ib of evidence with recommendation a to use aspirin desensitization in aspirin - sensitive patients , although the evidence and recommendation to treat crswnp patients is still low . leukotriene modifier drugs interrupt the leukotriene pathway and have an established impact in the therapy of asthma and allergic rhinitis . two classes of leukotriene modifier drugs have been approved for asthma treatment : the cysteinyl leukotriene 1 receptor antagonist ( montelukast , pranlukast , zafilrukast ) and the 5- lipoxagenase ( 5lo- ) inhibitor ( zileuton ) . both have been widely prescribed for symptom control of the upper and lower airways of patients classified by aerd [ 8385 ] . if added to standard medication ( including steroids ) of patients suffering from crs with or without nasal polyps , leukotriene modifier drugs will result in an overall improvement in nasal symptoms scores by 72% , thereby producing side effects in 11% . patients suffering from crswnp and treated with montelukast revealed an improvement only in some symptom scores and health - related quality - of - life parameters , whereas the nasal polyp scores and the ecp levels were not significantly altered [ 87 , 88 ] . a subjective improvement in nasal symptoms was documented in 64% of aspirin - tolerant and in 50% of aspirin - sensitive patients . as significant improvements were observed only in aspirin - tolerant patients , the selective role of anti - leukotrienes in patients suffering from crs with aspirin sensitivity was questioned . sinus symptoms improved in 60% of the patients treated with antileukotrienes , and an overall benefit was seen in 80% of patients suffering from samter 's triad . montelukast in addition to steroids significantly reduced headache , facial pain , and sneezing after eight weeks of treatment . however , no significant effect were observed on the overall symptom score , nasal blockage , hyposmia , or nasal discharge . reduced eosinophilic inflammation , viability , and cytokine production in nasal polyps following montelukast therapy postoperative treatment schemes with leukotriene inhibitor revealed similar results , and no significant differences were found one year after surgery . another study investigating the postoperative effects of montelukast and intranasal mometasone medication in patients suffering from crswnp revealed most likely complementary results . both treatments caused a significant reduction in the snot-22 scores and in the rate of nasal polyps , but only a marginal advantage of montelukast . these findings point to a possible role of leukotriene modifier drugs in the treatment of specific subpopulations , but significant scientific evidence is still lacking . therefore , leukotriene modifier drugs reveal a limited level of efficacy ( iii ) and have a low degree of recommendation ( c ) in patients suffering from crswnp . therefore , surgical procedures are often viewed as adjunctive to medical therapy [ 64 , 94 ] . the greatest review on sinus surgery with 11,147 patients has been published by dalziel et al . who screened 444 articles and evaluated 33 articles published between 1978 and 2001 . in 7595% of the cases patients consistently evaluated their symptoms to be improved or greatly improved . additionally , surgical procedures with 1.4% overall complications for functional endoscopic sinus surgery ( fess ) compared with 0.8% for conventional procedures proved to be safe . approximately two - thirds of the 3,128 patients participating in the national comparative audit suffered from crswnp but had no differences in clinical parameters , drug use , or general health when compared to crsw / onp . irrespective of the extent of surgery in the whole group of patients , a clinically significant improvement in snot-22 scores was demonstrated up to 36 months postoperatively . also significantly improved lung function and reduced systemic steroid use in patients with crswnp and concomitant asthma , whereas this was not the case in aspirin - sensitive asthma patients . however , nasal breathing and quality of life improved in most patients . the most recent prospective study investigated the effects of sinus surgery as well as fluticasone propionate nasal drops 400 g twice daily on nasal and lower airway parameters in asthmatics with crswnp . fess significantly improved mean asthma symptom scores and daily peak expiratory flow rate ( pefr ) and all nasal parameters , including subjective and objective olfaction tests . fess improved nasal and asthma symptoms in patients with np . taken together , sinus surgery has been proven to be an effective tool to treat crswnp after first - line medical therapies . patients with crswnp had higher total il-5 and ige levels in nasal secretions , nasal polyp homogenisates , and blood serum than in controls . anti - ige or anti - il-5 antibody only showed minimal beneficial effects on symptoms , eosinophilia and peak nasal inspiratory flow ( pnif ) [ 99104 ] . based on current data , the evidence for efficacy of available anti - ige and anti - il-5 antibodies on the market is very low , and more studies are needed in order to recommend their use in the treatment of crswnp patients . accordingly , when specific ige directed against staphylococcus aureus enterotoxins ( sae ) was found in nasal polyp tissue homogenates for the first time , a new paradigm was proposed , which indicated that these superantigens may be involved in the pathogenesis of eosinophilic crswnp . by their superantigenic activity indeed , nasal application of staphylococcus aureus enterotoxin b is capable of aggravating experimental allergic asthma . interestingly , an increased colonization rate of staphylococcus aureus and ige to saes was reported in nasal polyps , specifically in subjects with asthma and aerd . thereby , ige to saes was also coincident with higher levels of il-5 , eotaxin and eosinophil cationic protein which are known to potentiate and prolong the eosinophilic inflammation leading to polyps ' development . additionally , the presence of ige against saes in crswnp correlated with an increased number of t cells expressing the tcr -chain variable region known to be induced by microbial superantigens giving a link to the clinical importance of sae ige in polyps . moreover , new insights were gained into the modulatory effects of staphylococcus aureus enterotoxin b ( saeb ) on nasal polyp tissue . sae directed the mucosal inflammation to a th2-driven pattern contributing to persistent inflammation by suppression of treg lymphocytes . interestingly , sae might only locally activate b cells , because a significant increase of local ige antibodies can be observed in polyp patients , while independent of serum ige levels of the same patients . nasal polyps typically show upregulation of proinflammatory plts and downregulation of pge2 ( see prior section for more details ) . in tissue of crswnp patients with an immune response to sae , the production of plts , ltb4 and lxa4 is further upregulated , while saeb significantly downregulated pge2 , cox2 , and prostanoid receptor ep2 mrna expression in fibroblasts , pointing to a direct role of sae in regulating eicosanoids as a possible mechanism of sae inflammatory reaction . the latest nomenclature of aerd considers the obvious pathogenic association of chronic rhinosinusitis with / without polyps and asthma following the exposure to nsaid , which is usually last diagnosed . especially , the coexistence of crswnp elicits a more severe clinical appearance characterized by a prolonged and fatal course leading to a higher prevalence of recurrences and a coincidence with other atopic diseases . aerds are a distinct clinical entity characterized by acute nsaid triggered respiratory reactions like chronic hyperplastic rhinosinusitis with / without eosinophilia , formation of nasal polyps , or asthma . significant morbidity and even mortality , particularly if higher doses of nsaids are ingested , may occur , if aerd is not recognized and appropriately treated . concerned patients should be educated regarding nsaids and their avoidance preventing life - threatening asthma attacks . the standard procedure confirming the diagnosis of aerd is still in vivo aspirin challenge , completed by latest blood testing , for example , in patients where in vivo provocation tests are contraindicated , including patients with unstable asthma , asthma nonresponsive to corticosteroid therapy , patients with -blockers , and patients with potential compliance problems . future areas of investigation should focus on the identification of further biomarkers improving early diagnosis using various diagnostic techniques . the treatment of crs and nasal polyps is essential to effectively control asthma and to prevent secondary infections . an adjusted medication , including aspirin desensitization , will have a positive impact on course of the disease and the patients ' quality of life . moreover , children of parents with aerd suffer from crswnp and rhinosinusitis more often than children of healthy parents . this might point to a genetic background in terms of polymorphisms of cox-1 , cox-2 , 5-lo , and/or 15-lo pathways and/or receptors which need to be elucidated . our current knowledge on aerd focuses on a pathogenic concept based on decreased pge2 and increased levels of plt associated with overexpression of ltc4-synthase , which is accompanied by an altered transcellular metabolism of mediators of the eicosanoid cascade . this also involves local production of ige directed to staphylococcus aureus enterotoxins and overproduction of il-5 . there is a clear need to understand the implication of the metabolites of the eicosanoid cascade , their receptors , enzymes , and genes , the physiological and pathological impact of microbes , as well as the local and systemic function of dendritic cells , mast cells , and treg cells . the therapeutic interventions concerning the treatment of patients classified by aerd should include aspirin desensitization and medication with leukotriene modifier drugs being the most promising drugs at the moment . after failure of conservative treatment and/or in case of a severe phenotype , functional sinus surgery is an important option completed by further conservative therapies . finally , therapeutic approaches treating staphylococcus aureus and sae effects by antibiotics or appropriate vaccination are promising .

aspirin - exacerbated respiratory disease ( aerd ) refers to aspirin sensitivity , chronic rhinosinusitis ( crs ) , nasal polyposis , asthma , eosinophil inflammation in the upper and lower airways , urticaria , angioedema , and anaphylaxis following the ingestion of nsaids . epidemiologic and pathophysiological links between these diseases are established . the precise pathogenesis remains less defined , even though there is some progress in the understanding of several molecular mechanisms . nevertheless , these combinations of diseases in patients classified by aerd constitute a fatal combination and may be difficult to treat with standard medical and surgical interventions . this paper reviews in brief the epidemiology , clinical features , diagnosis , molecular pathogenesis , and specific therapies of patients classified by aerd and postulates future attempts to gain new insights into this disease .

1. Introduction 2. Epidemiology 3. Clinical Features 4. Diagnostic Approaches 5. Molecular Pathogenesis 6. Therapies 7. Future Attempts 8. Conclusions

patients suffering from nasal polyps remain one of the more challenging groups of patients to manage . however , inflammation of the upper and lower airways is well documented , and epidemiologic and pathophysiological links between chronic rhinosinusitis ( crs ) without or with nasal polyps , asthma , and/or eosinophilic inflammation have been established by recent investigation [ 13 ] . the association of nasal polyps , asthma , and hypersensitivity to aspirin was first described by widal et.al in 1922 and thereafter popularized by samter and beers in 1968 thoroughly characterizing the clinical picture . severe cutaneous and systemic adverse reactions upon ingestion of aspirin were first documented in 1902 by hirschberg , shortly after the market launch of aspirin . the diverse terms used in medical literature describing the adverse reactions upon ingestion of nonsteroidal anti - inflammatory drugs ( nsaids ) had been recently reviewed and summarized . this subset of patients with recurrent nasal polyps , asthma , and nsaids remains one of the more challenging groups of patients . the term aspirin - exacerbated respiratory disease ( aerd ) refers to the clinical syndromes of chronic rhinosinusitis ( crs ) , nasal polyps , bronchoconstriction in asthmatics , and/or eosinophil inflammation in the upper and lower airways , urticaria , angioedema , and anaphyalxis following the ingestion of nsaids blocking the cox-1 enzyme . aerd comprises the description of physical reactions , underlying airway - related diseases , and inhibitors of cyclooxygenase ( cox ) . aerd is subdivided , based on physical reactions , to ( 1 ) nsaid - induced rhinitis and asthma , ( 2 ) nsaid - induced urticaria / angioedema , ( 3 ) multiple - drug - induced urticaria / angioedema , ( 4 ) single - drug - induce anaphylaxis , and ( 5 ) single - drug- or nsaid - induced blended reaction ; by definition , there are none underlying diseases concerning the subclassification ( 3 ) to ( 5 ) . the appearance of diseases mentioned above in combination with the intake of nsaids constitutes a fatal combination for some patients . therefore , current epidemiology , clinical features , diagnostic approaches , molecular pathogenesis , and aerd specific therapies will be elaborated and postulates of future attempts to gain new insights into this disease will be presented . the frequency of symptoms associated with aerd published in literature is 510% with rhinitis , 530% with nasal polyps , 10% with bronchial asthma , 2530% with nasal polyps and bronchial asthma , and 510% with urticaria / quincke 's edema [ 1 , 2 , 913 ] . the estimation of prevalence of aerd varies depending on the determination through questionnaire ( 1120% ) , medical record ( ~3% ) , or oral provocation test ( 21% ) . ethnic preferences are not described and only rare familial associations were mentioned [ 2 , 912 ] . crs is estimated as the most frequent chronic diseases worldwide with an intense impact on healthcare system and on the quality of life of patients . the prevalence of crs is difficult to estimate due to different diagnostic criteria , heterogenous group of patients , treatment by different medical professions , and inconsistent definitions but is assumed to reveal 5% ranging from 1 to 19% . up to 70% of patients with crs also suffer from asthma and aspirin sensitivity [ 14 , 1823 ] . crswnp was observed in 7% of asthmatics but rises to 15% of patients with crswnp and aspirin sensitivity [ 13 , 27 , 28 ] . these tremendous discrepancies of aspirin - sensitive patients who suffer from nasal polyps have most likely been caused by the diagnostic techniques which have been used . in patients classified by aerd rhinitis appears first during the third decade with concomitant onset of nasal congestion , hyposmia , chronic rhinorrhea , and progress to chronic pansinusitis followed by nasal polyps which frequently relapses after surgery . finally , the disease results in nsaid - triggered hypersensitivity of the lower airways with the symptoms of asthma . about fifty percent of the patients demonstrate chronic , severe , corticoid - dependent asthma , often accompanied by systemic anaphylactoid reactions . based on our recent knowledge , once appeared , aerd remains throughout life , even though sporadic disappearance of intolerance has been reported [ 13 , 2931 ] . although there are typical clinical features , aerd is most likely underdiagnosed as exemplified above . atopy is present in approximately 30% of patients classified by aerd , which was significantly higher in patients with positive rather than negative skin tests . the formation of nasal polyps in patients suffering from aerd follows an aggressive course filling the nasal cavity , often protruding anteriorly in the face or posteriorly into the nasopharynx . the diagnosis of aerd is a major challenge not only in patients suffering from crs with / without nasal polyps and/or bronchial asthma , but also in individuals without known underlying airway - related diseases [ 13 ] . in europe , inhalation , nasal , or oral challenge are used ; in the united states oral aspirin challenge is performed . , forced expiratory volume in the first second of expiration ( fev1 ) is > 70% of expected value , with a variability of < 10% ) . increasing challenge doses of aspirin are administered ( oral : ~20 to 500 mg , inhalation : ~0.2 to 182 mg cumulative dose , or lower , depending on the route of administration and severity of reported symptoms ) , spaced by 1.5 to 2 hours ( or shorter ) . hypersensitivity emerged in general within 1 to 4 hours after intake of nsaids but may occur within minutes or takes up to 24 hours . , pregnancy , children of young age ) , unstable asthma , asthma nonresponsive to corticosteroids , patients on -blocker , anatomical alterations ( e.g. , massive nasal polyps ) , missing compliance of the patient ( e.g. , asthmatic experiences and therefore fear of life threatening symptoms ) , unavailability of specific technical and/or medical equipment ( e.g. , measurement of respiratory function , appropriate emergency unit ) , or inadequately trained staff [ 1 , 38 ] . furthermore , long - term developments such as crs with and without polyps or gastrointestinal complications can not be adequately followed or if a prognostic goal has to be considered in patients without typical aspirin - exacerbated respiratory symptoms . medical history revealed best positive and negative predictive values in comparison to oral challenges , but high false - positive and negative rates were also reported . the most promising in vitro tests are analysing peripheral blood leukocytes ( pbls ) focusing on genes , receptors , and metabolites of the eicosanoid cascade . the diagnosis of aerd is crucial for the onset of an appropriate therapy , but aspirin sensitivity usually is diagnosed very late during the chronological sequence of the disease . as outlined above , aspirin sensitivity might become obvious in several organs until all symptoms of aerd will have been developed ( for more details , see ) . the theories trying to explain aerd include ( i ) alteration of the arachidonic metabolism and its receptors / enzymes , ( ii ) release of inflammatory mediators and cytokines , and ( iii ) microorganisms such as virus and bacteria . driven by the known inhibitory action of nsaids on a subset of the eicosanoid pathway , that is , the cyclooxygenase pathway , szczeklik and colleagues speculated in an early paper in 1975 that the cyclooxygenases and their metabolic products , mainly prostaglandin e ( pge ) , reflect a central modulating role in patients classified by aerd . a decade later he presented his theory , that a viral respiratory infection may be an inciting event that starts the inflammatory processes that lead to respiratory inflammation and aerd in genetically susceptible individuals . the bronchoprotective effects of pge2 were confirmed by pavord and tattersfield in 1992 , and elevated peptido leukotrienes ( plt ) in nasal polyps were documented in 1996 by baenkler and colleagues . the interdependence of the pathways of cyclooxygenase and 5-lipoxygenase elucidated and exemplified first in 1999 by schfer and colleagues . they demonstrated that a profile of eicosanoids ( i.e. , pge2 and plt ) is specific for aerd and might play a role in the pathogenesis of aspirin exacerbated asthma . furthermore , an altered profile of expression , synthesis , and metabolic activity of receptors and enzymes was hypothesised . subsequent studies confirmed this concept of an aerd - specific eicosanoid pattern investigating the synthesis of eicosanoid mediators as well as the expression of receptors and enzymes in nasal secretion , urinary excretion , polypous , mucosal , and bronchial tissues in respect to the cyclooxygenase and lipoxygenase pathways [ 5457 ] . to adumbrate this complex concept in brief , pge as well as the corresponding receptors and enzymes are diminished even before nsaid challenge , whereas the plt and the corresponding receptors and selected enzymes are elevated . this imbalance is potentiated upon intake of nsaids ( for further details , see and references therein ) . recent studies also have implicated interleukin-10 and tumor growth factor-1 polymorphism through gene interaction in aerd and rhinosinusitis , and cyclooxygenases are modulated by cytokines . patients suffering from asthma and aspirin sensitivity as well as expressing the hla a1/b8 phenotype have a higher incidence of aerd . elevated release of inflammatory mediators , for example , tryptase , histamine , ecp , il-5 , gm - csf , rantes , and eotaxin predominantly by mast cells and eosinophils , were described in patients suffering from crswnp and aspirin sensitivity [ 6171 ] . the overproduction of il-5 might intensify eosinophilic inflammation in aspirin - sensitive patients and is correlated to increased levels of ige and staphylococcus aureus enterotoxins(sae ) present in nasal polyp tissue but is not specific for patients suffering from crswnp and aspirin sensitivity . in summary , there is a complex network of molecular pathomechanisms and transcellular metabolism of eicosanoids which are implicated in the pathogenesis of crswnp and aspirin sensitivity [ 7 , 66 , 67 , 73 , 74 ] . several reviews and meta - analyses during the last decade point to slight differences in the pathogenesis of crswnp in association with aspirin intolerance compared to crs . for more details aspirin desensitization might be beneficial for some patients classified with aerd demonstrating upper and lower airway inflammation . aspirin desensitization is recommended for those aerd individuals with corticoid - dependent asthma or those requiring daily nsaid therapy for other medical reasons , for example , coronary artery diseases or chronic arthritis . oral administration may reveal definitive improvements in both lower and upper airways in most patients with aspirin sensitivity . however , the synthesis of plt by pbls and nasal mucosa was reduced after desensitization [ 42 , 77 ] . focusing on the clinical symptoms , long - term aspirin desensitization ( 1 to 6 years ) reduced significantly the use of oral corticoid treatment for asthma , the dosage of nasal corticosteroids , the numbers of sinus infections , sinus surgery per year , and hospitalization and also improved olfaction . furthermore , the rate of recurrence of nasal polyp in patients undergoing desensitization after one and six years was 6.9% and 65% , whereas without aspirin desensitization the rate was 51.3% and 93.5% , respectively . intranasal aspirin treatment in patients with bilateral crswnp resulted in delayed polyps recurrence , and 8 of 16 patients remained without symptoms for 15 months . furthermore , endoscopy and acoustic rhinomanometry indicated a lower polyp size on the aspirin - treated nostril . a double - blind , randomized , placebo - controlled trial revealed no effect on nasal airway using 16 mg of intranasal aspirin every 48 h after 6 months of treatment , but significantly decreased expression of the cys - lt1 receptor in the turbinate mucosa of aspirin treated patients . intranasal lysine - aspirin ( up to 50 mg / d ) in addition to routine therapy reduced polyp size without adverse effects in the lower airways . therefore , currently there is level ib of evidence with recommendation a to use aspirin desensitization in aspirin - sensitive patients , although the evidence and recommendation to treat crswnp patients is still low . leukotriene modifier drugs interrupt the leukotriene pathway and have an established impact in the therapy of asthma and allergic rhinitis . both have been widely prescribed for symptom control of the upper and lower airways of patients classified by aerd [ 8385 ] . if added to standard medication ( including steroids ) of patients suffering from crs with or without nasal polyps , leukotriene modifier drugs will result in an overall improvement in nasal symptoms scores by 72% , thereby producing side effects in 11% . a subjective improvement in nasal symptoms was documented in 64% of aspirin - tolerant and in 50% of aspirin - sensitive patients . as significant improvements were observed only in aspirin - tolerant patients , the selective role of anti - leukotrienes in patients suffering from crs with aspirin sensitivity was questioned . sinus symptoms improved in 60% of the patients treated with antileukotrienes , and an overall benefit was seen in 80% of patients suffering from samter 's triad . montelukast in addition to steroids significantly reduced headache , facial pain , and sneezing after eight weeks of treatment . however , no significant effect were observed on the overall symptom score , nasal blockage , hyposmia , or nasal discharge . reduced eosinophilic inflammation , viability , and cytokine production in nasal polyps following montelukast therapy postoperative treatment schemes with leukotriene inhibitor revealed similar results , and no significant differences were found one year after surgery . another study investigating the postoperative effects of montelukast and intranasal mometasone medication in patients suffering from crswnp revealed most likely complementary results . both treatments caused a significant reduction in the snot-22 scores and in the rate of nasal polyps , but only a marginal advantage of montelukast . these findings point to a possible role of leukotriene modifier drugs in the treatment of specific subpopulations , but significant scientific evidence is still lacking . therefore , leukotriene modifier drugs reveal a limited level of efficacy ( iii ) and have a low degree of recommendation ( c ) in patients suffering from crswnp . who screened 444 articles and evaluated 33 articles published between 1978 and 2001 . additionally , surgical procedures with 1.4% overall complications for functional endoscopic sinus surgery ( fess ) compared with 0.8% for conventional procedures proved to be safe . approximately two - thirds of the 3,128 patients participating in the national comparative audit suffered from crswnp but had no differences in clinical parameters , drug use , or general health when compared to crsw / onp . irrespective of the extent of surgery in the whole group of patients , a clinically significant improvement in snot-22 scores was demonstrated up to 36 months postoperatively . also significantly improved lung function and reduced systemic steroid use in patients with crswnp and concomitant asthma , whereas this was not the case in aspirin - sensitive asthma patients . however , nasal breathing and quality of life improved in most patients . the most recent prospective study investigated the effects of sinus surgery as well as fluticasone propionate nasal drops 400 g twice daily on nasal and lower airway parameters in asthmatics with crswnp . fess improved nasal and asthma symptoms in patients with np . taken together , sinus surgery has been proven to be an effective tool to treat crswnp after first - line medical therapies . patients with crswnp had higher total il-5 and ige levels in nasal secretions , nasal polyp homogenisates , and blood serum than in controls . based on current data , the evidence for efficacy of available anti - ige and anti - il-5 antibodies on the market is very low , and more studies are needed in order to recommend their use in the treatment of crswnp patients . accordingly , when specific ige directed against staphylococcus aureus enterotoxins ( sae ) was found in nasal polyp tissue homogenates for the first time , a new paradigm was proposed , which indicated that these superantigens may be involved in the pathogenesis of eosinophilic crswnp . by their superantigenic activity indeed , nasal application of staphylococcus aureus enterotoxin b is capable of aggravating experimental allergic asthma . moreover , new insights were gained into the modulatory effects of staphylococcus aureus enterotoxin b ( saeb ) on nasal polyp tissue . in tissue of crswnp patients with an immune response to sae , the production of plts , ltb4 and lxa4 is further upregulated , while saeb significantly downregulated pge2 , cox2 , and prostanoid receptor ep2 mrna expression in fibroblasts , pointing to a direct role of sae in regulating eicosanoids as a possible mechanism of sae inflammatory reaction . the latest nomenclature of aerd considers the obvious pathogenic association of chronic rhinosinusitis with / without polyps and asthma following the exposure to nsaid , which is usually last diagnosed . especially , the coexistence of crswnp elicits a more severe clinical appearance characterized by a prolonged and fatal course leading to a higher prevalence of recurrences and a coincidence with other atopic diseases . significant morbidity and even mortality , particularly if higher doses of nsaids are ingested , may occur , if aerd is not recognized and appropriately treated . the standard procedure confirming the diagnosis of aerd is still in vivo aspirin challenge , completed by latest blood testing , for example , in patients where in vivo provocation tests are contraindicated , including patients with unstable asthma , asthma nonresponsive to corticosteroid therapy , patients with -blockers , and patients with potential compliance problems . our current knowledge on aerd focuses on a pathogenic concept based on decreased pge2 and increased levels of plt associated with overexpression of ltc4-synthase , which is accompanied by an altered transcellular metabolism of mediators of the eicosanoid cascade . there is a clear need to understand the implication of the metabolites of the eicosanoid cascade , their receptors , enzymes , and genes , the physiological and pathological impact of microbes , as well as the local and systemic function of dendritic cells , mast cells , and treg cells . the therapeutic interventions concerning the treatment of patients classified by aerd should include aspirin desensitization and medication with leukotriene modifier drugs being the most promising drugs at the moment . finally , therapeutic approaches treating staphylococcus aureus and sae effects by antibiotics or appropriate vaccination are promising .

conventional cancer treatments , such as tumor - targeting radiotherapy and systematic chemotherapy , mainly target the observable mass of tumor cells . however , the origin of cancer , that is , the origin of the first tumor cell and the subsequent sustained tumor growth and renewal , remains elusive . the two main models that have been proposed for cancer development are the stochastic model , and the hierarchical model ( figure 1 ) . in theory , cscs follow the latter model , which suggests that tumors are hierarchical in structure and heterogeneous in nature . in this model , a subpopulation of cells ( cscs ) are said to exhibit high self - renewal capacity and differentiation potential , giving rise to daughter cells with less potential , hence sustaining tumor development . the core stem cell - like properties commonly recognized among cscs in different types of cancer include the ability to self - renew , the potential to differentiate and give rise to daughter cells , the capacity to sustain tumor propagation in serial transplantation , and the exhibition of drug resistance , , . these stem cell - like biological features suggest that cscs are likely to be responsible for the initiation , propagation , and dissemination of metastatic tumor cells and the survival and relapse of tumors . the tumor - initiating role of cscs also makes them remarkable targets for cancer therapy research . since the identification of cscs in leukemia , there has been a plethora of studies on identifying cscs in solid tumors , such as ovarian cancer , renal carcinoma , head and neck squamous cell carcinoma , and bladder cancer . direct evidence of tumor regeneration from csc populations has been demonstrated in studies using genetic tracing , and linear tracing . a considerable number of publications have also been published on csc research in npc . tumors arise from an event , such as a mutation , in a certain cell that enables the cell to out - grow other subpopulations , which leads to clonal selection . b , however , the hierarchical model suggests that there is heterogeneity among the cells and that only subpopulations with high clonogenic potential are able to develop into a tumor mass with the same parental composition . the hierarchical model and the stochastic model may not necessarily be contradictory , as mutations and clonal selection may still occur within the hierarchical model . c , the concept of cscs follows the hierarchical model in that only a certain subpopulation of cells possesses sufficient potential to propagate and give rise to a tumor with the original composition . in the past few years , numerous studies have sought to identify and characterize the csc subpopulation in ebv - associated npc cell lines and primary tumors . a variety of conventional approaches used to examine npc cscs were examined in these studies . the expression of potential csc and embryonic stem cell ( esc ) markers in the cell lines and primary tumors were analyzed by multiparameter flow cytometry and immunohistochemical staining , respectively , . the anchorage - independent sphere - forming tumor cells or sphe - roids were isolated as a functional subpopulation with csc properties , . these experimental methods aimed to determine whether the target tumor cell population exhibited the commonly recognized properties of a csc population . by using fluorescence - activated cell sorting ( facs ) based on the hoechst 33342 efflux , wang et al . were the first group to demonstrate the existence of a csc side population ( sp ) in npc cell lines . however , sp cells were detected in 0.7%-6.8% of ebv - negative npc cell lines whereas the ebv - positive c666 - 1 cells contained only a scant amount of sp cells ( 0.1% ) . because the ability of sp cells to efflux out of cells is based on the atp - binding cassette ( abc ) subfamily g , member 2 ( abcg2 ) , the low expression of abcg2 in cscs may be responsible for the sparse sp in the ebv - associated npc cell line . our recent study showed that there was an absence of abcg2 up - regulation in either the cd44 or sphere - forming cells in c666 - 1 cells . in 2 npc cases , immunohistochemical staining showed relatively higher abcg2 expression in recurrent tumors than in corresponding primary tumors . nevertheless , this study did not provide sufficient evidence of the usefulness of abcg2 as a csc marker in npc . a strategy for using the sp approach to enumerate and isolate cscs in ebv - associated npc is still needed to further elucidate a panel of primary tumors . cscs in various solid tumors were successfully isolated and characterized using cell surface markers , such as cd24 , cd44 , cd90 , cd117 , and cd133 . among these markers , cd44 is a prominent antigen found in cscs in ebv - positive npc . as a hyaluronan receptor , cd44 was shown to be a csc marker in head and neck squamous cell carcinoma and has been shown to be an important molecule for anti - metastasis therapy . in the ebv - positive npc cell line c666 - 1 , the cd44 cscs in npc were demonstrated to have the exclusive ability to self - renew , differentiate , and initiate tumors in vivo and were shown to have higher resistance to 5-fluorouracil ( 5-fu ) treatment , . these findings provide solid evidence for the existence of cancer stem cells in ebv - associated npc . using flow cytometry analysis , approximately 8%-20% cd44 cells were detected in c666 - 1 cells and patient - derived xenografts . because identifying the precise csc subpopulation is crucial for further characterizing their properties and developing effective csc - targeting therapies , cd44 alone may not be sufficient to define this specific subpopulation . accordingly , a combination of cd44 with esc transcription factors may help to delineate the precise csc subpopulation . to elucidate the role of esc transcription factors in npc cscs , the expression of multiple esc transcription factors was examined in sphere - forming cells and the cd44 subpopulation . transcription factors such as octamer - binding transcription factor 4 ( oct4 ) and nanog are essential for maintaining the pluripotent phenotype of escs . these esc cd44 npc cscs were found to be enriched in transcription factors oct4 , nanog , and sex - determining region y ( sry)-box 2 ( sox2 ) . sox2 was found expressed mostly in cd44 cells , whereas less than 5% of cd44 cells expressed sox2 . sox2 is a member of the sry - related high mobility group transcription factor family and is crucial for promoting and maintaining pluripotency in embryonic and adult tissue - specific stem cells . the enriched expression of sox2 in cd44 npc cells suggests this subpopulation of cells possesses pluripotent potential . furthermore , human npc primary tumor cells expressing esc markers were also shown to be able to form secondary tumors in mouse models . cscs expressing the esc markers sox2 , oct4 , nanaog , and nestin were also found located at the invasive front in npc , which were associated with the disease aggressive behavior ( t , n , m classification and clinical stage ) and patient survival . aldehyde dehydrogenase isoform 1 ( aldh1 ) is another recognized marker of cscs , such as those in breast cancer . in our study on sphere - forming cells , significant enrichment of aldh1 expression notably , the aldh1 cells isolated from the c666 - 1 cell line demonstrated csc properties . coincidentally , strong aldh1 expression was detected at the npc invasive front , which was associated with the expression of epithelial - to - mesenchymal transition ( emt)-related biomarkers . in addition , aldh1-expressing cscs demonstrated strong association with npc tumor budding , disease aggressiveness , and poor patient survival . in addition to these markers , several cell surface markers including c - c chemokine receptor type 7 ( ccr7 ) and cd109 were found to be highly up - regulated in the sphere - forming cells using a microarray assay . ccr7 is a chemokine receptor that mediates cell migration in response to its ligand ccl21 , whereas cd109 is a glycosyl - phosphatidylinositol - anchored glycoprotein that inhibits tumor growth factor-1 ( tgf-1)-induced growth . the enrichment of the ccr7 and cd109 cell populations in the sphere - forming cells was confirmed by flow cytometry . importantly , the sphere - forming ability of npc cells was abolished after treatment with the ccr7-blocking antibody . the role of ccr7 as a new surface marker of npc cscs is currently being validated . the complex interplay between ebv and genetic aberrations in nasopharyngeal epithelial cells during tumor initiation and progression poses great challenges to npc research . our earlier studies demonstrated that several genetic aberrations were involved in the initiation of npc , which were suggested to predispose these npc cells to subsequent ebv infection . in ebv - associated npc and precancerous lesions , the persistence of the clonal ebv genome indicated that ebv latent infection facilitated tumor initiation and the transformation of nasopharyngeal epithelial cells , . recent studies have suggested that ebv - encoded proteins could induce stem cell - like properties in epithelial cells. in some precancerous lesions , it was also noted that ebv - infected cells only occur in or immediately adjacent to the basal layer of the nasopharyngeal epithelia . it has been speculated that npc originates from clonal ebv - infected basal stem cells . therefore , modulating or maintaining the stem cell properties may be a critical function of ebv in npc initiation . the significant increase in the copy number of ebv dna and the expression of ebv latent genes in isolated npc cscs further support this hypothesis . all ebv - infected npc cells exhibit type ii latency , with the expression of epstein - barr virus nuclear antigen 1 ( ebna1 ) , lmp1 , lmp2a , epstein - barr virus - encoded small rnas ( ebers ) , barts , and a number of ebv - encoded micrornas ( mirnas ) , . among these latent genes , lmp1 encodes a viral oncoprotein , which is responsible for altering multiple cellular mechanisms and signaling pathways in host cells . there is increasing evidence that lmp1 plays a role in acquiring the properties of stem cell - like or progenitor - like cells in npc . it is well known that lmp1 induces emt via twist family bhlh transcription factor 1 ( twist ) or snail family zinc finger 1 ( snail ) in npc cells . recently , kondo et al . demonstrated that lmp1-mediated emt induced the cd44 cd24 phenotype and the self - renewal properties of nasopharyngeal epithelial cells . furthermore , the special at - rich binding protein 1 ( satb1 ) up - regulated by lmp1 may contribute to cancer stemness by mediating the proper stem cell differentiation and negatively regulating the pluripotent genes . in an early study , lmp1 was proposed to induce a cancer progenitor cell ( cpc)-like rather than a csc - like phenotype due to the lack of induction of pluripotent stem cell - like genes ( e.g. , oct4 and nanog ) in lmp1-expressing nasopharyngeal epithelial cells . however , a recent study reported the induction of a sp and some csc markers such as oct4 , nanaog , b - cell - specific moloney murine leukemia virus insertion site 1 ( bmi-1 ) , and sox2 in the ebv - negative npc cell line expressing lmp1 as shown in figure 2 , lmp1 gene is highly expressed in a small subpopulation of cells in most ebv - positive npc cell lines and primary tumors . to clarify whether lmp1 induces the csc or cpc phenotypes , in addition to lmp1 , lmp2a is suspected to induce cscs in ebv - positive npc due to its function in altering the notch and hedgehog ( hh ) signaling pathways . the predominant lmp2a expression at the invasive tumor front also suggests that it plays a role in promoting tumor invasion and recurrence . demonstrated that lmp2a expression up - regulated the stem cell markers bmi1 , nanog , and sox2 and increased the size of the sp in ebv - negative npc cell lines . notably , the overexpression of lmp2a induced emt and stem - like characteristics in these cells . furthermore , the predominant expression of lmp2a at the invasive tumor front also suggested its role as a marker of csc and for predicting npc progression . ebv , epstein - barr virus ; lmp , latent membrane protein ; ccr7 , c - c chemokine receptor type 7 ; oct4 , octamer - binding transcription factor 4 ; sox2 , sex - determining region y ( sry)-box 2 ; abc , atp - binding cassette ; abcg2 , atp - binding cassette subfamily g , member 2 ; abcc3 , atp - binding cassette subfamily c , member 3 ; abcc11 , atp - binding cassette subfamily c , member 11 . in addition to the ebv latent genes , several stem cell - related pathways such as the notch and hh pathways were found to be involved in the survival and functioning of npc cscs . the notch signaling pathways are not only important in normal development , cell fate determination , and stem cell renewal , they also play a crucial role in tumorigenesis and contribute to the csc functioning in various cancer types . in npc , an early study demonstrated that a subpopulation of npc cells in primary tumors co - expressing sox2 and oct4 were involved in an activated notch1 pathway , with increased expression of the downstream effector molecule hes1 . in our study , notch3 was found to be consistently overexpressed in npc tumor cells and to be responsible for cisplatin resistance . the silencing of notch3 in npc demonstrated the suppression of stem cell - like properties such as oct4 expression , spheroid formation , and in vivo tumorigenicity . this finding implies that notch3 is a potential therapeutic target for the depletion of cscs in npc . the antitumor effects of notch receptor - specific antibodies and recombinant notch3 peptides have been demonstrated in multiple studies. integrating these targeting agents into the current therapeutic strategies may greatly enhance the outcomes for npc patients . notch4 has been identified as a crucial regulator in breast cscs , and its inhibition was shown to abolish tumor initiation . however , the functional roles of notch4 in npc cscs have yet to be elucidated . the hh signaling pathway is implicated in the maintenance of stem cells and is commonly dysregulated in ebv - associated npc . unlike notch3 signaling , the hh signaling pathway is activated by ebv through autocrine induction of the sonic hedgehog ( shh ) ligand . port et al . demonstrated that the ebv - encoded lmp1 and lmp2a proteins induced the expression of various stemness - related gene products ( bmi1 and sox2 ) and stem cell surface markers ( cd44v6 and cd133 ) via activation of the hh signaling pathway . consistent with these findings , our study also noted that the expression of gli family zinc finger 1 ( gli1 ) , an effector of the hh signaling pathway , and the ebv latent genes were enriched in npc cscs . these findings reveal the detailed mechanisms of ebv - induced csc phenotypes in npc . in npc , lymph node metastasis according to the csc theory , metastasis involves the migration of cscs from the original site to a distant organ . for chemotaxis of cells to occur , chemokine receptors are required to be present on chemotactic cells along with chemokines in the microenvironment . among the plethora of chemokines , ccr7 was shown to be related to lymph node metastasis in several types of cancers. as mentioned above , the up - regulation of ccr7 expression was found in npc cscs and may mediate cell migration in response to its ligand ccl21 in lymph nodes , . in addition to enhancing lymph node metastasis , ccr7 is crucial for the survival of cscs . we found that the blocking of this receptor with a neutralizing antibody resulted in a significant reduction in the sphere - forming ability of c666 - 1 cells . in addition to ccr7 , we noted that the chemokine ( c - x - c motif ) ligand 8 ( cxcl8 ) was highly up - regulated in the csc subpopulation . using the inhibitor of cxcl8 or its receptor cxcr2 , the contribution of cxcl8 and cxcr2 in the tumor sphere - forming properties via the phosphatidylinositol 3 kinase ( pi3k)/akt signaling pathway in npc c666 - 1 cells was determined . these findings indicate the important roles that chemokine - mediated signaling plays in the maintenance of cscs in npc . aberrant mirna expression is a key post - transcriptional regulatory mechanism in the tumorigenesis process . it has been reported that mir-200a contributes to the epithelial - mesenchymal to stem - like transition in npc cells via the targeting of zinc finger e - box - binding homeobox 2 ( zeb2 ) and catenin ( cadherin - associated protein ) , beta 1 ( ctnnb1 ) . the inhibitory effect of mir-200a expression on the properties of stem - like cells , including tumor sphere formation and in vivo tumorigenicity , and on stem cell marker expression was demonstrated in ebv - positive c666 - 1 cells . using the microrna microarray , we also noted the significant repression of multiple stemness - repressing mirnas , such as mir-96 and mir-183 , in the sphere - forming cells ( unpublished finding ) . these findings suggest that mirnas play a functional role in regulating the properties of cscs in ebv - associated npc . the failure to eradicate drug - resistant cscs has been proposed to be the core reason for tumor relapse after treatment , . the drug - resistant properties of cscs have been shown to be due to the overexpression of abc transporters , which are responsible for the efflux of therapeutic drugs , . abc transporters have been shown to be overexpressed in other cscs , such as abcb5 in hepatocellular carcinoma ( hcc ) cscs . in npc cscs , several abc transporters , such as atp - binding cassette subfamily c , member 3 ( abcc3 ) and abcc11 , were found to be highly up - regulated . furthermore , the cd44 npc cscs , which are resistant to cisplatin and doxorubicin , exhibited overexpression of abcc3 and abcc11 . these findings suggest that the overexpression of these abc transporters is responsible for the drug resistance in npc cscs . notably , abcc11 directly induces resistance to 5-fu , which is a main chemotherapeutic drug for treating npc patients . the overexpression of abcc11 in npc cscs may reduce the survival of this subpopulation during 5-fu treatment and lead to future tumor relapse . the resistance of npc cscs to radiotherapy was demonstrated in a study on the therapeutic potential of a mutant vesicular stomatitis virus ( vsv51 ) for treating ebv - positive npc . the vsv51 effectively eradicated the sphere - forming cells in multiple ebv - positive npc models and greatly enhanced their sensitivity to treatment with ionizing radiation . interestingly , depletion of the stem cell - associated gene bmi-1 was also found to significantly sensitize npc cells to radiation . furthermore , the aberrant signaling pathways ( e.g. , notch3 and pi3k / akt ) may promote the survival of the csc subpopulation in npc . because cscs resist the conventional treatments for npc , the development of novel therapies targeting cscs may provide an efficient strategy for improving patient outcomes . although few studies have investigated the targeting of the cscs in ebv - positive npc , the encouraging findings demonstrate its potential as a new clinical intervention for npc patients . as mentioned above , notch3 inhibition and the vsv51 treatment greatly enhanced the efficiency of chemotherapy and radiotherapy , respectively , in the in vitro and in vivo ebv - positive npc models , , . at13387 , a heat shock protein 90 ( hsp90 ) inhibitor , is currently undergoing clinical trials for treating various human cancers . a recent study demonstrated its potential effect in targeting the csc subpopulation in ebv - positive npc . the at13387 treatment effectively inhibited the formation of tumor spheres , reduced the expression of csc markers , and suppressed in vivo tumorigenicity of c666 - 1 cells . recently , short hairpin rna ( shrna ) approach has been used to target bmi-1 and enhance the radiotherapy sensitivity of cd44 npc cscs . thus , a similar shrna approach can be used to suppress csc - related makers and signaling molecules. notably , the loss of phosphatase and tensin homolog deleted on chromosome ten ( pten ) gene can lead to the emergence and proliferation of csc clones and reveal the potential of therapeutic targeting of tumors that lack pten expression . a similar treatment strategy can be used for npcs showing reduced ib kinase ( ikk ) expression . these findings provide preclinical evidence supporting the use of csc - targeting agents as novel drugs for the systematic treatment of npc . further elucidation of the inhibitors of the signaling pathways associated with cscs , such as the notch , wnt , and hh signaling pathways , or shrna targeting csc markers , including ccr7 and cd44 , are necessary for the development of efficient therapeutic approaches for treating npc . the application of antisense strategies for npc therapeutics has been of particular interest , especially the potential therapeutic approach in targeting ebv genes in npc . studies have demonstrated that the suppression of lmp1 in c666 - 1 cells resulted in the inhibition in tumor cell motility , adhesion , and transmembrane invasion ability , whereas the suppression of lmp2a in c666 - 1 cells resulted in the inhibition in cell proliferation and colony formation . these findings further confirmed that ebv - encoded lmps are potential therapeutic targets for ebv - associated malignancies . a recent study demonstrated that the radiosensitivity of npc was increased by using dnazyme 1 ( dz1 ) , a deoxyribozyme that was specifically designed to target lmp1 mrna . dnazyme is a small , in vitro selected dna enzyme that targets on rna substrates . shed light on the clinical use of targeting lmp1 by dnazyme in treating npc patients . ebv latent infection is not only a critical molecular event in the initiation of non - keratinizing tumors . its appearance in all tumor cells suggests that it also plays an essential role during the development of npc . the identification of the cscs in ebv - associated npc cell lines reveals that their phenotype is distinct from that in ebv - negative cell lines . the involvement of csc properties in nasopharyngeal epithelial cells by ebv - encoded lmp1 and lmp2a proteins implies that the induction and maintenance of the csc subpopulation is an important role of ebv in npc tumorigenesis . nevertheless , the precise role of ebv in cscs still needs to be demonstrated in more ebv - positive npc models or primary tumors . in most npc cases , lmp1 and lmp2a characterizing the csc properties of the subpopulations with high expression of lmp1 and lmp2a proteins may provide direct evidence of the role of ebv . furthermore , in addition to unveiling the unique features of cscs in npc , studies demonstrated that the targeted csc subpopulation have the potential to greatly enhance the efficiency of conventional radiotherapy and chemotherapy in the in vitro and in vivo models . however , only a few preclinical studies on csc - targeting agents have been conducted for ebv - associated npc . more therapeutic agents or inhibitors targeting the csc - associated signaling pathways need to be elucidated in preclinical studies and clinical trials involving npc patients . we envision that collaboration between clinicians and scientists in the fields of ebv , npc , and stem cell biology will facilitate the quest for a complete cure of npc .

although the epstein - barr virus ( ebv ) has spread to all populations in the world , ebv - associated nasopharyngeal carcinoma ( npc ) is prevalent only in south china and southeast asia . the role of ebv in the malignant transformation of nasopharyngeal epithelium is the main focus of current researches . radiotherapy and chemoradiotherapy have been successful in treating early stage npc , but the recurrence rates remain high . unfortunately , local relapse and metastasis are commonly unresponsive to conventional treatments . these recurrent and metastatic lesions are believed to arise from residual or surviving cells that have the properties of cancer stem cells . these cancer stem - like cells ( cscs ) have the ability to self - renew , differentiate , and sustain propagation . they are also chemo - resistant and can form spheres in anchorage - independent environments . this review summarizes recent researches on the cscs in ebv - associated npc , including the findings regarding cell surface markers , stem cell - related transcription factors , and various signaling pathways . in particular , the review focuses on the roles of ebv latent genes [ latent membrane protein 1 ( lmp1 ) and latent membrane protein 2a ( lmp2a ) ] , cellular micrornas , and adenosine triphosphate ( atp)-binding cassette chemodrug transporters in contributing to the properties of cscs , including the epithelial - mesenchymal transition , stem - like transition , and chemo - resistance . novel therapeutics that enhance the efficacy of radiotherapy and chemoradiotherapy and inhibitors that suppress the properties of cscs are also discussed .

The Concept of CSCs CSCs in NPC Roles of EBV in NPC CSCs CSC-associated Signaling Pathways Aberrant miRNA expression CSCs as Potential Therapeutic Targets Conclusions

conventional cancer treatments , such as tumor - targeting radiotherapy and systematic chemotherapy , mainly target the observable mass of tumor cells . however , the origin of cancer , that is , the origin of the first tumor cell and the subsequent sustained tumor growth and renewal , remains elusive . the two main models that have been proposed for cancer development are the stochastic model , and the hierarchical model ( figure 1 ) . in this model , a subpopulation of cells ( cscs ) are said to exhibit high self - renewal capacity and differentiation potential , giving rise to daughter cells with less potential , hence sustaining tumor development . the core stem cell - like properties commonly recognized among cscs in different types of cancer include the ability to self - renew , the potential to differentiate and give rise to daughter cells , the capacity to sustain tumor propagation in serial transplantation , and the exhibition of drug resistance , , . these stem cell - like biological features suggest that cscs are likely to be responsible for the initiation , propagation , and dissemination of metastatic tumor cells and the survival and relapse of tumors . the tumor - initiating role of cscs also makes them remarkable targets for cancer therapy research . since the identification of cscs in leukemia , there has been a plethora of studies on identifying cscs in solid tumors , such as ovarian cancer , renal carcinoma , head and neck squamous cell carcinoma , and bladder cancer . direct evidence of tumor regeneration from csc populations has been demonstrated in studies using genetic tracing , and linear tracing . tumors arise from an event , such as a mutation , in a certain cell that enables the cell to out - grow other subpopulations , which leads to clonal selection . c , the concept of cscs follows the hierarchical model in that only a certain subpopulation of cells possesses sufficient potential to propagate and give rise to a tumor with the original composition . in the past few years , numerous studies have sought to identify and characterize the csc subpopulation in ebv - associated npc cell lines and primary tumors . the expression of potential csc and embryonic stem cell ( esc ) markers in the cell lines and primary tumors were analyzed by multiparameter flow cytometry and immunohistochemical staining , respectively , . the anchorage - independent sphere - forming tumor cells or sphe - roids were isolated as a functional subpopulation with csc properties , . by using fluorescence - activated cell sorting ( facs ) based on the hoechst 33342 efflux , wang et al . however , sp cells were detected in 0.7%-6.8% of ebv - negative npc cell lines whereas the ebv - positive c666 - 1 cells contained only a scant amount of sp cells ( 0.1% ) . because the ability of sp cells to efflux out of cells is based on the atp - binding cassette ( abc ) subfamily g , member 2 ( abcg2 ) , the low expression of abcg2 in cscs may be responsible for the sparse sp in the ebv - associated npc cell line . a strategy for using the sp approach to enumerate and isolate cscs in ebv - associated npc is still needed to further elucidate a panel of primary tumors . cscs in various solid tumors were successfully isolated and characterized using cell surface markers , such as cd24 , cd44 , cd90 , cd117 , and cd133 . among these markers , cd44 is a prominent antigen found in cscs in ebv - positive npc . in the ebv - positive npc cell line c666 - 1 , the cd44 cscs in npc were demonstrated to have the exclusive ability to self - renew , differentiate , and initiate tumors in vivo and were shown to have higher resistance to 5-fluorouracil ( 5-fu ) treatment , . these findings provide solid evidence for the existence of cancer stem cells in ebv - associated npc . accordingly , a combination of cd44 with esc transcription factors may help to delineate the precise csc subpopulation . to elucidate the role of esc transcription factors in npc cscs , the expression of multiple esc transcription factors was examined in sphere - forming cells and the cd44 subpopulation . transcription factors such as octamer - binding transcription factor 4 ( oct4 ) and nanog are essential for maintaining the pluripotent phenotype of escs . these esc cd44 npc cscs were found to be enriched in transcription factors oct4 , nanog , and sex - determining region y ( sry)-box 2 ( sox2 ) . sox2 is a member of the sry - related high mobility group transcription factor family and is crucial for promoting and maintaining pluripotency in embryonic and adult tissue - specific stem cells . cscs expressing the esc markers sox2 , oct4 , nanaog , and nestin were also found located at the invasive front in npc , which were associated with the disease aggressive behavior ( t , n , m classification and clinical stage ) and patient survival . aldehyde dehydrogenase isoform 1 ( aldh1 ) is another recognized marker of cscs , such as those in breast cancer . in our study on sphere - forming cells , significant enrichment of aldh1 expression notably , the aldh1 cells isolated from the c666 - 1 cell line demonstrated csc properties . coincidentally , strong aldh1 expression was detected at the npc invasive front , which was associated with the expression of epithelial - to - mesenchymal transition ( emt)-related biomarkers . in addition , aldh1-expressing cscs demonstrated strong association with npc tumor budding , disease aggressiveness , and poor patient survival . in addition to these markers , several cell surface markers including c - c chemokine receptor type 7 ( ccr7 ) and cd109 were found to be highly up - regulated in the sphere - forming cells using a microarray assay . the enrichment of the ccr7 and cd109 cell populations in the sphere - forming cells was confirmed by flow cytometry . importantly , the sphere - forming ability of npc cells was abolished after treatment with the ccr7-blocking antibody . the role of ccr7 as a new surface marker of npc cscs is currently being validated . our earlier studies demonstrated that several genetic aberrations were involved in the initiation of npc , which were suggested to predispose these npc cells to subsequent ebv infection . in ebv - associated npc and precancerous lesions , the persistence of the clonal ebv genome indicated that ebv latent infection facilitated tumor initiation and the transformation of nasopharyngeal epithelial cells , . recent studies have suggested that ebv - encoded proteins could induce stem cell - like properties in epithelial cells. in some precancerous lesions , it was also noted that ebv - infected cells only occur in or immediately adjacent to the basal layer of the nasopharyngeal epithelia . it has been speculated that npc originates from clonal ebv - infected basal stem cells . therefore , modulating or maintaining the stem cell properties may be a critical function of ebv in npc initiation . the significant increase in the copy number of ebv dna and the expression of ebv latent genes in isolated npc cscs further support this hypothesis . all ebv - infected npc cells exhibit type ii latency , with the expression of epstein - barr virus nuclear antigen 1 ( ebna1 ) , lmp1 , lmp2a , epstein - barr virus - encoded small rnas ( ebers ) , barts , and a number of ebv - encoded micrornas ( mirnas ) , . among these latent genes , lmp1 encodes a viral oncoprotein , which is responsible for altering multiple cellular mechanisms and signaling pathways in host cells . there is increasing evidence that lmp1 plays a role in acquiring the properties of stem cell - like or progenitor - like cells in npc . demonstrated that lmp1-mediated emt induced the cd44 cd24 phenotype and the self - renewal properties of nasopharyngeal epithelial cells . furthermore , the special at - rich binding protein 1 ( satb1 ) up - regulated by lmp1 may contribute to cancer stemness by mediating the proper stem cell differentiation and negatively regulating the pluripotent genes . in an early study , lmp1 was proposed to induce a cancer progenitor cell ( cpc)-like rather than a csc - like phenotype due to the lack of induction of pluripotent stem cell - like genes ( e.g. however , a recent study reported the induction of a sp and some csc markers such as oct4 , nanaog , b - cell - specific moloney murine leukemia virus insertion site 1 ( bmi-1 ) , and sox2 in the ebv - negative npc cell line expressing lmp1 as shown in figure 2 , lmp1 gene is highly expressed in a small subpopulation of cells in most ebv - positive npc cell lines and primary tumors . to clarify whether lmp1 induces the csc or cpc phenotypes , in addition to lmp1 , lmp2a is suspected to induce cscs in ebv - positive npc due to its function in altering the notch and hedgehog ( hh ) signaling pathways . demonstrated that lmp2a expression up - regulated the stem cell markers bmi1 , nanog , and sox2 and increased the size of the sp in ebv - negative npc cell lines . notably , the overexpression of lmp2a induced emt and stem - like characteristics in these cells . ebv , epstein - barr virus ; lmp , latent membrane protein ; ccr7 , c - c chemokine receptor type 7 ; oct4 , octamer - binding transcription factor 4 ; sox2 , sex - determining region y ( sry)-box 2 ; abc , atp - binding cassette ; abcg2 , atp - binding cassette subfamily g , member 2 ; abcc3 , atp - binding cassette subfamily c , member 3 ; abcc11 , atp - binding cassette subfamily c , member 11 . in addition to the ebv latent genes , several stem cell - related pathways such as the notch and hh pathways were found to be involved in the survival and functioning of npc cscs . the notch signaling pathways are not only important in normal development , cell fate determination , and stem cell renewal , they also play a crucial role in tumorigenesis and contribute to the csc functioning in various cancer types . the silencing of notch3 in npc demonstrated the suppression of stem cell - like properties such as oct4 expression , spheroid formation , and in vivo tumorigenicity . this finding implies that notch3 is a potential therapeutic target for the depletion of cscs in npc . integrating these targeting agents into the current therapeutic strategies may greatly enhance the outcomes for npc patients . notch4 has been identified as a crucial regulator in breast cscs , and its inhibition was shown to abolish tumor initiation . however , the functional roles of notch4 in npc cscs have yet to be elucidated . the hh signaling pathway is implicated in the maintenance of stem cells and is commonly dysregulated in ebv - associated npc . demonstrated that the ebv - encoded lmp1 and lmp2a proteins induced the expression of various stemness - related gene products ( bmi1 and sox2 ) and stem cell surface markers ( cd44v6 and cd133 ) via activation of the hh signaling pathway . consistent with these findings , our study also noted that the expression of gli family zinc finger 1 ( gli1 ) , an effector of the hh signaling pathway , and the ebv latent genes were enriched in npc cscs . these findings reveal the detailed mechanisms of ebv - induced csc phenotypes in npc . in npc , lymph node metastasis according to the csc theory , metastasis involves the migration of cscs from the original site to a distant organ . for chemotaxis of cells to occur , chemokine receptors are required to be present on chemotactic cells along with chemokines in the microenvironment . as mentioned above , the up - regulation of ccr7 expression was found in npc cscs and may mediate cell migration in response to its ligand ccl21 in lymph nodes , . in addition to enhancing lymph node metastasis , ccr7 is crucial for the survival of cscs . we found that the blocking of this receptor with a neutralizing antibody resulted in a significant reduction in the sphere - forming ability of c666 - 1 cells . in addition to ccr7 , we noted that the chemokine ( c - x - c motif ) ligand 8 ( cxcl8 ) was highly up - regulated in the csc subpopulation . using the inhibitor of cxcl8 or its receptor cxcr2 , the contribution of cxcl8 and cxcr2 in the tumor sphere - forming properties via the phosphatidylinositol 3 kinase ( pi3k)/akt signaling pathway in npc c666 - 1 cells was determined . these findings indicate the important roles that chemokine - mediated signaling plays in the maintenance of cscs in npc . aberrant mirna expression is a key post - transcriptional regulatory mechanism in the tumorigenesis process . it has been reported that mir-200a contributes to the epithelial - mesenchymal to stem - like transition in npc cells via the targeting of zinc finger e - box - binding homeobox 2 ( zeb2 ) and catenin ( cadherin - associated protein ) , beta 1 ( ctnnb1 ) . the inhibitory effect of mir-200a expression on the properties of stem - like cells , including tumor sphere formation and in vivo tumorigenicity , and on stem cell marker expression was demonstrated in ebv - positive c666 - 1 cells . using the microrna microarray , we also noted the significant repression of multiple stemness - repressing mirnas , such as mir-96 and mir-183 , in the sphere - forming cells ( unpublished finding ) . these findings suggest that mirnas play a functional role in regulating the properties of cscs in ebv - associated npc . the failure to eradicate drug - resistant cscs has been proposed to be the core reason for tumor relapse after treatment , . the drug - resistant properties of cscs have been shown to be due to the overexpression of abc transporters , which are responsible for the efflux of therapeutic drugs , . abc transporters have been shown to be overexpressed in other cscs , such as abcb5 in hepatocellular carcinoma ( hcc ) cscs . in npc cscs , several abc transporters , such as atp - binding cassette subfamily c , member 3 ( abcc3 ) and abcc11 , were found to be highly up - regulated . furthermore , the cd44 npc cscs , which are resistant to cisplatin and doxorubicin , exhibited overexpression of abcc3 and abcc11 . the resistance of npc cscs to radiotherapy was demonstrated in a study on the therapeutic potential of a mutant vesicular stomatitis virus ( vsv51 ) for treating ebv - positive npc . interestingly , depletion of the stem cell - associated gene bmi-1 was also found to significantly sensitize npc cells to radiation . furthermore , the aberrant signaling pathways ( e.g. because cscs resist the conventional treatments for npc , the development of novel therapies targeting cscs may provide an efficient strategy for improving patient outcomes . although few studies have investigated the targeting of the cscs in ebv - positive npc , the encouraging findings demonstrate its potential as a new clinical intervention for npc patients . as mentioned above , notch3 inhibition and the vsv51 treatment greatly enhanced the efficiency of chemotherapy and radiotherapy , respectively , in the in vitro and in vivo ebv - positive npc models , , . a recent study demonstrated its potential effect in targeting the csc subpopulation in ebv - positive npc . the at13387 treatment effectively inhibited the formation of tumor spheres , reduced the expression of csc markers , and suppressed in vivo tumorigenicity of c666 - 1 cells . recently , short hairpin rna ( shrna ) approach has been used to target bmi-1 and enhance the radiotherapy sensitivity of cd44 npc cscs . thus , a similar shrna approach can be used to suppress csc - related makers and signaling molecules. notably , the loss of phosphatase and tensin homolog deleted on chromosome ten ( pten ) gene can lead to the emergence and proliferation of csc clones and reveal the potential of therapeutic targeting of tumors that lack pten expression . further elucidation of the inhibitors of the signaling pathways associated with cscs , such as the notch , wnt , and hh signaling pathways , or shrna targeting csc markers , including ccr7 and cd44 , are necessary for the development of efficient therapeutic approaches for treating npc . studies have demonstrated that the suppression of lmp1 in c666 - 1 cells resulted in the inhibition in tumor cell motility , adhesion , and transmembrane invasion ability , whereas the suppression of lmp2a in c666 - 1 cells resulted in the inhibition in cell proliferation and colony formation . these findings further confirmed that ebv - encoded lmps are potential therapeutic targets for ebv - associated malignancies . a recent study demonstrated that the radiosensitivity of npc was increased by using dnazyme 1 ( dz1 ) , a deoxyribozyme that was specifically designed to target lmp1 mrna . shed light on the clinical use of targeting lmp1 by dnazyme in treating npc patients . ebv latent infection is not only a critical molecular event in the initiation of non - keratinizing tumors . the identification of the cscs in ebv - associated npc cell lines reveals that their phenotype is distinct from that in ebv - negative cell lines . the involvement of csc properties in nasopharyngeal epithelial cells by ebv - encoded lmp1 and lmp2a proteins implies that the induction and maintenance of the csc subpopulation is an important role of ebv in npc tumorigenesis . nevertheless , the precise role of ebv in cscs still needs to be demonstrated in more ebv - positive npc models or primary tumors . in most npc cases , lmp1 and lmp2a characterizing the csc properties of the subpopulations with high expression of lmp1 and lmp2a proteins may provide direct evidence of the role of ebv . furthermore , in addition to unveiling the unique features of cscs in npc , studies demonstrated that the targeted csc subpopulation have the potential to greatly enhance the efficiency of conventional radiotherapy and chemotherapy in the in vitro and in vivo models . however , only a few preclinical studies on csc - targeting agents have been conducted for ebv - associated npc . more therapeutic agents or inhibitors targeting the csc - associated signaling pathways need to be elucidated in preclinical studies and clinical trials involving npc patients . we envision that collaboration between clinicians and scientists in the fields of ebv , npc , and stem cell biology will facilitate the quest for a complete cure of npc .

the cost of delivering health care in the united states has become one of the most important public policy issues of the 21st century . the percentage of the us gross domestic product ( gdp ) that has been consumed by health care has grown from 5% in 1960 to a staggering 16% in 2007 . according to latest projections by the congressional budget office , unless drastic measures are undertaken to control healthcare spending in the united states , total national spending on health care will claim more than 30% of the gdp by 2035 and approximately 50% by 2082 . although the problem is clearly multifactorial , many health policy analysts have pointed to the adoption of new and advanced healthcare technologies as one of the primary drivers of cost . the united states leads the world in biomedical research , and we have shown we can develop the most sophisticated care to tackle the most complex medical problems . however , our ability to deliver care effectively and efficiently to all who need it is woefully inadequate as evidenced by the high number of un- and underinsured citizens and our low ranking in measures of public health relative to other developed countries . thus , both the future health and viability of the us economy and its population are intimately related to the thoughtful and responsible adoption of new healthcare technologies . given past utilization rates and future demographic trends , the field of orthopaedic surgery can expect to be particularly impacted by this impending economic crisis . according to national healthcare statistics , musculoskeletal disorders and disease are the leading cause of disability and account for more than half of all chronic conditions in people over the age of 50 . during the period 2002 to 2004 , over 83 million americans reported one or more spine conditions , and 42 million americans reported arthritis and joint pain [ 19 , 26 ] . this resulted in an increase in ambulatory physician visits for musculoskeletal care from 426 million in 1996 to 1998 to 508 million in 2002 to 2004 as well as a 69% increase in total non - md ambulatory care visits over that same time period . the sum of direct and indirect expenditures for the burden of musculoskeletal ailments was $ 849 billion , or 7.7% of the gdp . moreover , in the next 20 years , the estimated increase of 35 million north americans over the age of 55 will further amplify the burden of musculoskeletal disease . nearly 50% of inpatient procedure volume is driven by individuals over the age of 65 , and it is estimated that by the year 2030 , close to 20% of the us population will have entered this age cohort . compounding the demographic trend of an aging baby boomer population , this unique cohort in american history has higher expectations of the healthcare system than any generation before them . although the future of the american healthcare system is wrought with both demographic and economic challenges , neither need prove insurmountable . in his research on disruptive innovations in health care , harvard business school professor clayton christensen and his colleagues provide valuable insight into one possible strategy to address this challenging , seemingly intractable problem . documents their precedence in and relation to the healthcare system , and provides specific examples from the field of orthopaedic surgery . in our concluding remarks , we address the inherent cultural and regulatory barriers to adoption of these disruptive innovations and offer some possible strategies to overcome these obstacles . in his book , christensen defines disruptive innovations as cheaper , simpler , more convenient products or services that start by meeting the needs of less - demanding customers . although the dominant players in a particular market sector focus on improving the functionality of their products or services to meet the needs of the most sophisticated customers at the high end of the market , they often miss the simpler , more convenient , and less costly alternatives initially designed to appeal to the least demanding customers at the low end of the market . over time , the simpler ( frequently technologically inferior ) products and services get better and are eventually able to meet the needs of the vast majority of consumers . sustaining innovations are those products and services marketed toward consumers at the higher end of the market . to remain leaders in their field , industries invest in research and development to create a progressively more sophisticated product , which is characterized by a steadily increasing trajectory of technologic improvement ( fig . 1 , top solid line ) . there are numerous examples of sustaining innovations in orthopaedics such as alternative hip replacement bearing surfaces , computer - assisted surgical navigation tools , total disk arthroplasty , locking plates , and bone graft substitutes . although these new technologies represent important developments that may advance the field of orthopaedics , expand the indications for orthopaedic procedures , and improve patient outcomes , they do not necessarily address the needs of the majority of our patients nor do they help address the problems of limited accessibility and affordability of musculoskeletal care.fig . 1the performance trajectory over time of disruptive versus sustaining innovations the area between the dashed lines outlines the rate of improvement consumers can absorb over time . the pace of sustaining innovation nearly always outstrips the ability of customers to absorb it , thereby creating the potential for the introduction of disruptive innovations . ( reprinted with permission from christensen cm , bohmer r , kenagy j. will disruptive innovations cure health care ? the area between the dashed lines outlines the rate of improvement consumers can absorb over time . the pace of sustaining innovation nearly always outstrips the ability of customers to absorb it , thereby creating the potential for the introduction of disruptive innovations . ( reprinted with permission from christensen cm , bohmer r , kenagy j. will disruptive innovations cure health care ? consumers ability to absorb or use the technologic advances of any innovation is finite ( fig . 1 , area between the dashed lines ) . furthermore , the pace of sustaining innovation nearly always outstrips the ability of consumers to absorb the improvements in functionality . this creates the potential for the introduction of disruptive innovations that start by meeting the needs of the least demanding consumers at the low end of the market ( fig . 1 , the performance trajectory of these disruptive innovations has a distinct but similar vector of technologic improvement compared with sustaining innovations ( fig . 1 , bottom solid line ) . over time , the functionality of these technologies improves , and eventually , they are capable of meeting the needs of the vast majority of consumers . christensen goes on to argue that the phenomenon of overshooting the needs of the average consumer and creating the potential for disruption quite accurately describes the current situation faced by the healthcare industry . sustaining innovations such as emerging technologies , specialist physicians , and academic teaching hospitals constantly strive to improve their functionality to address the needs of the sickest , most complex patients with the most demanding healthcare problems . no one would argue that these entities are contributing to the progress in the field of medicine . however , in a world of limited healthcare resources and a nation where the most medically complex patients make up a minority of the population , it could be argued that our focus should be redirected toward identifying and developing disruptive innovations . this could start by addressing the needs of less medically complex patients but over time could be used to facilitate higher - quality , more convenient , more accessible , and less costly care to the majority of healthcare consumers in the united states . the field of orthopaedics is constantly undergoing rapid development through ongoing basic science and clinical investigation . currently , the focus of most biomedical researchers and the pharmaceutical , medical device , and biotechnology industries is disproportionately centered on sustaining innovations , because of organizational structures and financial incentives . for these reasons , the impact of disruptive innovations on the future of orthopaedics and the health care of the nation can only be realized with a change in perspective and a reallocation of healthcare dollars . in the next sections , examples of disruptive innovations in four areas of orthopaedic care delivery diagnostics , we focus on the introduction and popularization of the mini - fluoroscan as a means of facilitating and increasing access to real - time imaging . in surgical techniques and technologies , the surgical implant generation network ( sign ) intramedullary nail is analyzed as a low - tech treatment option to improve the level and access of orthopaedic fracture care in the developing world . the expanding role and contributions of physician assistants ( pas ) and nurse practitioners ( nps ) to the orthopaedic team is the topic of the discussion on disruptive innovations in , we analyze the various ways in which institutions have attempted to implement the paradigm of patient - centered care by modifying existing programs and care delivery models , focusing specifically on the rise of ambulatory surgery centers . mini - fluoroscans , or mini c - arms , are mobile fluoroscopic imaging systems designed for point - of - service , real - time images that have the potential to increase access and decrease costs in the delivery of high - quality orthopaedic care . they are gaining popularity in operating rooms , emergency departments , and physician offices because of their mobility , simplicity of use , lower cost , and lower - dose radiation compared with their full - size counterparts . clearly , they do not currently offer the same image quality or functionality that full - scale fluoroscopy units or digital radiography equipment can provide , and they would not be appropriate for use in many complex procedures such as pedicle screw placement or open reduction internal fixation of a long - bone fracture . however , technologic advances have enabled these devices to provide adequate image quality for less complicated interventions such as evaluating the alignment of a distal radius fracture after closed reduction , allowing dynamic evaluation of small joint stability , or assessing the adequacy of reduction and hardware placement in the operative treatment of certain fractures . as a disruptive innovation , mini - fluoroscans offer the ability to reduce the costs associated with using radiology technicians and radiologists while at the same time increasing the autonomy of the orthopaedic surgeon to obtain and interpret his or her own radiographs . in addition , the ability to obtain real - time images cuts down on the cumulative time and money spent waiting for conventional radiographs to be obtained , processed , and then either printed or uploaded onto a picture archiving and communication system . as the technology reliably improves such as image resolution and cordless foot pedals , it is expected that the indications for use of the mini c - arm will increase . currently , there are two types of mini c - arm units available in the united states : the fluoroscan ( hologic , inc , bedford , ma ) and the xitec xiscan ( f&m control , sl , vitoria , spain ) . in a study comparing the mini - fluoroscan with a conventional mobile c - arm on its ability to maintain image quality while ( 1 ) delivering the lowest possible radiation exposure to both patient and physician ; and ( 2 ) minimizing operator effort and inconvenience , the authors of the study rated the mini c - arm as acceptable in both categories . ideally , increased use of mini c - arms could result in improved patient outcomes and increased autonomy of the orthopaedic surgeon while reducing the need for radiology technicians to operate conventional fluoroscopy units , thus lowering overhead costs for hospitals and surgery centers . the mini c - arm allows orthopaedic surgeons , emergency department physicians , and nonphysician providers to obtain fluoroscopic images , view the images in real time on a monitor , save and retrieve them later , or have a printout immediately available during the closed management of fractures . with the increased volume of patients presenting to physician offices , urgent care clinics , and emergency departments and the high use of plain radiography , the ability to substitute the use of the mini c - arm for the use of conventional fluoroscopy and digital radiography could lead to reliable cost savings and a decreased burden on the healthcare workforce . fluoroscan , a division of hologic , inc , illustrates the natural progression of disruptive innovations to a technology sector and specifically within a single company . within a year of expanding their product line by introducing the premier mini c - arm to their already established conventional c - arm market , the success of the premier resulted in the discontinuation of its larger , standard - sized c - arm . hologic executives attribute the success to the user - friendly interface of the mini c - arm and the advent of a proprietary beam alignment technology called laser aiming device ( lad ) . the lad enables the user to position the desired part of the anatomy in the center of the imaging field when the input surface of the image intensifier is obscured by drapes , arm boards , or other objects present in the operating room . currently , the company is focusing on improving their product by making the system even easier to use and streamlining system housekeeping functions like image storage and transfer . this example illustrates how a relatively simple technology that was originally inferior in functionality improved over time and was eventually able to meet the needs of most mainstream consumers , thus displacing its higher functionality predecessor ( sustaining innovation ) for certain indications . the sign system was developed in 1999 to address the orthopaedic needs of the developing world and represents a surgical technique and technology as a disruptive innovation . its founder , dr lewis zirkle , explains the sign intramedullary nail was founded to create equality of fracture care throughout the world . rather than incorporating the newest technologic advancements or design modifications to address the needs of the higher end of the fracture care market , the nail was specifically designed to be low tech , easy to use , and inexpensive to manufacture . specifically , the sign system relies on hand drills and reamers as well as a rigid slotted jig for placement of interlocking screws . this response to the recognized lack of access to fluoroscopy and reliable power instrumentation in many countries in the developing world represents the way in which a disruptive innovation can increase access to treatment . currently , approximately 40,000 sign nail procedures have been performed in over 140 programs in 40 countries . in addition to a surgical technique adapted for a resource - poor healthcare system , the sign system itself was developed to be inexpensive to manufacture , highly versatile , and widely available . a set of 100 nails , complete with the instrumentation , is sold for an initial cost of $ 20,000 . the nails themselves can be used for the humerus , tibia , and femur and inserted either in antegrade or retrograde fashion . furthermore , the philosophy behind the sign system is to provide not only equitable , but sustainable fracture care . as such , a replacement nail and the interlocking screws are sent free of charge for each one that is used as long as the treating surgeon systematically reports the details of the clinical case , implants used , and postoperative radiographs on the sign web site s surgical database . there are two published , peer - reviewed journal articles on clinical and radiographic outcomes after treatment with the sign nail system . one manuscript reviews the short- to midterm results of the sign nail for treatment of open tibia fractures in kathmandu , nepal , whereas the other reports on a consecutive series of patients who were treated with sign nails for extremity fractures in nigeria [ 12 , 24 ] . both studies report a high degree of clinical success with a low incidence of complications . in nepal , shah et al . were able to demonstrate that 86% of ( 31 of 36 ) open tibia fractures healed within 6 months with only an 8% infection rate . similarly , ikem and colleagues reported a mean time to union of 3 months , no missed interlocking screws , and a low complication rate . in their series of 40 patients , there were two cases of superficial infection , one interlocking screw failure from osteoporotic bone , and two delayed unions . like all disruptive innovations , the sign intramedullary nail system is not a static entity , but rather is evolving into other areas of fracture care . recently , dr zirkle developed a sign system for internal fixation of hip fractures that does not rely on power instruments or fluoroscopy , which is called the sign hip construct . the first clinical case in which this system was used occurred at wazir akbar khan hospital in kabul , afghanistan , in a patient who sustained an intertrochanteric hip fracture . the translation of the sign surgical technique and instrumentation from long bone fractures to hip fractures clearly demonstrates the manner in which the performance trajectory of a disruptive innovation can improve over time and begin to meet the needs of more demanding consumers ( fig . 1 , solid bottom line ) . the increased role of physician assistants ( pas ) and nurse practitioners ( nps ) in orthopaedic surgical practice is an example of healthcare providers as a disruptive innovation , because they are able to help facilitate increased access and lower costs associated with delivering high - quality orthopaedic care . although the establishment and development of the first formal pa and np educational programs and professional organizations occurred in the mid-1960s , it was not until the 1980s and 1990s that there was a rapid expansion of pa and np training programs . at that time , healthcare legislation was revised to reduce barriers to use pa and np services in a variety of healthcare settings . hmos recognized the important roles of pas and nps in controlling costs , and restrictions on resident duty hours spurred employment and postgraduate learning opportunities in hospital inpatient settings . twenty - three percent ( 11,500 ) of the estimated 50,000 clinically practicing pas work in surgical specialties or subspecialties , and there are currently 5000 pas employed in the field of orthopaedics in the united states . christensen et al . have noted that many of the most potent disruptive innovations in health care achieved success by enabling a larger population of less - skilled people to do in a more convenient , less costly setting things that historically could be performed only by expensive specialists in centralized , inconvenient locations . for pas and nps in orthopaedics , common responsibilities include taking evening and weekend calls as well as emergency department coverage , performing admission history and physical examinations , first - assisting in the operating room , providing patients with preoperative information and postoperative instructions , and performing dressing and cast changes . many of these tasks and roles that used to be performed by surgeons are clearly within the capabilities of certified pas and nps and for a fraction of the cost . moreover , according to the medical group management association ( mgma ) , pas and nps often generate revenue that more than covers their compensation . according to 2002 data , for pas in surgical practices , the employer pays 32 cents for every dollar of charges generated . this cost savings is experienced both at the practice and national level . as of january 1 , 1998 , medicare pays pas 85% of the physician fee schedule , which also holds true for first - assisting during surgery . for a surgical procedure , this translates to approximately 14% of the primary surgeon s fees ( ie , 85% of physician first assistant fees , which is 16% of the surgeon s fees ) . when an appropriate level of responsibility is bestowed on the pa or np , quality of care is not sacrificed . in fact , studies of patient satisfaction have shown a high level of satisfaction with care provided by pas and nps . in 1994 , the federal advisory group on physician assistants and the workforce concluded that published research since the profession began consistently found a high level of patient acceptance . furthermore , a comprehensive 1995 to 1996 kaiser study evaluating patient satisfaction with pas , nps , certified nurse midwives , and physicians in a managed care setting concluded that patient satisfaction with interpersonal care appears to depend on communication style and not on type of provider . with the anticipated changes in demographics in the coming decades , it can be expected that pas and nps will play an increasingly important role in the orthopaedic team by assuming a larger share of the nonoperative aspects of the orthopaedic patients care , thus allowing orthopaedic surgeons to focus on the surgical management of musculoskeletal disease . although technological advancements and the expanding role of ancillary staff may contribute to improved access and care , the greatest opportunities to improve the healthcare system as a whole and the care provided to the population is to focus on and modify the healthcare delivery systems currently in place . recently , there has been increased awareness that the current hospital system in the united states fails the individual patient in many regards as a result of the fragmented provision of care by multiple providers in a myriad of settings . in response to this , there has been an emphasis on patient - centered or patient - focused care as a low - technology solution to the existing problems . single - specialty hospitals and ambulatory surgery centers ( ascs ) have consistently enjoyed high patient volumes and high satisfaction rates , in part because of their ability to deliver focused , efficient , high - quality patient - centered care . in a recently published study , cram and colleagues reported that even after adjusting for numerous important facility and patient characteristics such as bed size , teaching status , and patient comorbidities , specialty orthopaedic hospitals delivered higher - quality care to orthopaedic patients than their general hospital counterparts . in response to this , general hospitals have had to modify their service lines and implement operational efficiencies in an attempt to compete for orthopaedic patients seeking high - quality , convenient , accessible care . as noted by christensen et al . , the basis for competition for many less complicated procedures such as elective orthopaedic surgery has changed to reward reliability , accessibility , convenience , and low cost rather than higher functionality . ambulatory surgery centers satisfy the criteria for a disruptive innovation in the field of care delivery in that they are a cheaper , simpler , more convenient alternative to hospital operating rooms , and they were initially designed to appeal to the low end of the market . an asc is a facility that by definition provides surgical treatment that does not require hospitalization . orthopaedic subspecialties amenable to ambulatory surgery include arthroscopy , sports - related injuries , hand , some spine as well as foot / ankle procedures , and the list is expanding . many of the initial ascs struggled after their debut in 1972 as a result of inadequate reimbursement and the need to establish themselves in the eyes of investors as a high - quality , profitable investment . after 1982 and the approval of medicare reimbursement for ascs , there has been substantial growth in the number of ascs in the united states . today there are more than 4000 ascs throughout the united states compared with 275 in 1980 and 1450 in 1990 [ 2 , 14 ] . from 1996 to 2003 , the number of ascs increased by 50% , from 2425 to 3646 , whereas the number of hospital - based outpatient surgery centers dropped by 16% . one of the primary reasons for the success of ascs when compared with hospital - based outpatient surgery centers is their profitability . the pennsylvania health cost containment council reports that free - standing ascs have an average net margin of 12% , whereas hospital outpatient surgery centers are on average barely profitable at 2% . these differences in profitability have led to payment reforms that have limited payment for ascs to 65% of the payment for hospital - based outpatient surgery centers for the same procedures . these types of misguided payment reforms incentivize less efficient care and threaten the future viability of ascs to deliver high - quality , cost - efficient care . the literature supports the fact that increased volume and profits have not come at the expense of quality of care . in fact , the american association for accreditation of ambulatory surgery facilities , in its ongoing effort to improve patient care , has developed an internet - based quality improvement and peer review program to analyze outcomes for the surgery centers it accredits . each surgeon must report all unanticipated sequelae , and at least six random cases are reviewed by an accepted peer review group biannually . in a study conducted over 2 years ( 2001 to 2002 ) , one unanticipated sequelae occurred in every 299 procedures ( incidence of 0.33% ) , and a death occurred in one in 58,810 procedures ( 0.0017% ) , which was comparable whether the procedure was performed in an accredited ambulatory surgery facility or a hospital - based surgical facility . thus , it appears that ascs , rather than being static entities , are constantly innovating , streamlining , and incorporating technologic advances in surgical devices , techniques , and anesthesia protocols to expand the conditions for which same - day surgery is safe and appropriate . disruptive innovations in health care , in theory , offer lower cost , more efficient and convenient alternatives while maintaining , if not improving , the quality of care provided to patients . therefore , it might seem unusual that there would be any resistance to their adoption . yet as christensen aptly notes , healthcare may be the most entrenched , change - averse industry in the united states . from physicians to hospitals , health plans to implant manufacturers and regulators , it appears many stakeholders in the us healthcare system support the status quo to maintain their positions and profits . although no one would blame the fact that most healthcare stakeholders feel a keen sense of defensiveness to protect their piece of an ever diminishing pie , without thoughtful , voluntary sacrifice , we risk losing control of how our piece is divided . it is at these crossroads specifically where providers can regain some control of the practice of medicine . the rise of managed care , changes in reimbursement rates from third - party payers , and the diffusion of new technologies to the market have intensified turf wars between the different professional guilds of medical practitioners . disruptive innovations , some would argue , are only stoking the flames , because they allow procedures to be performed more efficiently in a more convenient location by providers who had previously been unable to provide such a service . in orthopaedic surgery , examples of turf wars include the ongoing battle with radiologists over the use of in - office diagnostic imaging and conflicts with podiatrists and physical therapists over the scope of their practice . these disputes are provider - centric conflicts that consume valuable resources and energy that could be redirected to enhance the value of care we provide by increasing quality and reducing costs . hospitals , by virtue of their size , associated overhead , and their large investments in the latest sustaining technologies , are similarly wary of disruptive innovations , because they may threaten their long - term financial viability . the rise in ascs and single - specialty hospitals have shown investors and physicians alike that high - quality care can be provided with a much higher return on investment when facilities are patient - oriented , care delivery and operations are streamlined , and physicians and personnel are well integrated . clearly , general hospitals are at a distinct disadvantage when it comes to competing for profits with standalone ascs and single - specialty hospitals , because the burden of the uninsured and underinsured falls squarely on the shoulders of the former and the scope of practice differs greatly between the various healthcare delivery systems . however , comparisons can and should be drawn between the greater operating efficiencies of the ambulatory surgery centers as compared with hospital - based outpatient surgery centers . the margin differentials that exist between these two delivery systems can not be explained simply by the patient population cared for or services provided . although it may be true that asc s are guilty of cherry picking patients , an analysis of the systems in place in these facilities may provide valuable lessons to improve the efficiencies of the hospital - based outpatient surgery centers . unfortunately , it appears many hospitals are ill - equipped to compete in today s flexible , highly competitive healthcare delivery market . commercial health plans and other third - party payers , with their strong focus on profit margins , would seem to be an ally of disruptive innovations in that these technologies hold the promise of providing cost savings without sacrificing quality of care . however , commercial payers only reimburse for procedures that have been clinically validated , and many disruptive innovations , by virtue of being recent entrants to the market , do not yet have peer - reviewed research to support their efficacy compared with so - called gold standard treatment interventions . by labeling many new potentially disruptive procedures as investigational , private insurers stifle growth and innovation that could lead to higher - quality , lower - cost care in the long run . the rise in direct - to - consumer advertising of hip and knee replacement implants , primarily by implant manufacturers , but also by hospitals and surgeons , to gain a more competitive position in the lucrative and expanding arthroplasty market also poses a formidable institutional barrier to the adoption of disruptive innovations . it is the sustaining innovations that currently fuel the research and development of the orthopaedic device manufacturers such as alternative bearings for use in tha , computer - assisted surgical navigation tools , and new suture fixation devices . although these devices have the potential to improve patient outcomes , they are consistently introduced into the market at a substantial cost premium and before validation of their clinical benefit and cost - effectiveness through well - designed , prospective clinical studies . patients , as consumers , are shielded from the costs of orthopaedic implants and are often presented with misleading information about the benefits of particular products , which in turn may lead to inappropriate demands for newer , more costly , unproven technologies , strain the physician - patient relationship , and potentially contribute to the rising cost of health care . regulators , as opposed to physicians , care delivery systems , and third - party payers , have no financial incentive to resist the diffusion of disruptive innovations . although originally established to protect both the consumer and society from unforeseeable risk as well as enforce the standards established by physicians , regulatory bodies often indirectly curb the diffusion of disruptive innovations by protecting and enforcing the status quo , which happens to be on the side of sustaining innovations . one of the main obstacles to diffusion of disruptive innovations stems from a dearth of published research demonstrating safety and efficacy . the lengthy and cumbersome fda approval process for investigational devices and the bureaucratically complex hipaa regulations are two key examples of how regulators indirectly impede the diffusion of disruptive innovations . additionally , the enforcement of stringent intellectual property laws has the unintended consequence of limiting the introduction of generic drugs and device alternatives . in his book , the innovator s dilemma , christensen defines disruptive innovations as cheaper , simpler , more convenient products or services that start by meeting the needs of less - demanding customers . although the dominant players in a particular market sector focus on improving the functionality of their products or services to meet the needs of the most sophisticated customers at the high end of the market , they often miss the simpler , more convenient , and less costly alternatives initially designed to appeal to the least demanding customers at the low end of the market . over time , the simpler ( frequently technologically inferior ) products and services get better and are eventually able to meet the needs of the vast majority of consumers . sustaining innovations are those products and services marketed toward consumers at the higher end of the market . to remain leaders in their field , industries invest in research and development to create a progressively more sophisticated product , which is characterized by a steadily increasing trajectory of technologic improvement ( fig . 1 , top solid line ) . there are numerous examples of sustaining innovations in orthopaedics such as alternative hip replacement bearing surfaces , computer - assisted surgical navigation tools , total disk arthroplasty , locking plates , and bone graft substitutes . although these new technologies represent important developments that may advance the field of orthopaedics , expand the indications for orthopaedic procedures , and improve patient outcomes , they do not necessarily address the needs of the majority of our patients nor do they help address the problems of limited accessibility and affordability of musculoskeletal care.fig . 1the performance trajectory over time of disruptive versus sustaining innovations the area between the dashed lines outlines the rate of improvement consumers can absorb over time . the pace of sustaining innovation nearly always outstrips the ability of customers to absorb it , thereby creating the potential for the introduction of disruptive innovations . ( reprinted with permission from christensen cm , bohmer r , kenagy j. will disruptive innovations cure health care ? the area between the dashed lines outlines the rate of improvement consumers can absorb over time . the pace of sustaining innovation nearly always outstrips the ability of customers to absorb it , thereby creating the potential for the introduction of disruptive innovations . ( reprinted with permission from christensen cm , bohmer r , kenagy j. will disruptive innovations cure health care ? consumers ability to absorb or use the technologic advances of any innovation is finite ( fig . 1 , area between the dashed lines ) . furthermore , the pace of sustaining innovation nearly always outstrips the ability of consumers to absorb the improvements in functionality . this creates the potential for the introduction of disruptive innovations that start by meeting the needs of the least demanding consumers at the low end of the market ( fig . 1 , bottom dashed line ) . the performance trajectory of these disruptive innovations has a distinct but similar vector of technologic improvement compared with sustaining innovations ( fig . 1 , bottom solid line ) . over time , the functionality of these technologies improves , and eventually , they are capable of meeting the needs of the vast majority of consumers . christensen goes on to argue that the phenomenon of overshooting the needs of the average consumer and creating the potential for disruption quite accurately describes the current situation faced by the healthcare industry . sustaining innovations such as emerging technologies , specialist physicians , and academic teaching hospitals constantly strive to improve their functionality to address the needs of the sickest , most complex patients with the most demanding healthcare problems . no one would argue that these entities are contributing to the progress in the field of medicine . however , in a world of limited healthcare resources and a nation where the most medically complex patients make up a minority of the population , it could be argued that our focus should be redirected toward identifying and developing disruptive innovations . this could start by addressing the needs of less medically complex patients but over time could be used to facilitate higher - quality , more convenient , more accessible , and less costly care to the majority of healthcare consumers in the united states . the field of orthopaedics is constantly undergoing rapid development through ongoing basic science and clinical investigation . currently , the focus of most biomedical researchers and the pharmaceutical , medical device , and biotechnology industries is disproportionately centered on sustaining innovations , because of organizational structures and financial incentives . for these reasons , the impact of disruptive innovations on the future of orthopaedics and the health care of the nation can only be realized with a change in perspective and a reallocation of healthcare dollars . in the next sections , examples of disruptive innovations in four areas of orthopaedic care delivery illustrate various manners in which they may impact the healthcare system . in diagnostics , we focus on the introduction and popularization of the mini - fluoroscan as a means of facilitating and increasing access to real - time imaging . in surgical techniques and technologies , the surgical implant generation network ( sign ) intramedullary nail is analyzed as a low - tech treatment option to improve the level and access of orthopaedic fracture care in the developing world . the expanding role and contributions of physician assistants ( pas ) and nurse practitioners ( nps ) to the orthopaedic team is the topic of the discussion on disruptive innovations in , we analyze the various ways in which institutions have attempted to implement the paradigm of patient - centered care by modifying existing programs and care delivery models , focusing specifically on the rise of ambulatory surgery centers . mini - fluoroscans , or mini c - arms , are mobile fluoroscopic imaging systems designed for point - of - service , real - time images that have the potential to increase access and decrease costs in the delivery of high - quality orthopaedic care . they are gaining popularity in operating rooms , emergency departments , and physician offices because of their mobility , simplicity of use , lower cost , and lower - dose radiation compared with their full - size counterparts . clearly , they do not currently offer the same image quality or functionality that full - scale fluoroscopy units or digital radiography equipment can provide , and they would not be appropriate for use in many complex procedures such as pedicle screw placement or open reduction internal fixation of a long - bone fracture . however , technologic advances have enabled these devices to provide adequate image quality for less complicated interventions such as evaluating the alignment of a distal radius fracture after closed reduction , allowing dynamic evaluation of small joint stability , or assessing the adequacy of reduction and hardware placement in the operative treatment of certain fractures . as a disruptive innovation , mini - fluoroscans offer the ability to reduce the costs associated with using radiology technicians and radiologists while at the same time increasing the autonomy of the orthopaedic surgeon to obtain and interpret his or her own radiographs . in addition , the ability to obtain real - time images cuts down on the cumulative time and money spent waiting for conventional radiographs to be obtained , processed , and then either printed or uploaded onto a picture archiving and communication system . as the technology reliably improves such as image resolution and cordless foot pedals , it is expected that the indications for use of the mini c - arm will increase . currently , there are two types of mini c - arm units available in the united states : the fluoroscan ( hologic , inc , bedford , ma ) and the xitec xiscan ( f&m control , sl , vitoria , spain ) . in a study comparing the mini - fluoroscan with a conventional mobile c - arm on its ability to maintain image quality while ( 1 ) delivering the lowest possible radiation exposure to both patient and physician ; and ( 2 ) minimizing operator effort and inconvenience , the authors of the study rated the mini c - arm as acceptable in both categories . ideally , increased use of mini c - arms could result in improved patient outcomes and increased autonomy of the orthopaedic surgeon while reducing the need for radiology technicians to operate conventional fluoroscopy units , thus lowering overhead costs for hospitals and surgery centers . the mini c - arm allows orthopaedic surgeons , emergency department physicians , and nonphysician providers to obtain fluoroscopic images , view the images in real time on a monitor , save and retrieve them later , or have a printout immediately available during the closed management of fractures . with the increased volume of patients presenting to physician offices , urgent care clinics , and emergency departments and the high use of plain radiography , the ability to substitute the use of the mini c - arm for the use of conventional fluoroscopy and digital radiography could lead to reliable cost savings and a decreased burden on the healthcare workforce . fluoroscan , a division of hologic , inc , illustrates the natural progression of disruptive innovations to a technology sector and specifically within a single company . within a year of expanding their product line by introducing the premier mini c - arm to their already established conventional c - arm market , the success of the premier resulted in the discontinuation of its larger , standard - sized c - arm . hologic executives attribute the success to the user - friendly interface of the mini c - arm and the advent of a proprietary beam alignment technology called laser aiming device ( lad ) . the lad enables the user to position the desired part of the anatomy in the center of the imaging field when the input surface of the image intensifier is obscured by drapes , arm boards , or other objects present in the operating room . currently , the company is focusing on improving their product by making the system even easier to use and streamlining system housekeeping functions like image storage and transfer . this example illustrates how a relatively simple technology that was originally inferior in functionality improved over time and was eventually able to meet the needs of most mainstream consumers , thus displacing its higher functionality predecessor ( sustaining innovation ) for certain indications . the sign system was developed in 1999 to address the orthopaedic needs of the developing world and represents a surgical technique and technology as a disruptive innovation . its founder , dr lewis zirkle , explains the sign intramedullary nail was founded to create equality of fracture care throughout the world . rather than incorporating the newest technologic advancements or design modifications to address the needs of the higher end of the fracture care market , the nail was specifically designed to be low tech , easy to use , and inexpensive to manufacture specifically , the sign system relies on hand drills and reamers as well as a rigid slotted jig for placement of interlocking screws . this response to the recognized lack of access to fluoroscopy and reliable power instrumentation in many countries in the developing world represents the way in which a disruptive innovation can increase access to treatment . currently , approximately 40,000 sign nail procedures have been performed in over 140 programs in 40 countries . in addition to a surgical technique adapted for a resource - poor healthcare system , the sign system itself was developed to be inexpensive to manufacture , highly versatile , and widely available . a set of 100 nails , complete with the instrumentation , is sold for an initial cost of $ 20,000 . the nails themselves can be used for the humerus , tibia , and femur and inserted either in antegrade or retrograde fashion . furthermore , the philosophy behind the sign system is to provide not only equitable , but sustainable fracture care . as such , a replacement nail and the interlocking screws are sent free of charge for each one that is used as long as the treating surgeon systematically reports the details of the clinical case , implants used , and postoperative radiographs on the sign web site s surgical database . there are two published , peer - reviewed journal articles on clinical and radiographic outcomes after treatment with the sign nail system . one manuscript reviews the short- to midterm results of the sign nail for treatment of open tibia fractures in kathmandu , nepal , whereas the other reports on a consecutive series of patients who were treated with sign nails for extremity fractures in nigeria [ 12 , 24 ] . both studies report a high degree of clinical success with a low incidence of complications . in nepal , shah et al . were able to demonstrate that 86% of ( 31 of 36 ) open tibia fractures healed within 6 months with only an 8% infection rate . similarly , ikem and colleagues reported a mean time to union of 3 months , no missed interlocking screws , and a low complication rate . in their series of 40 patients , there were two cases of superficial infection , one interlocking screw failure from osteoporotic bone , and two delayed unions . like all disruptive innovations , the sign intramedullary nail system is not a static entity , but rather is evolving into other areas of fracture care . recently , dr zirkle developed a sign system for internal fixation of hip fractures that does not rely on power instruments or fluoroscopy , which is called the sign hip construct . the first clinical case in which this system was used occurred at wazir akbar khan hospital in kabul , afghanistan , in a patient who sustained an intertrochanteric hip fracture . the translation of the sign surgical technique and instrumentation from long bone fractures to hip fractures clearly demonstrates the manner in which the performance trajectory of a disruptive innovation can improve over time and begin to meet the needs of more demanding consumers ( fig . 1 , solid bottom line ) . the increased role of physician assistants ( pas ) and nurse practitioners ( nps ) in orthopaedic surgical practice is an example of healthcare providers as a disruptive innovation , because they are able to help facilitate increased access and lower costs associated with delivering high - quality orthopaedic care . although the establishment and development of the first formal pa and np educational programs and professional organizations occurred in the mid-1960s , it was not until the 1980s and 1990s that there was a rapid expansion of pa and np training programs . at that time , healthcare legislation was revised to reduce barriers to use pa and np services in a variety of healthcare settings . hmos recognized the important roles of pas and nps in controlling costs , and restrictions on resident duty hours spurred employment and postgraduate learning opportunities in hospital inpatient settings . twenty - three percent ( 11,500 ) of the estimated 50,000 clinically practicing pas work in surgical specialties or subspecialties , and there are currently 5000 pas employed in the field of orthopaedics in the united states . christensen et al . have noted that many of the most potent disruptive innovations in health care achieved success by enabling a larger population of less - skilled people to do in a more convenient , less costly setting things that historically could be performed only by expensive specialists in centralized , inconvenient locations . for pas and nps in orthopaedics , common responsibilities include taking evening and weekend calls as well as emergency department coverage , performing admission history and physical examinations , first - assisting in the operating room , providing patients with preoperative information and postoperative instructions , and performing dressing and cast changes . many of these tasks and roles that used to be performed by surgeons are clearly within the capabilities of certified pas and nps and for a fraction of the cost . moreover , according to the medical group management association ( mgma ) , pas and nps often generate revenue that more than covers their compensation . according to 2002 data , for pas in surgical practices , the employer pays 32 cents for every dollar of charges generated . this cost savings is experienced both at the practice and national level . as of january 1 , 1998 , medicare pays pas 85% of the physician fee schedule , which also holds true for first - assisting during surgery . for a surgical procedure , this translates to approximately 14% of the primary surgeon s fees ( ie , 85% of physician first assistant fees , which is 16% of the surgeon s fees ) . when an appropriate level of responsibility is bestowed on the pa or np , quality of care is not sacrificed . in fact , studies of patient satisfaction have shown a high level of satisfaction with care provided by pas and nps . in 1994 , the federal advisory group on physician assistants and the workforce concluded that published research since the profession began consistently found a high level of patient acceptance . furthermore , a comprehensive 1995 to 1996 kaiser study evaluating patient satisfaction with pas , nps , certified nurse midwives , and physicians in a managed care setting concluded that patient satisfaction with interpersonal care appears to depend on communication style and not on type of provider . with the anticipated changes in demographics in the coming decades , it can be expected that pas and nps will play an increasingly important role in the orthopaedic team by assuming a larger share of the nonoperative aspects of the orthopaedic patients care , thus allowing orthopaedic surgeons to focus on the surgical management of musculoskeletal disease . although technological advancements and the expanding role of ancillary staff may contribute to improved access and care , the greatest opportunities to improve the healthcare system as a whole and the care provided to the population is to focus on and modify the healthcare delivery systems currently in place . recently , there has been increased awareness that the current hospital system in the united states fails the individual patient in many regards as a result of the fragmented provision of care by multiple providers in a myriad of settings . in response to this , there has been an emphasis on patient - centered or patient - focused care as a low - technology solution to the existing problems . single - specialty hospitals and ambulatory surgery centers ( ascs ) have consistently enjoyed high patient volumes and high satisfaction rates , in part because of their ability to deliver focused , efficient , high - quality patient - centered care . in a recently published study , cram and colleagues reported that even after adjusting for numerous important facility and patient characteristics such as bed size , teaching status , and patient comorbidities , specialty orthopaedic hospitals delivered higher - quality care to orthopaedic patients than their general hospital counterparts . in response to this , general hospitals have had to modify their service lines and implement operational efficiencies in an attempt to compete for orthopaedic patients seeking high - quality , convenient , accessible care . as noted by christensen et al . , the basis for competition for many less complicated procedures such as elective orthopaedic surgery has changed to reward reliability , accessibility , convenience , and low cost rather than higher functionality . ambulatory surgery centers satisfy the criteria for a disruptive innovation in the field of care delivery in that they are a cheaper , simpler , more convenient alternative to hospital operating rooms , and they were initially designed to appeal to the low end of the market . an asc is a facility that by definition provides surgical treatment that does not require hospitalization . orthopaedic subspecialties amenable to ambulatory surgery include arthroscopy , sports - related injuries , hand , some spine as well as foot / ankle procedures , and the list is expanding . many of the initial ascs struggled after their debut in 1972 as a result of inadequate reimbursement and the need to establish themselves in the eyes of investors as a high - quality , profitable investment . after 1982 and the approval of medicare reimbursement for ascs , there has been substantial growth in the number of ascs in the united states . today there are more than 4000 ascs throughout the united states compared with 275 in 1980 and 1450 in 1990 [ 2 , 14 ] . from 1996 to 2003 , the number of ascs increased by 50% , from 2425 to 3646 , whereas the number of hospital - based outpatient surgery centers dropped by 16% . one of the primary reasons for the success of ascs when compared with hospital - based outpatient surgery centers is their profitability . the pennsylvania health cost containment council reports that free - standing ascs have an average net margin of 12% , whereas hospital outpatient surgery centers are on average barely profitable at 2% . these differences in profitability have led to payment reforms that have limited payment for ascs to 65% of the payment for hospital - based outpatient surgery centers for the same procedures . these types of misguided payment reforms incentivize less efficient care and threaten the future viability of ascs to deliver high - quality , cost - efficient care . the literature supports the fact that increased volume and profits have not come at the expense of quality of care . in fact , the american association for accreditation of ambulatory surgery facilities , in its ongoing effort to improve patient care , has developed an internet - based quality improvement and peer review program to analyze outcomes for the surgery centers it accredits . each surgeon must report all unanticipated sequelae , and at least six random cases are reviewed by an accepted peer review group biannually . in a study conducted over 2 years ( 2001 to 2002 ) , one unanticipated sequelae occurred in every 299 procedures ( incidence of 0.33% ) , and a death occurred in one in 58,810 procedures ( 0.0017% ) , which was comparable whether the procedure was performed in an accredited ambulatory surgery facility or a hospital - based surgical facility . thus , it appears that ascs , rather than being static entities , are constantly innovating , streamlining , and incorporating technologic advances in surgical devices , techniques , and anesthesia protocols to expand the conditions for which same - day surgery is safe and appropriate . disruptive innovations in health care , in theory , offer lower cost , more efficient and convenient alternatives while maintaining , if not improving , the quality of care provided to patients . therefore , it might seem unusual that there would be any resistance to their adoption . yet as christensen aptly notes , healthcare may be the most entrenched , change - averse industry in the united states . from physicians to hospitals , health plans to implant manufacturers and regulators , it appears many stakeholders in the us healthcare system support the status quo to maintain their positions and profits . although no one would blame the fact that most healthcare stakeholders feel a keen sense of defensiveness to protect their piece of an ever diminishing pie , without thoughtful , voluntary sacrifice , we risk losing control of how our piece is divided . it is at these crossroads specifically where providers can regain some control of the practice of medicine . the rise of managed care , changes in reimbursement rates from third - party payers , and the diffusion of new technologies to the market have intensified turf wars between the different professional guilds of medical practitioners . disruptive innovations , some would argue , are only stoking the flames , because they allow procedures to be performed more efficiently in a more convenient location by providers who had previously been unable to provide such a service . in orthopaedic surgery , examples of turf wars include the ongoing battle with radiologists over the use of in - office diagnostic imaging and conflicts with podiatrists and physical therapists over the scope of their practice . these disputes are provider - centric conflicts that consume valuable resources and energy that could be redirected to enhance the value of care we provide by increasing quality and reducing costs . hospitals , by virtue of their size , associated overhead , and their large investments in the latest sustaining technologies , are similarly wary of disruptive innovations , because they may threaten their long - term financial viability . the rise in ascs and single - specialty hospitals have shown investors and physicians alike that high - quality care can be provided with a much higher return on investment when facilities are patient - oriented , care delivery and operations are streamlined , and physicians and personnel are well integrated . clearly , general hospitals are at a distinct disadvantage when it comes to competing for profits with standalone ascs and single - specialty hospitals , because the burden of the uninsured and underinsured falls squarely on the shoulders of the former and the scope of practice differs greatly between the various healthcare delivery systems . however , comparisons can and should be drawn between the greater operating efficiencies of the ambulatory surgery centers as compared with hospital - based outpatient surgery centers . the margin differentials that exist between these two delivery systems can not be explained simply by the patient population cared for or services provided . although it may be true that asc s are guilty of cherry picking patients , an analysis of the systems in place in these facilities may provide valuable lessons to improve the efficiencies of the hospital - based outpatient surgery centers . unfortunately , it appears many hospitals are ill - equipped to compete in today s flexible , highly competitive healthcare delivery market . commercial health plans and other third - party payers , with their strong focus on profit margins , would seem to be an ally of disruptive innovations in that these technologies hold the promise of providing cost savings without sacrificing quality of care . however , commercial payers only reimburse for procedures that have been clinically validated , and many disruptive innovations , by virtue of being recent entrants to the market , do not yet have peer - reviewed research to support their efficacy compared with so - called gold standard treatment interventions . by labeling many new potentially disruptive procedures as investigational , private insurers stifle growth and innovation that could lead to higher - quality , lower - cost care in the long run . the rise in direct - to - consumer advertising of hip and knee replacement implants , primarily by implant manufacturers , but also by hospitals and surgeons , to gain a more competitive position in the lucrative and expanding arthroplasty market also poses a formidable institutional barrier to the adoption of disruptive innovations . it is the sustaining innovations that currently fuel the research and development of the orthopaedic device manufacturers such as alternative bearings for use in tha , computer - assisted surgical navigation tools , and new suture fixation devices . although these devices have the potential to improve patient outcomes , they are consistently introduced into the market at a substantial cost premium and before validation of their clinical benefit and cost - effectiveness through well - designed , prospective clinical studies . patients , as consumers , are shielded from the costs of orthopaedic implants and are often presented with misleading information about the benefits of particular products , which in turn may lead to inappropriate demands for newer , more costly , unproven technologies , strain the physician - patient relationship , and potentially contribute to the rising cost of health care . regulators , as opposed to physicians , care delivery systems , and third - party payers , have no financial incentive to resist the diffusion of disruptive innovations . although originally established to protect both the consumer and society from unforeseeable risk as well as enforce the standards established by physicians , regulatory bodies often indirectly curb the diffusion of disruptive innovations by protecting and enforcing the status quo , which happens to be on the side of sustaining innovations . one of the main obstacles to diffusion of disruptive innovations stems from a dearth of published research demonstrating safety and efficacy . the lengthy and cumbersome fda approval process for investigational devices and the bureaucratically complex hipaa regulations are two key examples of how regulators indirectly impede the diffusion of disruptive innovations . additionally , the enforcement of stringent intellectual property laws has the unintended consequence of limiting the introduction of generic drugs and device alternatives . the us healthcare system is clearly in crisis , and current strategies have proven inadequate . many healthcare institutions and healthcare technologies have overshot the needs of most patients with nonlife - threatening conditions who require elective surgical treatment such as many orthopaedic patients . as christensen et al . have noted in their work , the market for simple , elective surgical procedures no longer rewards higher functionality , but rather rewards convenience , accessibility , low cost , and reliability . in this article , we have applied the concept of disruptive innovations popularized by christensen to the field of orthopaedics to illustrate examples in which simpler , cheaper alternatives have improved access of care , increased patient satisfaction , and empowered clinicians to provide higher - quality , more convenient and efficient care to their patients . although we chose to focus on the specific examples of disruptive innovations described , there are many other potential examples related to musculoskeletal care delivery , including calcium phosphate cement for the treatment of distal radius fractures in the elderly , musculoskeletal ultrasound , and medical tourism . one of the defining characteristics of a disruptive innovation is a performance trajectory that allows it to eventually meet the demands of more sophisticated consumers . each of our examples has the potential to continually evolve , improve , and thereby become more disruptive over time . specifically , the technology underlying the mini - fluoroscans may improve to the point that the image quality achieved is comparable to that of conventional c - arms . furthermore , although a number of the larger c - arms now are able to create fluoroscopic computed tomography reformats for three - dimensional detail , one could imagine that the mini c - arms may incorporate this feature in the years to come . this would allow for an expansion of current indications such as evaluation of complex , intraarticular fractures by these smaller imaging machines . additionally , less expensive radiographic printers may be developed to allow for point - of - service printing of formal radiographs , thereby obviating any dependence on the radiology department . in regard to increasing the role of pas and nps , these so - called midlevel providers have become an integral part of most orthopaedic practices , functioning as primary care musculoskeletal providers and thus allowing orthopaedic surgeons to focus on the surgical management of musculoskeletal disease . similarly , ascs will continue to grow their market share as surgeons and anesthesiologists collaborate to develop perioperative protocols that allow for more complex procedures to be performed as same day surgeries without compromising safety ( eg , improving preoperative education , including the proper use of assistive devices , providing appropriate preoperative education to patients and their families , and discharging patients home with indwelling pain catheters ) . the sustaining orthopaedic innovations that are repeatedly introduced into the marketplace will continue to benefit our patients and advance the field of orthopaedics . to date , health care in general and the field of orthopaedic surgery in particular has focused a disproportionate share of resources and effort on the problems of a medical minority . to improve the overall health of the nation , efforts and resources should be redirected toward technologies that improve access and tackle issues of affordability to address the needs of the majority . although it would be nave to believe all of the ills of the healthcare system could be reversed simply by the development and diffusion of disruptive innovations , not actively attempting to identify and cultivate them will lead us further down our current path toward unsustainable healthcare inflation and an ever increasing gap between the health care we are capable of delivering based on technologic innovation and the health care we are able to deliver based on limited resources . moreover , the concept of disruptive innovations offers the opportunity for clinicians to regain leadership in health care by working together with other healthcare leaders to deliver higher - quality , more convenient and accessible cost - effective care to our patients .

the us healthcare system is currently facing daunting demographic and economic challenges . because musculoskeletal disorders and disease represent a substantial and growing portion of this healthcare burden , novel approaches will be needed to continue to provide high - quality , affordable , and accessible orthopaedic care to our population . the concept of disruptive innovations , which has been studied and popularized by harvard business school professor clayton christensen , may offer a potential framework for developing strategies to improve quality and control costs associated with musculoskeletal care . the introduction of mobile fluoroscopic imaging systems , the development of the surgical implant generation network intramedullary nail for treatment of long bone fractures in the developing world , the expanding role and contributions of physician assistants and nurse practitioners to the orthopaedic team , and the rise of ambulatory surgery centers are all examples of disruptive innovations in the field of orthopaedics . although numerous cultural and regulatory barriers have limited the widespread adoption of these disruptive innovations , we believe they represent an opportunity for clinicians to regain leadership in health care while at the same time improving quality and access to care for patients with musculoskeletal disease .

Introduction Disruptive Innovations Diagnostics: Point-of-service Radiology: the Mini-fluoroscan Surgical Techniques & Technology: SIGN (Surgical Implant Generation Network) System to Create Equality of Fracture Care Throughout the World Process of Care: Physician Assistants and Nurse PractitionersColleagues, Not Competitors Healthcare Delivery Systems: The Rise of Ambulatory Surgery Centers Reasons for Nonadoption: Barriers to Progress Discussion

thus , both the future health and viability of the us economy and its population are intimately related to the thoughtful and responsible adoption of new healthcare technologies . although the future of the american healthcare system is wrought with both demographic and economic challenges , neither need prove insurmountable . in his research on disruptive innovations in health care , harvard business school professor clayton christensen and his colleagues provide valuable insight into one possible strategy to address this challenging , seemingly intractable problem . documents their precedence in and relation to the healthcare system , and provides specific examples from the field of orthopaedic surgery . in our concluding remarks , we address the inherent cultural and regulatory barriers to adoption of these disruptive innovations and offer some possible strategies to overcome these obstacles . although the dominant players in a particular market sector focus on improving the functionality of their products or services to meet the needs of the most sophisticated customers at the high end of the market , they often miss the simpler , more convenient , and less costly alternatives initially designed to appeal to the least demanding customers at the low end of the market . although these new technologies represent important developments that may advance the field of orthopaedics , expand the indications for orthopaedic procedures , and improve patient outcomes , they do not necessarily address the needs of the majority of our patients nor do they help address the problems of limited accessibility and affordability of musculoskeletal care.fig . the pace of sustaining innovation nearly always outstrips the ability of customers to absorb it , thereby creating the potential for the introduction of disruptive innovations . this creates the potential for the introduction of disruptive innovations that start by meeting the needs of the least demanding consumers at the low end of the market ( fig . sustaining innovations such as emerging technologies , specialist physicians , and academic teaching hospitals constantly strive to improve their functionality to address the needs of the sickest , most complex patients with the most demanding healthcare problems . no one would argue that these entities are contributing to the progress in the field of medicine . this could start by addressing the needs of less medically complex patients but over time could be used to facilitate higher - quality , more convenient , more accessible , and less costly care to the majority of healthcare consumers in the united states . currently , the focus of most biomedical researchers and the pharmaceutical , medical device , and biotechnology industries is disproportionately centered on sustaining innovations , because of organizational structures and financial incentives . for these reasons , the impact of disruptive innovations on the future of orthopaedics and the health care of the nation can only be realized with a change in perspective and a reallocation of healthcare dollars . in the next sections , examples of disruptive innovations in four areas of orthopaedic care delivery diagnostics , we focus on the introduction and popularization of the mini - fluoroscan as a means of facilitating and increasing access to real - time imaging . in surgical techniques and technologies , the surgical implant generation network ( sign ) intramedullary nail is analyzed as a low - tech treatment option to improve the level and access of orthopaedic fracture care in the developing world . the expanding role and contributions of physician assistants ( pas ) and nurse practitioners ( nps ) to the orthopaedic team is the topic of the discussion on disruptive innovations in , we analyze the various ways in which institutions have attempted to implement the paradigm of patient - centered care by modifying existing programs and care delivery models , focusing specifically on the rise of ambulatory surgery centers . mini - fluoroscans , or mini c - arms , are mobile fluoroscopic imaging systems designed for point - of - service , real - time images that have the potential to increase access and decrease costs in the delivery of high - quality orthopaedic care . as a disruptive innovation , mini - fluoroscans offer the ability to reduce the costs associated with using radiology technicians and radiologists while at the same time increasing the autonomy of the orthopaedic surgeon to obtain and interpret his or her own radiographs . with the increased volume of patients presenting to physician offices , urgent care clinics , and emergency departments and the high use of plain radiography , the ability to substitute the use of the mini c - arm for the use of conventional fluoroscopy and digital radiography could lead to reliable cost savings and a decreased burden on the healthcare workforce . the sign system was developed in 1999 to address the orthopaedic needs of the developing world and represents a surgical technique and technology as a disruptive innovation . this response to the recognized lack of access to fluoroscopy and reliable power instrumentation in many countries in the developing world represents the way in which a disruptive innovation can increase access to treatment . one manuscript reviews the short- to midterm results of the sign nail for treatment of open tibia fractures in kathmandu , nepal , whereas the other reports on a consecutive series of patients who were treated with sign nails for extremity fractures in nigeria [ 12 , 24 ] . like all disruptive innovations , the sign intramedullary nail system is not a static entity , but rather is evolving into other areas of fracture care . the increased role of physician assistants ( pas ) and nurse practitioners ( nps ) in orthopaedic surgical practice is an example of healthcare providers as a disruptive innovation , because they are able to help facilitate increased access and lower costs associated with delivering high - quality orthopaedic care . twenty - three percent ( 11,500 ) of the estimated 50,000 clinically practicing pas work in surgical specialties or subspecialties , and there are currently 5000 pas employed in the field of orthopaedics in the united states . have noted that many of the most potent disruptive innovations in health care achieved success by enabling a larger population of less - skilled people to do in a more convenient , less costly setting things that historically could be performed only by expensive specialists in centralized , inconvenient locations . in 1994 , the federal advisory group on physician assistants and the workforce concluded that published research since the profession began consistently found a high level of patient acceptance . with the anticipated changes in demographics in the coming decades , it can be expected that pas and nps will play an increasingly important role in the orthopaedic team by assuming a larger share of the nonoperative aspects of the orthopaedic patients care , thus allowing orthopaedic surgeons to focus on the surgical management of musculoskeletal disease . although technological advancements and the expanding role of ancillary staff may contribute to improved access and care , the greatest opportunities to improve the healthcare system as a whole and the care provided to the population is to focus on and modify the healthcare delivery systems currently in place . single - specialty hospitals and ambulatory surgery centers ( ascs ) have consistently enjoyed high patient volumes and high satisfaction rates , in part because of their ability to deliver focused , efficient , high - quality patient - centered care . ambulatory surgery centers satisfy the criteria for a disruptive innovation in the field of care delivery in that they are a cheaper , simpler , more convenient alternative to hospital operating rooms , and they were initially designed to appeal to the low end of the market . many of the initial ascs struggled after their debut in 1972 as a result of inadequate reimbursement and the need to establish themselves in the eyes of investors as a high - quality , profitable investment . in fact , the american association for accreditation of ambulatory surgery facilities , in its ongoing effort to improve patient care , has developed an internet - based quality improvement and peer review program to analyze outcomes for the surgery centers it accredits . disruptive innovations in health care , in theory , offer lower cost , more efficient and convenient alternatives while maintaining , if not improving , the quality of care provided to patients . the rise of managed care , changes in reimbursement rates from third - party payers , and the diffusion of new technologies to the market have intensified turf wars between the different professional guilds of medical practitioners . hospitals , by virtue of their size , associated overhead , and their large investments in the latest sustaining technologies , are similarly wary of disruptive innovations , because they may threaten their long - term financial viability . the rise in ascs and single - specialty hospitals have shown investors and physicians alike that high - quality care can be provided with a much higher return on investment when facilities are patient - oriented , care delivery and operations are streamlined , and physicians and personnel are well integrated . however , commercial payers only reimburse for procedures that have been clinically validated , and many disruptive innovations , by virtue of being recent entrants to the market , do not yet have peer - reviewed research to support their efficacy compared with so - called gold standard treatment interventions . the rise in direct - to - consumer advertising of hip and knee replacement implants , primarily by implant manufacturers , but also by hospitals and surgeons , to gain a more competitive position in the lucrative and expanding arthroplasty market also poses a formidable institutional barrier to the adoption of disruptive innovations . it is the sustaining innovations that currently fuel the research and development of the orthopaedic device manufacturers such as alternative bearings for use in tha , computer - assisted surgical navigation tools , and new suture fixation devices . regulators , as opposed to physicians , care delivery systems , and third - party payers , have no financial incentive to resist the diffusion of disruptive innovations . the lengthy and cumbersome fda approval process for investigational devices and the bureaucratically complex hipaa regulations are two key examples of how regulators indirectly impede the diffusion of disruptive innovations . although the dominant players in a particular market sector focus on improving the functionality of their products or services to meet the needs of the most sophisticated customers at the high end of the market , they often miss the simpler , more convenient , and less costly alternatives initially designed to appeal to the least demanding customers at the low end of the market . although these new technologies represent important developments that may advance the field of orthopaedics , expand the indications for orthopaedic procedures , and improve patient outcomes , they do not necessarily address the needs of the majority of our patients nor do they help address the problems of limited accessibility and affordability of musculoskeletal care.fig . the pace of sustaining innovation nearly always outstrips the ability of customers to absorb it , thereby creating the potential for the introduction of disruptive innovations . the pace of sustaining innovation nearly always outstrips the ability of customers to absorb it , thereby creating the potential for the introduction of disruptive innovations . this creates the potential for the introduction of disruptive innovations that start by meeting the needs of the least demanding consumers at the low end of the market ( fig . sustaining innovations such as emerging technologies , specialist physicians , and academic teaching hospitals constantly strive to improve their functionality to address the needs of the sickest , most complex patients with the most demanding healthcare problems . no one would argue that these entities are contributing to the progress in the field of medicine . this could start by addressing the needs of less medically complex patients but over time could be used to facilitate higher - quality , more convenient , more accessible , and less costly care to the majority of healthcare consumers in the united states . currently , the focus of most biomedical researchers and the pharmaceutical , medical device , and biotechnology industries is disproportionately centered on sustaining innovations , because of organizational structures and financial incentives . for these reasons , the impact of disruptive innovations on the future of orthopaedics and the health care of the nation can only be realized with a change in perspective and a reallocation of healthcare dollars . in the next sections , examples of disruptive innovations in four areas of orthopaedic care delivery illustrate various manners in which they may impact the healthcare system . in diagnostics , we focus on the introduction and popularization of the mini - fluoroscan as a means of facilitating and increasing access to real - time imaging . in surgical techniques and technologies , the surgical implant generation network ( sign ) intramedullary nail is analyzed as a low - tech treatment option to improve the level and access of orthopaedic fracture care in the developing world . the expanding role and contributions of physician assistants ( pas ) and nurse practitioners ( nps ) to the orthopaedic team is the topic of the discussion on disruptive innovations in , we analyze the various ways in which institutions have attempted to implement the paradigm of patient - centered care by modifying existing programs and care delivery models , focusing specifically on the rise of ambulatory surgery centers . mini - fluoroscans , or mini c - arms , are mobile fluoroscopic imaging systems designed for point - of - service , real - time images that have the potential to increase access and decrease costs in the delivery of high - quality orthopaedic care . as a disruptive innovation , mini - fluoroscans offer the ability to reduce the costs associated with using radiology technicians and radiologists while at the same time increasing the autonomy of the orthopaedic surgeon to obtain and interpret his or her own radiographs . the sign system was developed in 1999 to address the orthopaedic needs of the developing world and represents a surgical technique and technology as a disruptive innovation . this response to the recognized lack of access to fluoroscopy and reliable power instrumentation in many countries in the developing world represents the way in which a disruptive innovation can increase access to treatment . one manuscript reviews the short- to midterm results of the sign nail for treatment of open tibia fractures in kathmandu , nepal , whereas the other reports on a consecutive series of patients who were treated with sign nails for extremity fractures in nigeria [ 12 , 24 ] . like all disruptive innovations , the sign intramedullary nail system is not a static entity , but rather is evolving into other areas of fracture care . the increased role of physician assistants ( pas ) and nurse practitioners ( nps ) in orthopaedic surgical practice is an example of healthcare providers as a disruptive innovation , because they are able to help facilitate increased access and lower costs associated with delivering high - quality orthopaedic care . twenty - three percent ( 11,500 ) of the estimated 50,000 clinically practicing pas work in surgical specialties or subspecialties , and there are currently 5000 pas employed in the field of orthopaedics in the united states . have noted that many of the most potent disruptive innovations in health care achieved success by enabling a larger population of less - skilled people to do in a more convenient , less costly setting things that historically could be performed only by expensive specialists in centralized , inconvenient locations . in 1994 , the federal advisory group on physician assistants and the workforce concluded that published research since the profession began consistently found a high level of patient acceptance . with the anticipated changes in demographics in the coming decades , it can be expected that pas and nps will play an increasingly important role in the orthopaedic team by assuming a larger share of the nonoperative aspects of the orthopaedic patients care , thus allowing orthopaedic surgeons to focus on the surgical management of musculoskeletal disease . although technological advancements and the expanding role of ancillary staff may contribute to improved access and care , the greatest opportunities to improve the healthcare system as a whole and the care provided to the population is to focus on and modify the healthcare delivery systems currently in place . ambulatory surgery centers satisfy the criteria for a disruptive innovation in the field of care delivery in that they are a cheaper , simpler , more convenient alternative to hospital operating rooms , and they were initially designed to appeal to the low end of the market . many of the initial ascs struggled after their debut in 1972 as a result of inadequate reimbursement and the need to establish themselves in the eyes of investors as a high - quality , profitable investment . these differences in profitability have led to payment reforms that have limited payment for ascs to 65% of the payment for hospital - based outpatient surgery centers for the same procedures . in fact , the american association for accreditation of ambulatory surgery facilities , in its ongoing effort to improve patient care , has developed an internet - based quality improvement and peer review program to analyze outcomes for the surgery centers it accredits . disruptive innovations in health care , in theory , offer lower cost , more efficient and convenient alternatives while maintaining , if not improving , the quality of care provided to patients . from physicians to hospitals , health plans to implant manufacturers and regulators , it appears many stakeholders in the us healthcare system support the status quo to maintain their positions and profits . the rise of managed care , changes in reimbursement rates from third - party payers , and the diffusion of new technologies to the market have intensified turf wars between the different professional guilds of medical practitioners . hospitals , by virtue of their size , associated overhead , and their large investments in the latest sustaining technologies , are similarly wary of disruptive innovations , because they may threaten their long - term financial viability . the rise in ascs and single - specialty hospitals have shown investors and physicians alike that high - quality care can be provided with a much higher return on investment when facilities are patient - oriented , care delivery and operations are streamlined , and physicians and personnel are well integrated . however , commercial payers only reimburse for procedures that have been clinically validated , and many disruptive innovations , by virtue of being recent entrants to the market , do not yet have peer - reviewed research to support their efficacy compared with so - called gold standard treatment interventions . the rise in direct - to - consumer advertising of hip and knee replacement implants , primarily by implant manufacturers , but also by hospitals and surgeons , to gain a more competitive position in the lucrative and expanding arthroplasty market also poses a formidable institutional barrier to the adoption of disruptive innovations . it is the sustaining innovations that currently fuel the research and development of the orthopaedic device manufacturers such as alternative bearings for use in tha , computer - assisted surgical navigation tools , and new suture fixation devices . patients , as consumers , are shielded from the costs of orthopaedic implants and are often presented with misleading information about the benefits of particular products , which in turn may lead to inappropriate demands for newer , more costly , unproven technologies , strain the physician - patient relationship , and potentially contribute to the rising cost of health care . regulators , as opposed to physicians , care delivery systems , and third - party payers , have no financial incentive to resist the diffusion of disruptive innovations . the lengthy and cumbersome fda approval process for investigational devices and the bureaucratically complex hipaa regulations are two key examples of how regulators indirectly impede the diffusion of disruptive innovations . the us healthcare system is clearly in crisis , and current strategies have proven inadequate . in this article , we have applied the concept of disruptive innovations popularized by christensen to the field of orthopaedics to illustrate examples in which simpler , cheaper alternatives have improved access of care , increased patient satisfaction , and empowered clinicians to provide higher - quality , more convenient and efficient care to their patients . although we chose to focus on the specific examples of disruptive innovations described , there are many other potential examples related to musculoskeletal care delivery , including calcium phosphate cement for the treatment of distal radius fractures in the elderly , musculoskeletal ultrasound , and medical tourism . the sustaining orthopaedic innovations that are repeatedly introduced into the marketplace will continue to benefit our patients and advance the field of orthopaedics . to date , health care in general and the field of orthopaedic surgery in particular has focused a disproportionate share of resources and effort on the problems of a medical minority . although it would be nave to believe all of the ills of the healthcare system could be reversed simply by the development and diffusion of disruptive innovations , not actively attempting to identify and cultivate them will lead us further down our current path toward unsustainable healthcare inflation and an ever increasing gap between the health care we are capable of delivering based on technologic innovation and the health care we are able to deliver based on limited resources . moreover , the concept of disruptive innovations offers the opportunity for clinicians to regain leadership in health care by working together with other healthcare leaders to deliver higher - quality , more convenient and accessible cost - effective care to our patients .

science aims to provide simple and general explanations for natural phenomena , and all sciences must deal with the problem of heterogeneity . variation and heterogeneity are the hallmarks of biological diversity and capture the attention of anyone interested in trying to unravel the mysteries of the biological world . explaining biological diversity was indeed the problem for which darwin provided a simple but revolutionary solution . variation and adaptation are the first two words that come to mind in relation to living organisms , and it was darwin 's genius that in using these two observations he was able to formulate the theory of natural selection to explain the diversity that we see reflected in the millions of different kinds of organisms or species on this planet . given its spectacular success in providing a causal explanation for organismic change ( evolution within lineages ) , it is equally remarkable that darwin was unable to provide a causal mechanism of speciation ( evolution between lineages ) . such a causal theory had to wait nearly a century after the publication of darwin 's origin of species and it materialized only after the evolutionary synthesis of the 1940s after population genetics had developed a theoretical framework [ 2 , 3 ] . diversity is a problem in biology in two ways : the most obvious of which is that diversity needs to be explained ; the other is that it can thwart our efforts in elucidating precise and simple explanations for the complexity of biological phenomena . as a consequence , we trade precision for generality and a rich theoretical base has been developed , which at first appears to ignore diversity . population genetics is a good example as it developed a significantly elaborate theory of how genes change in evolution without prior knowledge of the material / chemical basis of the gene or of genetic variation . the allopatric theory of speciation relied on geographic isolation and differentiation of populations and cumulative effects of gene differences and gene incompatibility [ 2 , 5 ] without specifying anything about the nature of the genes involved . it was therefore surprising that when gel electrophoresis made its debut in scientific methods , genetic differences between closely related species turned out to be minimal . much of this had to do with the fact that the genes being investigated by molecular biologists were of the general cell metabolism category ( i.e. , allozymes ) and had no direct relation to reproductive biology the crime scene of reproductive isolation . on the other hand , the mendelian approach to studying speciation focused on the right phenotype the mendelian approach therefore eventually became more successful in speciation research by uncovering regions of chromosomes and discrete genes with large effects that played a role in causing hybrid sterility / inviability . provided a glimpse into the nature and the variety of genes that effect postzygotic reproductive isolation , but by the virtue of deliberately being chosen as large effect genes ( for the ease of mapping ) , they may or may not represent the pool of genetic variation that is the basis of speciation . it is for this reason that another , parallel approach to investigating the nature of genetic variation affecting reproductive isolation was needed . to understand the genetic basis of reproductive isolation , a more systematic methodology was needed to screen genes and genetic variation associated with the reproductive system . a systematic genomic / proteomic approach was essential because not all genes in the reproductive system would affect reproductive isolation ; many are indeed essential for development and reproductive biology . we needed to find and target the genes and proteins in the reproductive system that matter genes with minor or large effects that are most likely involved in the early stages of species isolation . it was indeed this realization that led us to the idea of investigating genetic variation in the reproductive tracts of drosophila . the idea of using 2d electrophoresis to examine reproductive proteins seemed exciting but there was some hesitation due to the technical difficulties associated with the technique . mike coulthart , a graduate student at that time , was up to the task as he had what was needed : technical precision , patience , and perseverance . he investigated the levels of genetic variation and genetic differentiation , respectively , within and between sibling species of the drosophila melanogaster group . the 2d results were surprising and somewhat un - interpretable at the time . by separating over 250 protein spots from the reproductive tract and comparing them between species , mike found little genetic variation within species but high genetic divergence between species [ 810 ] . under the neutral theory we expected parity between the levels of within - species and between - species variation , which was indeed the case in the massive amount of data that had accumulated using one - dimensional gel electrophoresis [ 6 , 11 ] . the 2d results were therefore interesting and revolutionary given the dominant framework of neutrality and the expected constant and slow rate of evolution . this was mainly because reproductive tract proteins were considered essential to the organism and therefore not expected to evolve rapidly . the unusual nature of these results called for more investigations and research on sex and reproduction - related ( srr ) molecules began . the ensuing series of experiments involving 2d electrophoresis showed that ( 1 ) nonenzymatic , abundant proteins were generally less polymorphic than enzymes ; ( 2 ) reproductive tissue proteins were more diverged between species than nonreproductive tissue proteins , such as those of the brain ; ( 3 ) testes and ovary proteins showed higher levels of species divergence than nonreproductive proteins ; ( 4 ) reproductive proteins ( and reproductive morphological traits ) showed more gene expression breakdown in species hybrids than nonreproductive proteins [ 1316 ] . these data and particularly civetta and singh 's [ 13 , 14 ] research , which showed that sex and reproduction - related ( srr ) genes evolve faster than nonreproductive genes , unveiled the importance of studying the evolution of srr molecules in speciation research [ 14 , 1720 ] . while the average rates of evolutionary change per gene may be small , genes can evolve rapidly depending upon the environmental conditions and the selection pressure . the dynamics of selection acting on each locus will determine its rate and pattern of evolution . some groups of genes may evolve rapidly by virtue of their functions as is the case with the immune response genes in mammals . immune response genes are an example of a coevolutionary system where evolution of immunity or resistance in hosts is countered by the evolution of virulence in pathogens and/or parasites . immune system genes and virulence genes are locked in an antagonistic coevolutionary arms race and are expected to evolve rapidly . sexual system genes provide another example of a coupled coevolutionary system , in this case involving the interactions between males and females of the same species . advances in molecular technology particularly dna amplification and sequencing propelled srr gene research and a remarkable trend of pervasive rapid srr gene evolution emerged at several levels . some sex determining genes , assumed to be conserved due to their important functions during early development , were shown to evolve rapidly . when genes expressed in testis , ovary , and nonreproductive tissues were screened for rates of evolution it became clear that a substantial proportion of these genes evolved more rapidly than genes expressed in nonreproductive tissues [ 24 , 25 ] . a divergence trend of testis > ovary > somatic genes emerged suggesting male and female srr genes evolve under different selective pressures . microarray and computational methods using entire tissue - specific transcriptomes showed that testis that expressed srr genes were more likely to break down in species hybrids [ 27 , 28 ] . sperm proteins were shown to evolve rapidly and divergently in invertebrates and mammals [ 2932 ] . proteins in the seminal fluid of drosophila were also found to be evolving rapidly and were shown to confer specific physiological and behavioural modifications in the female [ 3337 ] . these studies not only indicate that sexual reproduction provides an opportunity for exerting constant selection pressure generation after generation but also that differences in the evolutionary dynamics of male and female reproductive systems are presumably due to intersexual selection pressure arising from male - female interaction in each generation . the relationship between rapid evolution of srr genes and reproductive isolation is attested by the fact that the known candidate speciation genes are either srr genes or autosomal genes that , via incompatible interactions with genes on the x chromosome , affect hybrid dysfunction ( reviewed in [ 3841 ] ) . in addition , genome - wide evidence of rapid evolution of srr genes provided a mechanistic framework to discuss the nature of genetic changes that may occur during speciation [ 14 , 17 , 42 ] . the discovery of jingwei and sdic opened up investigations into the origin of new srr genes marking yet another important step into understanding the evolutionary dynamics of genetic systems [ 4548 ] . novel genes arise through a variety of molecular mechanisms , including being derived from previously noncoding dna and may be important in functional diversification . what is extremely interesting is that the majority of novel genes or gene copies that have evolved novel functions have also evolved testis - specific expression . interestingly , odysseus ( odsh ) , a hybrid sterility gene , evolved as a duplicate of the neuron expressed unc-4 gene and has taken up a testicular expression and role [ 50 , 51 ] . another example is ms(3)k81 , a gene that evolved by duplication and retroposition from a previously ubiquitously expressed copy to acquire a male - germline - specific expression and function . ms(3)k81 is only found in the 9 species of the melanogaster subgroup and in its new functional role is crucial for zygote viability . accessory gland proteins ( acps ) are a prime example of male - specific genes in drosophila that have taken up a variety of reproduction - related functions and have important physiological effects in the female reproductive system [ 3437 , 5356 ] . retrotransposition is another important means of gene copying and shuffling that can be important in the evolution of new functions [ 45 , 46 ] . interestingly more genes moving from the x chromosome to autosomes have been retained ( active ) than genes moving in the opposite direction [ 7 , 47 , 5761 ] . again , testis - specific expression and rapid evolution appear to be common amongst retroposed genes . thus for some yet unexplained reason , it appears that the testes act as cauldrons of retaining , if not manufacturing , new / refugee genes . in fact , it turns out that not only the evolution of new genes but also gene loss ( loss of orthology ) is also elevated in male - biased genes as compared to female - biased genes in drosophila species . the genetic machinery of the sexual system shows faster rates of turnover and it points to the role of sexual selection acting preferentially through the males ( male - driven sexual selection ) [ 62 , 63 ] . the evolution of new genes and novel functions can be a potent driving force of reproductive isolation as exemplified by odysseus ( odsh ) . sexual selection , strictly speaking , is only a small part of the total selection pressure that the organism is exposed to in relation to sex . classical theories of sexual selection apply only to those traits and genes that are influenced , directly or indirectly , by female choice . on the contrary , selection in relation to sex , or what has been called sexual selection in the broad sense , applies to all aspects of reproductive biology from soliciting mates , courting , and mating , to production of offspring . a large proportion of the genome is involved in the development and maintenance of reproductive systems and a significant proportion of genes ( ~30% ) in the drosophila genome shows sex - biased gene expression , most of which is reproductive tissue specific [ 65 , 66 ] . this raises the possibility of an unexpected level of conflict between natural and sexual selection . current studies of sexual selection have expanded to different aspects of reproductive biology and constitute a major area of research in evolutionary biology . genetically identical ( with the exception of the y - chromosome ) , males and females are expected to differ in genes associated with primary sexual characteristics such as ovary , testes , and copulatory organs . traditionally thought to be associated to few genes on the sex chromosome , it turns out that the breadth and complexity of sexual interaction between the two sexes has become so elaborate that a large number of genes controlling a variety of traits have become associated in a sex - specific manner ( sex - biased and sex - enriched genes ) expanding the level of sexual dimorphism [ 66 , 67 ] . in the drosophila genome a substantial proportion of genes show sex - biased expression [ 65 , 66 ] . male - biased genes are underrepresented on the x chromosome and female - biased genes are enriched on the x chromosome . these genes are expressed in a tissue - specific manner ( e.g. , somatic tissues , ovary , and testis ) and they even show sex - specific elevated levels of movement between sex chromosome and autosomes [ 59 , 60 , 6772 ] . in the last decade , several theories including sexual antagonism , dosage compensation , meiotic sex chromosome inactivation , and meiotic drive have been proposed to explain the paucity of male - biased gene on the x chromosome and the driving force responsible for the evolution of sex chromosomes , their gene content , and expression patterns [ 58 , 67 , 7274 ] . meiotic sex chromosome inactivation ( msci ) pioneered first by lifschytz and lindsley and then shown at a genomic scale [ 58 , 72 ] appeared to be convincing in drosophila ; however , recent evidence shows otherwise and is currently under debate [ 74 , 7679 ] . while explaining the relocation and expression pattern of sex - biased genes will remain a prominent research area , it is noteworthy that , with respect to rates of evolution , genes with sex - biased expression and particularly male - biased sex genes show unusually high rates of evolution [ 67 , 80 , 81 ] . it is not surprising then that the sexes differ in their rates of evolution of sterility and inviability during speciation as pointed out by haldane . sea urchins have traditionally been a model organism for development biology and reproductive biology but have recently received considerable attention from an evolutionary standpoint particularly in the evolution of reproductive isolation to explain speciation in the sea [ 83 , 84 ] . in most internally fertilizing animals , specific courtship behaviours mediate male and female interactions ensuring species - specific copulation and fertilization . in contrast , in externally fertilizing organisms with little or no such mating behaviours ( e.g. , sea urchins ) gametes are shed into the sea where species - specific fertilization occurs . several molecules on the surface of gametes that mediate various stages of sperm - egg interaction have been characterized [ 8587 ] . studies on two important proteins exemplify the evolutionary dynamics of gametic molecules in externally fertilizing marine organisms ( see for a recent review ) . studies on the abalone sperm molecule lysin and its egg receptor verl demonstrate the fact that male and female gametic proteins coevolve species - specific structures and affinities to maintain species - specific interactions and avoid cross - fertilization [ 83 , 84 , 8890 ] . the sperm molecule bindin and its egg receptor are another classic example from sea urchins [ 83 , 84 , 88 , 91 ] . while the evolutionary dynamics of the egg receptor for bindin remains obscure , the sperm protein bindin evolves rapidly and divergently in some genera but not in others [ 83 , 84 ] . however , bindin does appear to have some involvement in reproductive isolation since its divergence correlates with the degree of gametic incompatibility between but not with time since species divergence . in all likelihood , other molecules must be involved and there is a need for further characterization of such gametic and other sex and reproduction - related molecules in broadcast spawners . once such molecules are identified , it will be interesting to correlate the evolution of egg - sperm interacting molecules to the patterns of hybrid incompatibility in these organisms . external fertilizing systems may provide a unique opportunity to assess the relative roles and genetic consequences of sexual selection and conflict in driving divergence of reproductive molecules and speciation . while research into reproductive molecules is at its inception in externally fertilizing systems , the sperm proteome of drosophila has opened up exciting venues of research in reproductive biology [ 9395 ] . as with other male - biased genes , sperm genes are underrepresented on the x chromosome and are nonrandomly clustered in the genome . while certain groups of sperm proteins , such as binding factors , do evolve rapidly , overall , the sperm proteome does not appear to be evolving fast , there is little evidence of positive selection , and there is widespread functional and structural constraint . this is in stark contrast to seminal fluid proteins that evolve fast and are under selection . the contrasting evolutionary patterns of the two groups of male ejaculate proteins are interesting and are indicative of the complexity of reproductive processes , where crucial sperm - egg interacting proteins are sheltered but seminal fluids that accompany them interact with the general environment of the female reproductive tract and proteins therein are under strong selection . future research on sperm - egg interacting proteins promises to increase our knowledge about the functional evolution of the male and female fertilization machinery and , more broadly , the evolutionary origins of sexual reproduction . haldane 's rule ( of speciation ) points to the preferential appearance of hybrid sterility and and/or inviability in the heterogametic sex . in flies and mammals , it is the males who are affected while in moths and birds it is the females [ 39 , 96 , 97 ] . the genetics of hybrid sterility and inviability have been of intense focus in speciation studies and a great deal of effort has been made in mapping and characterizing genes involved in hybrid breakdown [ 38 , 39 , 50 , 98106 ] . the evolution of hybrid sterility / inviability is explained by the bateson - dobzhansky - muller incompatibility model which states that incompatibility is the result of independent evolution of genes in isolated populations [ 7 , 106 ] . haerty and singh showed that the genes showing breakdown in the hybrids are preferentially sex - related genes and that these genes evolve faster both in sequence and gene expression [ 27 , 28 , 107 ] . in the light of this it is interesting to note that all but a handful of the so - called speciation genes and hybrid - sterility genes are characterized by high sequence divergence ; they are often sex - related genes or somatic genes that affect the sexual system [ 7 , 38 , 40 , 41 , 108 ] . the effects of genes are prone to change in response to incorporation of new mutants and during the course of speciation earlier mutations would have fewer interactions than older mutations ( cascade effect ) . speciation genes may have started as small - effect minor genes and have become elaborate in their genic interactions later through species - specific adaptation and evolution . in this scenario thus the effect of cascade evolution is not only that speciation would occur rapidly and precipitously but also that speciation genes would evolve in their average effect from being minor to major genes . so while in reality speciation may occur in an incremental manner through a combination of many minor genes , in postspeciation genetic investigations genes would often appear as major genes . this is an interesting scenario and we must find a way to approach this problem experimentally . srr genes provide new opportunities to mount comparative studies of the role of selection versus developmental constraints in evolution . for example , srr genes have provided an excellent example of testing alternative explanations for von baer 's third law . this was later interpreted to be the result of selection against changes in earlier stages of development , which could have cascading , deleterious developmental repercussions . darwin on the other hand explained the conservation of morphology in earlier stages as being due to lack of opportunity for natural selection to act . since natural selection results from changes in the environment , it follows that earlier , sessile stages that have not fully developed will have little opportunity to experience variation in the environment . darwin further pointed out that secondary / sex - specific sexual traits appear when they are needed and this can be seen in the secondary sexual traits in animals . two recent studies [ 28 , 107 ] explored the relationship between expression level over ontogeny and rates of divergence and found support for both selection against deleterious cascading effects and darwin 's hypothesis : genes expressed during early stages show reduced divergence . however , the more rapid divergence of later , adult stages , is dominated by genes expressed in adult males , which are , as noted above , presumably diverging under the effect of directional ( sexual ) selection . as a result of these observations , artieri et al . proposed a model of divergence involving two factors playing dominant roles during different periods of development : conservation early and opportunity late . more of such studies should shed light on the relationship between evolution and development [ 110113 ] as well as on the broader aspects of speciation and macroevolution beyond reproductive isolation . while srr research has provided a useful , complementary approach to study speciation , there is a need to explain the evolutionary forces driving pervasive rapid evolution of male and female reproductive genes . initially rapid srr gene evolution invoked the role of sexual selection by female choice but recent developments on parker 's original theses revived the role of sexual conflict in explaining evolutionary changes in sexual systems . sexual dimorphism in higher organisms leads to sex - specific life styles , reproductive behaviours , and investments in sexual interactions . it is expected that these differences will lead to conflicts of interest between males and females and this conflict may work as a stimulus for retaliatory evolutionary changes known as sexual arms races [ 115117 ] . a sexual arms race would require action and reaction on the part of both partners and thus it provides a test of the role of female choice , male - driven sexual selection , and of sexual arms race theories . increasing empirical evidence suggests that sexual conflict may be pervasive [ 90 , 115 , 118123 ] but much remains to be done particularly at the level of genes to substantiate how sexual conflict and sexual selection affect male and female genetic systems differently . genomics provides the means to explore the molecular consequences of sexual arms races and associated sexual selection theories . intersexual interactions , be they mutualistic or antagonistic , have the potential to drive population divergence in a self - accelerating manner and this may be one of the reasons why origins of diversity and speciation are much higher in higher organisms . evo - devo studies will also help to answer the perennial question : during evolution and speciation what comes first reproductive isolation or adaptive radiation ? since its inception 25 years ago , srr gene research has rapidly evolved into a large coherent field in evolutionary biology , particularly influencing reproductive biology and speciation . the focus on srr system studies has provided valuable mechanistic frameworks that directly relate to theories of how speciation occurs . it has emphasized the role of sexual selection in evolution , propelling research on how sexual selection and sexual conflict work at the population level . the genomics era has revolutionized srr gene research and resulted in the characterization of rates of evolution and patterns of gene expression in reproductive transcriptomes . rapid evolution is now commonly associated with reproductive genes but , in the future , work will be needed to understand the functions of rapidly evolving srr genes and details of why they evolve rapidly and how their rapid evolution affects the rest of organismal biology . it will call for an integrated approach , unifying disparate fields of science , particularly biochemistry , genetics , ecology , and molecular biology . a key issue will be to explore the relationship between changes in gene sequence , gene expression , protein syntheses , and protein function in reproductive systems . fundamental behavioural and ecological studies will be essential in explaining the nature of molecular changes associated with the reproductive systems . selection in relation to sex , encompassing sexual selection in the strict sense , and in the broad sense is a large and growing area of research in evolutionary biology . investigating the molecular consequences of sexual interaction and their role in speciation stands to open one of the most important areas of research bearing on the biology of sexual reproduction .

the protein electrophoresis revolution , nearly fifty years ago , provided the first glimpse into the nature of molecular genetic variation within and between species and showed that the amount of genetic differences between newly arisen species was minimal . twenty years later , 2d electrophoresis showed that , in contrast to general gene - enzyme variation , reproductive tract proteins were less polymorphic within species but highly diverged between species . the 2d results were interesting and revolutionary , but somewhat uninterpretable because , at the time , rapid evolution and selective sweeps were not yet part of the common vocabulary of evolutionary biologists . since then , genomic studies of sex and reproduction - related ( srr ) genes have grown rapidly into a large area of research in evolutionary biology and are shedding light on a number of phenomena . here we review some of the major and current fields of research that have greatly contributed to our understanding of the evolutionary dynamics and importance of srr genes and genetic systems in understanding reproductive biology and speciation .

1. Introduction 2. Speciation Genetics: Mendelian versus Molecular Approach 3. DNA Sequence Variation and Rates of Evolution 4. Evolution of Sex-Biased Genes: Role of Sexual Selection versus Selection in relation to Sex 5. Sexual Conflict, Sexual Arms Race, and Sexual Selection 6. Conclusion

science aims to provide simple and general explanations for natural phenomena , and all sciences must deal with the problem of heterogeneity . variation and heterogeneity are the hallmarks of biological diversity and capture the attention of anyone interested in trying to unravel the mysteries of the biological world . variation and adaptation are the first two words that come to mind in relation to living organisms , and it was darwin 's genius that in using these two observations he was able to formulate the theory of natural selection to explain the diversity that we see reflected in the millions of different kinds of organisms or species on this planet . given its spectacular success in providing a causal explanation for organismic change ( evolution within lineages ) , it is equally remarkable that darwin was unable to provide a causal mechanism of speciation ( evolution between lineages ) . such a causal theory had to wait nearly a century after the publication of darwin 's origin of species and it materialized only after the evolutionary synthesis of the 1940s after population genetics had developed a theoretical framework [ 2 , 3 ] . diversity is a problem in biology in two ways : the most obvious of which is that diversity needs to be explained ; the other is that it can thwart our efforts in elucidating precise and simple explanations for the complexity of biological phenomena . as a consequence , we trade precision for generality and a rich theoretical base has been developed , which at first appears to ignore diversity . population genetics is a good example as it developed a significantly elaborate theory of how genes change in evolution without prior knowledge of the material / chemical basis of the gene or of genetic variation . the allopatric theory of speciation relied on geographic isolation and differentiation of populations and cumulative effects of gene differences and gene incompatibility [ 2 , 5 ] without specifying anything about the nature of the genes involved . it was therefore surprising that when gel electrophoresis made its debut in scientific methods , genetic differences between closely related species turned out to be minimal . much of this had to do with the fact that the genes being investigated by molecular biologists were of the general cell metabolism category ( i.e. , allozymes ) and had no direct relation to reproductive biology the crime scene of reproductive isolation . on the other hand , the mendelian approach to studying speciation focused on the right phenotype the mendelian approach therefore eventually became more successful in speciation research by uncovering regions of chromosomes and discrete genes with large effects that played a role in causing hybrid sterility / inviability . provided a glimpse into the nature and the variety of genes that effect postzygotic reproductive isolation , but by the virtue of deliberately being chosen as large effect genes ( for the ease of mapping ) , they may or may not represent the pool of genetic variation that is the basis of speciation . it is for this reason that another , parallel approach to investigating the nature of genetic variation affecting reproductive isolation was needed . to understand the genetic basis of reproductive isolation , a more systematic methodology was needed to screen genes and genetic variation associated with the reproductive system . a systematic genomic / proteomic approach was essential because not all genes in the reproductive system would affect reproductive isolation ; many are indeed essential for development and reproductive biology . we needed to find and target the genes and proteins in the reproductive system that matter genes with minor or large effects that are most likely involved in the early stages of species isolation . it was indeed this realization that led us to the idea of investigating genetic variation in the reproductive tracts of drosophila . the idea of using 2d electrophoresis to examine reproductive proteins seemed exciting but there was some hesitation due to the technical difficulties associated with the technique . mike coulthart , a graduate student at that time , was up to the task as he had what was needed : technical precision , patience , and perseverance . he investigated the levels of genetic variation and genetic differentiation , respectively , within and between sibling species of the drosophila melanogaster group . the 2d results were surprising and somewhat un - interpretable at the time . by separating over 250 protein spots from the reproductive tract and comparing them between species , mike found little genetic variation within species but high genetic divergence between species [ 810 ] . under the neutral theory we expected parity between the levels of within - species and between - species variation , which was indeed the case in the massive amount of data that had accumulated using one - dimensional gel electrophoresis [ 6 , 11 ] . the 2d results were therefore interesting and revolutionary given the dominant framework of neutrality and the expected constant and slow rate of evolution . this was mainly because reproductive tract proteins were considered essential to the organism and therefore not expected to evolve rapidly . the unusual nature of these results called for more investigations and research on sex and reproduction - related ( srr ) molecules began . the ensuing series of experiments involving 2d electrophoresis showed that ( 1 ) nonenzymatic , abundant proteins were generally less polymorphic than enzymes ; ( 2 ) reproductive tissue proteins were more diverged between species than nonreproductive tissue proteins , such as those of the brain ; ( 3 ) testes and ovary proteins showed higher levels of species divergence than nonreproductive proteins ; ( 4 ) reproductive proteins ( and reproductive morphological traits ) showed more gene expression breakdown in species hybrids than nonreproductive proteins [ 1316 ] . these data and particularly civetta and singh 's [ 13 , 14 ] research , which showed that sex and reproduction - related ( srr ) genes evolve faster than nonreproductive genes , unveiled the importance of studying the evolution of srr molecules in speciation research [ 14 , 1720 ] . while the average rates of evolutionary change per gene may be small , genes can evolve rapidly depending upon the environmental conditions and the selection pressure . immune response genes are an example of a coevolutionary system where evolution of immunity or resistance in hosts is countered by the evolution of virulence in pathogens and/or parasites . immune system genes and virulence genes are locked in an antagonistic coevolutionary arms race and are expected to evolve rapidly . sexual system genes provide another example of a coupled coevolutionary system , in this case involving the interactions between males and females of the same species . advances in molecular technology particularly dna amplification and sequencing propelled srr gene research and a remarkable trend of pervasive rapid srr gene evolution emerged at several levels . some sex determining genes , assumed to be conserved due to their important functions during early development , were shown to evolve rapidly . a divergence trend of testis > ovary > somatic genes emerged suggesting male and female srr genes evolve under different selective pressures . microarray and computational methods using entire tissue - specific transcriptomes showed that testis that expressed srr genes were more likely to break down in species hybrids [ 27 , 28 ] . sperm proteins were shown to evolve rapidly and divergently in invertebrates and mammals [ 2932 ] . these studies not only indicate that sexual reproduction provides an opportunity for exerting constant selection pressure generation after generation but also that differences in the evolutionary dynamics of male and female reproductive systems are presumably due to intersexual selection pressure arising from male - female interaction in each generation . the relationship between rapid evolution of srr genes and reproductive isolation is attested by the fact that the known candidate speciation genes are either srr genes or autosomal genes that , via incompatible interactions with genes on the x chromosome , affect hybrid dysfunction ( reviewed in [ 3841 ] ) . in addition , genome - wide evidence of rapid evolution of srr genes provided a mechanistic framework to discuss the nature of genetic changes that may occur during speciation [ 14 , 17 , 42 ] . the discovery of jingwei and sdic opened up investigations into the origin of new srr genes marking yet another important step into understanding the evolutionary dynamics of genetic systems [ 4548 ] . novel genes arise through a variety of molecular mechanisms , including being derived from previously noncoding dna and may be important in functional diversification . what is extremely interesting is that the majority of novel genes or gene copies that have evolved novel functions have also evolved testis - specific expression . interestingly , odysseus ( odsh ) , a hybrid sterility gene , evolved as a duplicate of the neuron expressed unc-4 gene and has taken up a testicular expression and role [ 50 , 51 ] . another example is ms(3)k81 , a gene that evolved by duplication and retroposition from a previously ubiquitously expressed copy to acquire a male - germline - specific expression and function . ms(3)k81 is only found in the 9 species of the melanogaster subgroup and in its new functional role is crucial for zygote viability . accessory gland proteins ( acps ) are a prime example of male - specific genes in drosophila that have taken up a variety of reproduction - related functions and have important physiological effects in the female reproductive system [ 3437 , 5356 ] . again , testis - specific expression and rapid evolution appear to be common amongst retroposed genes . thus for some yet unexplained reason , it appears that the testes act as cauldrons of retaining , if not manufacturing , new / refugee genes . the genetic machinery of the sexual system shows faster rates of turnover and it points to the role of sexual selection acting preferentially through the males ( male - driven sexual selection ) [ 62 , 63 ] . the evolution of new genes and novel functions can be a potent driving force of reproductive isolation as exemplified by odysseus ( odsh ) . sexual selection , strictly speaking , is only a small part of the total selection pressure that the organism is exposed to in relation to sex . classical theories of sexual selection apply only to those traits and genes that are influenced , directly or indirectly , by female choice . on the contrary , selection in relation to sex , or what has been called sexual selection in the broad sense , applies to all aspects of reproductive biology from soliciting mates , courting , and mating , to production of offspring . a large proportion of the genome is involved in the development and maintenance of reproductive systems and a significant proportion of genes ( ~30% ) in the drosophila genome shows sex - biased gene expression , most of which is reproductive tissue specific [ 65 , 66 ] . current studies of sexual selection have expanded to different aspects of reproductive biology and constitute a major area of research in evolutionary biology . genetically identical ( with the exception of the y - chromosome ) , males and females are expected to differ in genes associated with primary sexual characteristics such as ovary , testes , and copulatory organs . traditionally thought to be associated to few genes on the sex chromosome , it turns out that the breadth and complexity of sexual interaction between the two sexes has become so elaborate that a large number of genes controlling a variety of traits have become associated in a sex - specific manner ( sex - biased and sex - enriched genes ) expanding the level of sexual dimorphism [ 66 , 67 ] . in the last decade , several theories including sexual antagonism , dosage compensation , meiotic sex chromosome inactivation , and meiotic drive have been proposed to explain the paucity of male - biased gene on the x chromosome and the driving force responsible for the evolution of sex chromosomes , their gene content , and expression patterns [ 58 , 67 , 7274 ] . meiotic sex chromosome inactivation ( msci ) pioneered first by lifschytz and lindsley and then shown at a genomic scale [ 58 , 72 ] appeared to be convincing in drosophila ; however , recent evidence shows otherwise and is currently under debate [ 74 , 7679 ] . while explaining the relocation and expression pattern of sex - biased genes will remain a prominent research area , it is noteworthy that , with respect to rates of evolution , genes with sex - biased expression and particularly male - biased sex genes show unusually high rates of evolution [ 67 , 80 , 81 ] . it is not surprising then that the sexes differ in their rates of evolution of sterility and inviability during speciation as pointed out by haldane . sea urchins have traditionally been a model organism for development biology and reproductive biology but have recently received considerable attention from an evolutionary standpoint particularly in the evolution of reproductive isolation to explain speciation in the sea [ 83 , 84 ] . in most internally fertilizing animals , specific courtship behaviours mediate male and female interactions ensuring species - specific copulation and fertilization . in contrast , in externally fertilizing organisms with little or no such mating behaviours ( e.g. , sea urchins ) gametes are shed into the sea where species - specific fertilization occurs . several molecules on the surface of gametes that mediate various stages of sperm - egg interaction have been characterized [ 8587 ] . studies on two important proteins exemplify the evolutionary dynamics of gametic molecules in externally fertilizing marine organisms ( see for a recent review ) . while the evolutionary dynamics of the egg receptor for bindin remains obscure , the sperm protein bindin evolves rapidly and divergently in some genera but not in others [ 83 , 84 ] . in all likelihood , other molecules must be involved and there is a need for further characterization of such gametic and other sex and reproduction - related molecules in broadcast spawners . once such molecules are identified , it will be interesting to correlate the evolution of egg - sperm interacting molecules to the patterns of hybrid incompatibility in these organisms . external fertilizing systems may provide a unique opportunity to assess the relative roles and genetic consequences of sexual selection and conflict in driving divergence of reproductive molecules and speciation . while research into reproductive molecules is at its inception in externally fertilizing systems , the sperm proteome of drosophila has opened up exciting venues of research in reproductive biology [ 9395 ] . as with other male - biased genes , sperm genes are underrepresented on the x chromosome and are nonrandomly clustered in the genome . while certain groups of sperm proteins , such as binding factors , do evolve rapidly , overall , the sperm proteome does not appear to be evolving fast , there is little evidence of positive selection , and there is widespread functional and structural constraint . this is in stark contrast to seminal fluid proteins that evolve fast and are under selection . the contrasting evolutionary patterns of the two groups of male ejaculate proteins are interesting and are indicative of the complexity of reproductive processes , where crucial sperm - egg interacting proteins are sheltered but seminal fluids that accompany them interact with the general environment of the female reproductive tract and proteins therein are under strong selection . future research on sperm - egg interacting proteins promises to increase our knowledge about the functional evolution of the male and female fertilization machinery and , more broadly , the evolutionary origins of sexual reproduction . the evolution of hybrid sterility / inviability is explained by the bateson - dobzhansky - muller incompatibility model which states that incompatibility is the result of independent evolution of genes in isolated populations [ 7 , 106 ] . haerty and singh showed that the genes showing breakdown in the hybrids are preferentially sex - related genes and that these genes evolve faster both in sequence and gene expression [ 27 , 28 , 107 ] . in the light of this it is interesting to note that all but a handful of the so - called speciation genes and hybrid - sterility genes are characterized by high sequence divergence ; they are often sex - related genes or somatic genes that affect the sexual system [ 7 , 38 , 40 , 41 , 108 ] . speciation genes may have started as small - effect minor genes and have become elaborate in their genic interactions later through species - specific adaptation and evolution . in this scenario thus the effect of cascade evolution is not only that speciation would occur rapidly and precipitously but also that speciation genes would evolve in their average effect from being minor to major genes . so while in reality speciation may occur in an incremental manner through a combination of many minor genes , in postspeciation genetic investigations genes would often appear as major genes . srr genes provide new opportunities to mount comparative studies of the role of selection versus developmental constraints in evolution . for example , srr genes have provided an excellent example of testing alternative explanations for von baer 's third law . this was later interpreted to be the result of selection against changes in earlier stages of development , which could have cascading , deleterious developmental repercussions . darwin on the other hand explained the conservation of morphology in earlier stages as being due to lack of opportunity for natural selection to act . since natural selection results from changes in the environment , it follows that earlier , sessile stages that have not fully developed will have little opportunity to experience variation in the environment . darwin further pointed out that secondary / sex - specific sexual traits appear when they are needed and this can be seen in the secondary sexual traits in animals . however , the more rapid divergence of later , adult stages , is dominated by genes expressed in adult males , which are , as noted above , presumably diverging under the effect of directional ( sexual ) selection . proposed a model of divergence involving two factors playing dominant roles during different periods of development : conservation early and opportunity late . more of such studies should shed light on the relationship between evolution and development [ 110113 ] as well as on the broader aspects of speciation and macroevolution beyond reproductive isolation . while srr research has provided a useful , complementary approach to study speciation , there is a need to explain the evolutionary forces driving pervasive rapid evolution of male and female reproductive genes . initially rapid srr gene evolution invoked the role of sexual selection by female choice but recent developments on parker 's original theses revived the role of sexual conflict in explaining evolutionary changes in sexual systems . sexual dimorphism in higher organisms leads to sex - specific life styles , reproductive behaviours , and investments in sexual interactions . a sexual arms race would require action and reaction on the part of both partners and thus it provides a test of the role of female choice , male - driven sexual selection , and of sexual arms race theories . increasing empirical evidence suggests that sexual conflict may be pervasive [ 90 , 115 , 118123 ] but much remains to be done particularly at the level of genes to substantiate how sexual conflict and sexual selection affect male and female genetic systems differently . intersexual interactions , be they mutualistic or antagonistic , have the potential to drive population divergence in a self - accelerating manner and this may be one of the reasons why origins of diversity and speciation are much higher in higher organisms . evo - devo studies will also help to answer the perennial question : during evolution and speciation what comes first reproductive isolation or adaptive radiation ? since its inception 25 years ago , srr gene research has rapidly evolved into a large coherent field in evolutionary biology , particularly influencing reproductive biology and speciation . the focus on srr system studies has provided valuable mechanistic frameworks that directly relate to theories of how speciation occurs . it has emphasized the role of sexual selection in evolution , propelling research on how sexual selection and sexual conflict work at the population level . the genomics era has revolutionized srr gene research and resulted in the characterization of rates of evolution and patterns of gene expression in reproductive transcriptomes . rapid evolution is now commonly associated with reproductive genes but , in the future , work will be needed to understand the functions of rapidly evolving srr genes and details of why they evolve rapidly and how their rapid evolution affects the rest of organismal biology . it will call for an integrated approach , unifying disparate fields of science , particularly biochemistry , genetics , ecology , and molecular biology . fundamental behavioural and ecological studies will be essential in explaining the nature of molecular changes associated with the reproductive systems . selection in relation to sex , encompassing sexual selection in the strict sense , and in the broad sense is a large and growing area of research in evolutionary biology . investigating the molecular consequences of sexual interaction and their role in speciation stands to open one of the most important areas of research bearing on the biology of sexual reproduction .

two major impacts of acl rupture , whether treated conservatively or surgically , are failure to return to pre - injury activity levels ( myklebust et al . , 2003 ; gobbi and francisco , 2006 ; strehl and eggli , 2007 ; ardern et al . , 2011a ) and future predisposition to osteoarthritis ( blagojevic et al . , 2010 ) . a review evaluating return to sport following acl injury indicated that up to 48% was not returning to their pre - injury sporting levels ( ardern et al . , 2011a ) . medio - lateral knee control is an important factor to assess in acl injured patients . besides adductor moments , clinical evidence suggests that fluency of movement is an aspect of medio - lateral knee control that is worth investigating . this study therefore investigated recovery of both these aspects of knee control in acl patients . regardless of whether injury is managed conservatively or surgically , current rehabilitation methods recommend strengthening , neuromuscular control , perturbation and plyometric exercise ( risberg et al . , 2009 ; eitzen et al . , 2010 ; hartigan et al . , 2010 ; escamilla et al . , 2012 ; 2012 ) but evidence is inconclusive on the biomechanical effect and clinical effectiveness of individual exercises ( escamilla et al . , 2012 ; button et al . , single leg hop is an exercise used in late stage rehabilitation and a tool to evaluate recovery and inform treatment selection ( ardern et al . this activity challenges knee stability by requiring large knee moments during take - off and landing and mimics some maneuvers encountered on return to sport . clinically the symmetry index of the injured and non - injured hop distance is frequently used to evaluate hop performance ( engelen - van melick et al . , however , reduced performance of the non - injured leg can exaggerate estimation of recovery ( button et al . , 2005 ) . there is no consensus in the literature on the recovery of hop distance for aclr individuals ( gokeler et al . , 2010 ; orishimo et al . , acld individuals on the other hand have been reported not to recover hop distance ( gauffin et al . , 1990 ; , 1999 ; button et al . , 2006 ; gustavsson et al . , 2006 ) . only a limited number of previous studies have analyzed 3d kinematic and kinetic hop performance of acl injured individuals . differences in hop performance have been reported between high and poor functioning acld individuals ( rudolph et al . , 2000 ) ; with high functioning acld having unchanged knee kinematics and an increased contribution of the ankle to the total support moment , and poor functioning acld using a smaller range of knee flexion , a lower peak vertical ground reaction force , lower knee extensor moments and greater contribution from the hip to the total support moment . compared to healthy controls , acld individuals performed a single leg hop for distance using higher moments at the ankle and hip , more forward trunk lean and a more anterior ground reaction force vector ( oberlnder et al . , 2012 ) . aclr individuals demonstrated a reduced knee range of motion during the landing phase in some ( orishimo et al . , 2010 and deneweth et al . , 2010 ) but not all studies ( gokeler et al . , 2010 ) . besides these kinematic and kinetic differences reported on single leg hopping in acl injured individuals , there are no studies investigating how this movement challenges motor control . recovery of motor control is essential for return to sports and therefore an important aspect of rehabilitation this study therefore investigated the movement strategies used during the landing phase of a single leg hop for distance . this landing phase consists of a phase where the forward velocity of the center of mass ( com ) is decelerated . this strategy requires high knee extensor moments and puts high demands on dynamic knee control . com deceleration can also be achieved by using a pendular strategy where com rotation around the ankle is controlled . this strategy requires smaller knee extensor moments and requires less medio - lateral knee control , but larger hip flexion and plantar flexion moments . a telescopic inverted pendulum ( tip ) analysis ( jacobs and van ingen schenau , 1992 ; papa and cappozzo 1999 ; van deursen and phillips , 2006 ) can be used to identify if the landing phase is predominantly telescopic or pendular . clearly a better understanding of knee control during functional movements is needed to be able to improve rehabilitation outcome . this study therefore investigated a single leg hop for distance , which challenges knee stability , with the aims of evaluating if:1)landing strategies in acld and aclr have been recovered to those of healthy control subjects and2)medio - lateral control has been recovered in acld and aclr . landing strategies in acld and aclr have been recovered to those of healthy control subjects and medio - lateral control has been recovered in acld and aclr . we hypothesized that acl injury would result in altered landing strategies and reduced medio - lateral knee control compared to healthy controls . in addition , we hypothesized that acld patients would be more affected in their landing strategies and medio - lateral knee control compared to aclr . 21 acld , 23 aclr and 20 healthy control ( cont ) subjects provided informed consent to participate in this study ( subject demographics are in table 1 ) . acl subjects were recruited from a typical clinical ( non - elite sporting ) population . ethical approval for this study was obtained from the south east wales research ethics committee . inclusion criteria were that patients were aged between 18 and 65 years , had an acl rupture ( acld group ) , or a primary acl reconstruction ( aclr group ) that may or may not be accompanied with a meniscal tear , collateral ligament sprain , or cartilage and sub - cortical bone bruises ; had finished their rehabilitation ; had no other pathology which affects their movement ; had no previous knee surgery and were able to provide informed consent independently . the typical population of patients seen in the hospital setting are not elite athletes and the distribution of injuries is mixed . for this study all of our subjects had mri scans taken and those were assessed by an expert clinician to decide whether they fit into the category of a typical injury . our approach has been to filter out individuals who had locked knees , fractures , mcl , pcl and posterior lateral corner complete ruptures . however , when we explored the number of subjects that have a singular acl injury , our finding was that this hardly ever occurs without at least some comorbidity . therefore , a representative sample of acl injured individuals has to include people with mcl sprains , meniscal tears , as well as cartilage and sub - cortical bone bruises . the acld did not have surgery because they were either copers ( as in they were functioning extremely well ) , adapters ( as in they were willing to adjust their activity level ) , non - copers waiting for surgery , or a decision about surgery had not yet been made . knee function was scored for acld and aclr using the international knee documentation subjective knee ( ikdc ) questionnaire ( irrgang et al . , 2001 ) . knee extensor ( skneeext ) and flexor ( skneeflex ) , and hip abductor ( shipabd ) and adductor ( shipadd ) isokinetic strength were measured at 90 /s and 45 /s respectively on a biodex system 4 pro dynamometer ( biodex medical systems inc , usa ) . this was measured on both legs , but presented for the injured ( aclr and acld ) and the dominant stance leg ( cont ) only . individuals were asked to hop their maximum single leg hop distance and regain their balance after landing . the hop distance was marked from the force platform and subjects were then asked to perform four single leg hops for maximum distance from this mark , as such that they would land on the force platform . all acl injured subjects hopped using their injured leg and the controls using their dominant stance leg . this was based on findings from a previous study that hopping in healthy subjects was virtually identical ( within about 5% ) for the dominant and non - dominant leg ( figure 6 , button et al . , 2005 ) . furthermore , in knee injured subjects the non - injured leg was affected and therefore can not be used for comparison . for each subject hopping trials were collected until at least four successful hopping trials were achieved where they landed on the force platform and were able to regain balance without touching the floor with the other foot . prior to this a static anatomical calibration trial was collected . kinematic data were collected at 250 hz using an eight camera vicon mx motion analysis system ( oxford metrics group ltd . , uk ) . reflective markers were placed using the plug - in - gait full body marker set . two additional markers were placed on the left and right lateral sides of the iliac crest ( lilc and rilc ) . ground reaction force data were collected using a kistler force plate ( kistler instruments ltd . , switzerland ) at 1000 hz . in some trials the trunk flexed as such that the markers on the left and right anterior superior iliac crests ( lasi and rasi ) were occluded ; these gaps were filled using a custom written program in vicon bodybuilder for biomechanics ( version 1.2 , oxford metrics group ltd . , uk ) and the data of the lilc and rilc markers . inverse dynamics calculations were performed within vicon nexus software ( version 1.6.1 ) and data were further processed and analyzed in matlab r2010b ( the mathworks inc . , usa ) . anthropometric measurements were recorded ( height , weight , leg length , knee width and ankle width ) and used for the inverse dynamic calculations . parameters used for hop performance were hopping distance ( dhop ) , com velocity along the axis of hopping movement prior to landing ( vcom ) and duration of the com deceleration phase during landing ( tdec ) . dhop was calculated as the distance the ankle joint center traveled along the axis of hopping . dhop was normalized to body height . to investigate the kinematics and kinetics , a telescopic inverted pendulum ( tip ) model approach was used ( fig . 1 ; jacobs and van ingen schenau , 1992 ; papa and cappozzo , 1999 ; van deursen and phillips , 2006 ) . hopping can be simulated by an inverted pendulum model where the stance limb is modeled as a rigid segment between the ankle and com that rotates around the ankle . the tip model approach will show whether acl injured individuals use a predominantly telescopic motion ( large change in the distance between the ankle and com ( lcom ) ) or predominately pendular motion ( large change of the approach angle of the com ( com ) ) . further output variables for the tip analysis were calculated in matlab using the kinematic and kinetic data . extension range of motion ( romknee ) , peak internal knee extensor moment ( mknee(max ) ) , knee flexion angle at mknee(max ) ( knee(mknee(max ) ) ) , peak hip moment ( mhip(max ) ) , peak ankle moment ( mankle(max ) ) , and trunk lean at the peak knee extensor moment , calculated as the sagittal plane angle of line connecting pelvis and shoulder markers ' average ( trunkap ) . the landing phase of the single leg hop was also analyzed in the coronal plane to investigate medio - lateral control of the knee joint with the following output variables : the peak external adductor moment ( madd(max ) ) , which was normalized using body weight ( bw ) and height , the maximum medio - lateral distance between the projection of the ankle and knee on the ground ( dknee ) , and trunk lean ( trunkml , calculated as trunkap but in the coronal plane ) . the peak external and not internal adductor moment was calculated , as this term is most commonly used in literature . fluency of the knee movement in the coronal plane was calculated by a method adapted from smeulders et al . it was defined as the number of times the velocity of the knee position in the coronal plane crossed zero , averaged per second . the inverse of this measure ( period ( s ) : t=1/f ) was used so that a larger value agreed with a more fluent movement . after checking for normal distribution , statistical differences for the output variables between the acl and control groups an alpha level of p<0.05 was used to evaluate statistically significant between - groups difference . when between - group differences were significant , a polynomial first order ( linear ) contrast post hoc test was performed to evaluate if either acld < aclr < cont or acld > aclr > cont ( alpha level of p<0.05 ) . if there was no significant linear contrast a secondary post hoc analysis using a simple contrast was performed to evaluate differences in acld and aclr from cont ( alpha level of p<0.025 ) . pearson 's correlations were used to explore the relation between output variables and subject characteristics . the subject groups were reasonably matched for age , height and mass ( table 1 ) . there was however a larger proportion of female subjects in the cont group ( cont : 9 females and 11 males ; aclr : 4 females and 19 males ; acld : 3 females and 18 males ; table 1 ) . acld scored on average lower in the ikdc questionnaire than aclr ( acld : 6512 ; acld : 869 ; table 1 ) , which means that they had lower knee function . the time since injury had a range of 3240 months in acld and 1083 months in aclr . there were two acld subjects who were 132 and 240 months post injury , otherwise acld ranged between 3 and 34 months post injury . visual inspection of the main output variables demonstrated that these subjects were not outliers and could be included in this study . relevant correlations between output variables and subject characteristics were not found significant ( e.g. dhop / time since injury : r=231 ; p=0.132 in acld ) . relative knee extensor ( skneeext ) strength was not significantly different between groups , while relative knee flexor ( skneeflex ) strength was significantly reduced in aclr and acld compared to cont ( cont : skneeext : 0.110.03 , skneeflex : 0.070.02 ; aclr : skneeext : 0.100.03 , skneeflex : 0.060.02 ; acld : skneeext : 0.100.02 , skneeflex : 0.060.02 ; table 1 ) . all aclr and cont subjects were able to hop , while five acld subjects were unable to . there was a significant between group difference in hop distance ( dhop ) ( p<0.001 ) and a significant linear contrast ( cont : 77.714.1 ; aclr : 75.117.8 ; acld : 57.114.1% height ; p<0.001 ; table 2 ) . the com velocity prior to landing ( vcom ) was significantly different between groups ( p<0.001 ) and had a significant linear contrast ( cont : 1.740.38 ; aclr : 1.710.40 ; acld : 1.280.34 m / s ; p<0.001 ; table 2 ) . vcom was taken as a covariate in further statistical analysis to take account of the difference in hop performance . the time taken to decelerate the com after landing ( tdec ) was not significantly different between the groups when vcom was taken into account ( cont : 0.0520.016 ; aclr : 0.0580.020 ; acld : 0.0500.018 s ; p=0.064 ; table 2 ) . tip model analysis ( fig . 2 shows that the kinematic strategy used by aclr is similar to that used by cont , while acld used a different strategy . acld landed with a more upright posture ( com closer to 90 ) than aclr and cont , and had a smaller change in lcom during the landing phase . this was further confirmed by a significant group difference in knee flexion / extension range of motion throughout landing ( romknee ) ( p=0.018 ) ; there was a significant linear contrast in romknee with the smallest range of motion in acld and the largest in cont ( cont : 69.015.7 ; aclr : 63.713.3 ; acld : 59.115.6 ; p=0.009 ; table 3 ) . there was a significant group difference in peak internal knee extensor moment ( mknee(max ) ) ( p<0.001 ) but there was no significant linear contrast ( p=0.056 ) . secondary post hoc analysis demonstrated aclr had a significantly reduced mknee(max ) compared to cont ( p<0.001 ) but there was no significant difference in mknee(max ) between acld and cont ( cont : 0.420.13 ; aclr : 0.310.16 ; acld : 0.320.14 bw.height ; p=0.056 ; table 3 ) . the peak hip flexion moment ( mhip(max ) ) was significantly different between groups ( p<0.001 ) ; there was a significant linear contrast ( cont : 0.500.18 ; aclr : 0.600.19 ; acld : 0.560.24 bw.height ; p<0.001 ; table 3 ) . plantar flexion moment ( mankle(max ) ) was significantly different between groups ( p<0.001 ) ; again the linear contrast was significant ( cont : 0.290.10 ; aclr : 0.310.10 ; acld : 0.360.10 bw.height ; p<0.001 ; table 3 ) . key features of posture at mknee(max ) were comparable in acld , aclr and cont ; there were no significant group differences in knee flexion angle at peak knee moment ( knee(mknee(max ) ) ) ( cont : 38.88.3 ; aclr : 36.310.7 ; acld : 36.210.2 ; p=0.207 ; table 3 ) ; and there was no significant difference in the forward lean of the trunk ( trunkap ) at peak knee moment ( cont : 12.77.2 ; aclr : 12.27.0 ; acld : 10.26.6 ; p=0.449 ; table 3 ) ; also the com angle at mknee(max ) ( com(mknee(max ) ) ) was not significantly different between groups ( cont : 81.94.3 ; aclr : 83.55.1 ; acld : 86.94.1 ; p=0.056 ; table 3 ) . fluency of the knee movement in the coronal plane was significantly different between groups ( p=0.006 ) and demonstrated a significant linear contrast ( cont : 0.170.41 ; aclr : 0.140.34 ; acld : 0.130.34 s ; p=0.006 ; table 4 ) . the maximum medio - lateral displacement of the knee relative to the ankle ( dknee(max ) ) showed significant group differences ( p=0.009 ) with a significant linear contrast ( cont : 0.0060.0039 ; aclr : 0.0230.038 ; acld : 0.0400.033 m ; p<0.003 ; table 4 ) . at peak knee moment there was however no group difference in medial displacement ( dknee(mknee(max ) ) ) ( cont : 0.0300.017 ; aclr : 0.0280.018 ; acld : 0.0280.016 m ; p=0.641 ; table 4 ) . peak knee adduction moments ( madd(max ) ) were significantly different between groups ( p<0.05 ) and there was a significant linear contrast with the highest madd(max ) in aclr and the lowest in cont ( cont : 0.300.01 ; aclr : 0.330.01 ; acld : 0.320.02 bw.height ; p=0.01 ; table 4 ) . there was a significant group difference for medio - lateral trunk lean at peak knee moment ( trunkml ) ( p=0.025 ) but no linear contrast ( p<0.628 ; table 4 ) . secondary post hoc analysis identified that aclr use significantly less trunkml than cont ( p=0.009 ) but there was no difference between acld and cont ( cont : 10.84.3 ; aclr : 9.14.1 ; acld : 8.93.4 ; p=0.628 ; table 4 ) . the difference in trunkml between aclr and cont was however only 1.7 and therefore not considered meaningful . this study investigated recovery of landing strategies and medio - lateral knee control during a single leg hop for distance in acld and aclr individuals . all cont and aclr subjects were able to hop whereas some acld were unable to . the acld that were able to hop did not perform as well as the other groups with a reduced hop distance ( dhop ) and com velocity prior to landing ( vcom ) . despite the decreased dhop and vcom acld required the same amount of time to decelerate ( tdec ) in the landing phase as cont . consequently a pattern of acld < aclr < cont does emerge for hop performance , which was consistent with our expectations . the reduced hop distance in acld and aclr could be partly caused by their reduced relative strength , as this was significantly correlated with hop distance ( 0.576 ; p<0.01 ) . the pattern of aclr having intermediate hop performance between acld and cont has not previously been shown . our findings that dhop was reduced in acld was in agreement with previous studies ( gauffin et al . , 1990 ; scavenius et al . , 1999 ; ( 2010 ) . to investigate recovery of landing strategies , tip analysis was used to evaluate sagittal plane kinematics and kinetics . 2 ) showed that acld used a more pendular strategy than aclr and cont as they used reduced knee flexion / extension range of motion ( romknee ) throughout landing ( table 3 ) . this pendular strategy coincided with decreased knee extensor moments and increased hip flexion and plantar flexion moments . cont used a more telescopic strategy with a greater romknee and a larger mknee(max ) ( table 3 ) . aclr also seemed to use a more telescopic strategy but performed the hop with a significantly smaller mknee(max ) than cont ( table 3 ) . they landed in a more upright posture which is a disadvantage as it leads to limited opportunities to decelerate the com . this constraint could explain that they did not show a reduced tdec despite a reduced dhop . we demonstrated that sagittal plane control during landing was intermediate in aclr between acld and cont . most of our findings on sagittal plane control of hop landing were in agreement with previous findings in literature . ( 2010 ) , but not with gokeler et al . , ( 2010 ) . in acld , rudolph et al . ( 2000 ) observed that low functioning individuals used a smaller range of knee flexion , smaller knee extensor moments and a greater contribution from the hip to the total support moment . ( 2012 ) also found that acld performed a hop using higher moments at the ankle and hip compared to cont . in an earlier study van deursen and phillips ( 2006 ) showed that the landing technique during a run and stop task was adapted in acld compared to healthy subjects and that acld used a knee avoidance technique . their findings with reduced knee extension and increased hip flexion and plantar flexion moments agreed with our findings for a single leg hop . these findings collectively support our conclusion that acld used a more pendular strategy , which coincides with a smaller romknee and mknee(max ) and larger mhip(max ) and mankle(max ) than aclr and cont . we investigated medio - lateral control and coronal plane kinetics . in respect of medio - lateral control , knee movement was least fluent in acld , intermediate in aclr and most fluent in cont ( table 4 ) . peak displacement of the knee ( dknee(max ) ) showed a similar relationship , with the largest knee displacement medial relative to the ankle in acld and the smallest in cont . at the peak extensor moment the knee was however at a similar position relative to the ankle in all groups . the knee movement was less fluent in the acl patients and showed larger medio - lateral excursions , which is interpreted as a sign of lack of knee control . either they were unable to provide sufficient motor control for an external adductor moment of the same magnitude as in cont , or it could be interpreted as a protective strategy to avoid challenging adductor moments altogether . although the peak knee moments were reduced , the lack of control could arguably result in loading of the articular cartilage on locations that are normally not loaded in such a manner and this presumed altered loading could have implications for early development of osteoarthritis ( blagojevic et al . , 2010 ) . our study is the first to demonstrate that knee control was not fully recovered in acld and aclr . there is no previous literature evaluating coronal plane control of the knee during single leg hop in acl patients . the cont group had a larger proportion of females than the acld and aclr groups . the main gender differences are due to height and mass and we therefore normalized our outcome variables to body height and weight to account for the gender differences . it could be expected that if the groups were perfectly matched with a larger proportion of male subjects in the cont group their mean maximum hop distance would have been increased , which would have exaggerated our results and increased significance . another limitation was that the acl injured patients had other accompanying injuries besides acl rupture . the typical population of patients seen in the hospital setting however has a mixed combination of injuries and an acl injury hardly ever occurs without at least some comorbidity . the participants in this study also had a relatively wide range according to time since injury and surgery . a proportion of the acld participants ( 20% ) were unable to hop and could not be included in the analysis . therefore those acld that were included in the analysis were a subset of better performing acld individuals . because of this some of the conclusions regarding acld could have been underestimated , as in that they were performing better as a group as would be expected . in spite of that effect we still found significant differences , which emphasizes that between group differences were present . this study demonstrated that to improve treatment outcome other aspects need to be evaluated , such as movement strategies and medio - lateral knee control . neuromuscular control and perturbation exercises are already recommended but need to be further developed . to optimize control of knee joint loading a telescopic deceleration strategy could be promoted by early introduction in less demanding tasks such as one legged squat , before progressing to more dynamic tasks . this study showed that acld and aclr patients had not recovered landing strategies and medio - lateral knee control . this altered control may result in altered stresses within the knee that could result in further damage and early onset osteoarthritis .

anterior cruciate ligament ( acl ) injury can result in failure to return to pre - injury activity levels and future osteoarthritis predisposition . single leg hop is used in late rehabilitation to evaluate recovery and inform treatment but biomechanical understanding of this activity is insufficient.this study investigated single leg hop for distance aiming to evaluate if acl patients had recovered : ( 1 ) landing strategies and ( 2 ) medio - lateral knee control . we hypothesized that patients with reconstructive surgery ( aclr ) would have more similar landing strategies and knee control to healthy controls than patients treated conservatively ( acld).16 acld and 23 aclr subjects were compared to 20 healthy controls ( cont ) . kinematic and ground reaction force data were collected while subjects hopped their maximum distance . the main output parameters were hop distance , peak knee flexor angles and extensor moments and fluency ( a measure introduced to represent medio - lateral knee control ) . statistical differences between acl and control groups were analyzed using a general linear model univariate analysis , with com velocity prior to landing as covariate.hop distance was the smallest for acld and largest for cont ( p<0.001 ; acld 57.114.1 ; aclr 75.117.8 ; cont 77.714.07% height ) . aclr used a similar kinematic strategy to cont , but had a reduced peak knee extensor moment ( p<0.001 ; acld 0.320.14 ; aclr 0.310.16 ; cont 0.420.13 bw.height ) . fluency was reduced in both acld and aclr ( p=0.006 ; acld 0.130.34 ; aclr 0.140.34 ; cont 0.170.41 s).clinical practice uses hopping distance to evaluate acl patients ' recovery . this study demonstrated that aspects such as movement strategies and knee control need to be evaluated .

Introduction Methods Results Discussion Conflict of interest statement

two major impacts of acl rupture , whether treated conservatively or surgically , are failure to return to pre - injury activity levels ( myklebust et al . a review evaluating return to sport following acl injury indicated that up to 48% was not returning to their pre - injury sporting levels ( ardern et al . medio - lateral knee control is an important factor to assess in acl injured patients . besides adductor moments , clinical evidence suggests that fluency of movement is an aspect of medio - lateral knee control that is worth investigating . this study therefore investigated recovery of both these aspects of knee control in acl patients . , single leg hop is an exercise used in late stage rehabilitation and a tool to evaluate recovery and inform treatment selection ( ardern et al . this activity challenges knee stability by requiring large knee moments during take - off and landing and mimics some maneuvers encountered on return to sport . clinically the symmetry index of the injured and non - injured hop distance is frequently used to evaluate hop performance ( engelen - van melick et al . , acld individuals on the other hand have been reported not to recover hop distance ( gauffin et al . , 2000 ) ; with high functioning acld having unchanged knee kinematics and an increased contribution of the ankle to the total support moment , and poor functioning acld using a smaller range of knee flexion , a lower peak vertical ground reaction force , lower knee extensor moments and greater contribution from the hip to the total support moment . compared to healthy controls , acld individuals performed a single leg hop for distance using higher moments at the ankle and hip , more forward trunk lean and a more anterior ground reaction force vector ( oberlnder et al . besides these kinematic and kinetic differences reported on single leg hopping in acl injured individuals , there are no studies investigating how this movement challenges motor control . recovery of motor control is essential for return to sports and therefore an important aspect of rehabilitation this study therefore investigated the movement strategies used during the landing phase of a single leg hop for distance . this strategy requires high knee extensor moments and puts high demands on dynamic knee control . com deceleration can also be achieved by using a pendular strategy where com rotation around the ankle is controlled . this strategy requires smaller knee extensor moments and requires less medio - lateral knee control , but larger hip flexion and plantar flexion moments . clearly a better understanding of knee control during functional movements is needed to be able to improve rehabilitation outcome . this study therefore investigated a single leg hop for distance , which challenges knee stability , with the aims of evaluating if:1)landing strategies in acld and aclr have been recovered to those of healthy control subjects and2)medio - lateral control has been recovered in acld and aclr . landing strategies in acld and aclr have been recovered to those of healthy control subjects and medio - lateral control has been recovered in acld and aclr . we hypothesized that acl injury would result in altered landing strategies and reduced medio - lateral knee control compared to healthy controls . in addition , we hypothesized that acld patients would be more affected in their landing strategies and medio - lateral knee control compared to aclr . 21 acld , 23 aclr and 20 healthy control ( cont ) subjects provided informed consent to participate in this study ( subject demographics are in table 1 ) . acl subjects were recruited from a typical clinical ( non - elite sporting ) population . ethical approval for this study was obtained from the south east wales research ethics committee . inclusion criteria were that patients were aged between 18 and 65 years , had an acl rupture ( acld group ) , or a primary acl reconstruction ( aclr group ) that may or may not be accompanied with a meniscal tear , collateral ligament sprain , or cartilage and sub - cortical bone bruises ; had finished their rehabilitation ; had no other pathology which affects their movement ; had no previous knee surgery and were able to provide informed consent independently . for this study all of our subjects had mri scans taken and those were assessed by an expert clinician to decide whether they fit into the category of a typical injury . knee function was scored for acld and aclr using the international knee documentation subjective knee ( ikdc ) questionnaire ( irrgang et al . knee extensor ( skneeext ) and flexor ( skneeflex ) , and hip abductor ( shipabd ) and adductor ( shipadd ) isokinetic strength were measured at 90 /s and 45 /s respectively on a biodex system 4 pro dynamometer ( biodex medical systems inc , usa ) . this was measured on both legs , but presented for the injured ( aclr and acld ) and the dominant stance leg ( cont ) only . individuals were asked to hop their maximum single leg hop distance and regain their balance after landing . the hop distance was marked from the force platform and subjects were then asked to perform four single leg hops for maximum distance from this mark , as such that they would land on the force platform . kinematic data were collected at 250 hz using an eight camera vicon mx motion analysis system ( oxford metrics group ltd . ground reaction force data were collected using a kistler force plate ( kistler instruments ltd . inverse dynamics calculations were performed within vicon nexus software ( version 1.6.1 ) and data were further processed and analyzed in matlab r2010b ( the mathworks inc . parameters used for hop performance were hopping distance ( dhop ) , com velocity along the axis of hopping movement prior to landing ( vcom ) and duration of the com deceleration phase during landing ( tdec ) . dhop was calculated as the distance the ankle joint center traveled along the axis of hopping . extension range of motion ( romknee ) , peak internal knee extensor moment ( mknee(max ) ) , knee flexion angle at mknee(max ) ( knee(mknee(max ) ) ) , peak hip moment ( mhip(max ) ) , peak ankle moment ( mankle(max ) ) , and trunk lean at the peak knee extensor moment , calculated as the sagittal plane angle of line connecting pelvis and shoulder markers ' average ( trunkap ) . the landing phase of the single leg hop was also analyzed in the coronal plane to investigate medio - lateral control of the knee joint with the following output variables : the peak external adductor moment ( madd(max ) ) , which was normalized using body weight ( bw ) and height , the maximum medio - lateral distance between the projection of the ankle and knee on the ground ( dknee ) , and trunk lean ( trunkml , calculated as trunkap but in the coronal plane ) . after checking for normal distribution , statistical differences for the output variables between the acl and control groups an alpha level of p<0.05 was used to evaluate statistically significant between - groups difference . when between - group differences were significant , a polynomial first order ( linear ) contrast post hoc test was performed to evaluate if either acld < aclr < cont or acld > aclr > cont ( alpha level of p<0.05 ) . if there was no significant linear contrast a secondary post hoc analysis using a simple contrast was performed to evaluate differences in acld and aclr from cont ( alpha level of p<0.025 ) . the subject groups were reasonably matched for age , height and mass ( table 1 ) . there was however a larger proportion of female subjects in the cont group ( cont : 9 females and 11 males ; aclr : 4 females and 19 males ; acld : 3 females and 18 males ; table 1 ) . acld scored on average lower in the ikdc questionnaire than aclr ( acld : 6512 ; acld : 869 ; table 1 ) , which means that they had lower knee function . the time since injury had a range of 3240 months in acld and 1083 months in aclr . visual inspection of the main output variables demonstrated that these subjects were not outliers and could be included in this study . relative knee extensor ( skneeext ) strength was not significantly different between groups , while relative knee flexor ( skneeflex ) strength was significantly reduced in aclr and acld compared to cont ( cont : skneeext : 0.110.03 , skneeflex : 0.070.02 ; aclr : skneeext : 0.100.03 , skneeflex : 0.060.02 ; acld : skneeext : 0.100.02 , skneeflex : 0.060.02 ; table 1 ) . there was a significant between group difference in hop distance ( dhop ) ( p<0.001 ) and a significant linear contrast ( cont : 77.714.1 ; aclr : 75.117.8 ; acld : 57.114.1% height ; p<0.001 ; table 2 ) . the com velocity prior to landing ( vcom ) was significantly different between groups ( p<0.001 ) and had a significant linear contrast ( cont : 1.740.38 ; aclr : 1.710.40 ; acld : 1.280.34 m / s ; p<0.001 ; table 2 ) . the time taken to decelerate the com after landing ( tdec ) was not significantly different between the groups when vcom was taken into account ( cont : 0.0520.016 ; aclr : 0.0580.020 ; acld : 0.0500.018 s ; p=0.064 ; table 2 ) . 2 shows that the kinematic strategy used by aclr is similar to that used by cont , while acld used a different strategy . acld landed with a more upright posture ( com closer to 90 ) than aclr and cont , and had a smaller change in lcom during the landing phase . this was further confirmed by a significant group difference in knee flexion / extension range of motion throughout landing ( romknee ) ( p=0.018 ) ; there was a significant linear contrast in romknee with the smallest range of motion in acld and the largest in cont ( cont : 69.015.7 ; aclr : 63.713.3 ; acld : 59.115.6 ; p=0.009 ; table 3 ) . there was a significant group difference in peak internal knee extensor moment ( mknee(max ) ) ( p<0.001 ) but there was no significant linear contrast ( p=0.056 ) . secondary post hoc analysis demonstrated aclr had a significantly reduced mknee(max ) compared to cont ( p<0.001 ) but there was no significant difference in mknee(max ) between acld and cont ( cont : 0.420.13 ; aclr : 0.310.16 ; acld : 0.320.14 bw.height ; p=0.056 ; table 3 ) . the peak hip flexion moment ( mhip(max ) ) was significantly different between groups ( p<0.001 ) ; there was a significant linear contrast ( cont : 0.500.18 ; aclr : 0.600.19 ; acld : 0.560.24 bw.height ; p<0.001 ; table 3 ) . plantar flexion moment ( mankle(max ) ) was significantly different between groups ( p<0.001 ) ; again the linear contrast was significant ( cont : 0.290.10 ; aclr : 0.310.10 ; acld : 0.360.10 bw.height ; p<0.001 ; table 3 ) . key features of posture at mknee(max ) were comparable in acld , aclr and cont ; there were no significant group differences in knee flexion angle at peak knee moment ( knee(mknee(max ) ) ) ( cont : 38.88.3 ; aclr : 36.310.7 ; acld : 36.210.2 ; p=0.207 ; table 3 ) ; and there was no significant difference in the forward lean of the trunk ( trunkap ) at peak knee moment ( cont : 12.77.2 ; aclr : 12.27.0 ; acld : 10.26.6 ; p=0.449 ; table 3 ) ; also the com angle at mknee(max ) ( com(mknee(max ) ) ) was not significantly different between groups ( cont : 81.94.3 ; aclr : 83.55.1 ; acld : 86.94.1 ; p=0.056 ; table 3 ) . fluency of the knee movement in the coronal plane was significantly different between groups ( p=0.006 ) and demonstrated a significant linear contrast ( cont : 0.170.41 ; aclr : 0.140.34 ; acld : 0.130.34 s ; p=0.006 ; table 4 ) . the maximum medio - lateral displacement of the knee relative to the ankle ( dknee(max ) ) showed significant group differences ( p=0.009 ) with a significant linear contrast ( cont : 0.0060.0039 ; aclr : 0.0230.038 ; acld : 0.0400.033 m ; p<0.003 ; table 4 ) . at peak knee moment there was however no group difference in medial displacement ( dknee(mknee(max ) ) ) ( cont : 0.0300.017 ; aclr : 0.0280.018 ; acld : 0.0280.016 m ; p=0.641 ; table 4 ) . peak knee adduction moments ( madd(max ) ) were significantly different between groups ( p<0.05 ) and there was a significant linear contrast with the highest madd(max ) in aclr and the lowest in cont ( cont : 0.300.01 ; aclr : 0.330.01 ; acld : 0.320.02 bw.height ; p=0.01 ; table 4 ) . there was a significant group difference for medio - lateral trunk lean at peak knee moment ( trunkml ) ( p=0.025 ) but no linear contrast ( p<0.628 ; table 4 ) . secondary post hoc analysis identified that aclr use significantly less trunkml than cont ( p=0.009 ) but there was no difference between acld and cont ( cont : 10.84.3 ; aclr : 9.14.1 ; acld : 8.93.4 ; p=0.628 ; table 4 ) . this study investigated recovery of landing strategies and medio - lateral knee control during a single leg hop for distance in acld and aclr individuals . all cont and aclr subjects were able to hop whereas some acld were unable to . the acld that were able to hop did not perform as well as the other groups with a reduced hop distance ( dhop ) and com velocity prior to landing ( vcom ) . the reduced hop distance in acld and aclr could be partly caused by their reduced relative strength , as this was significantly correlated with hop distance ( 0.576 ; p<0.01 ) . the pattern of aclr having intermediate hop performance between acld and cont has not previously been shown . our findings that dhop was reduced in acld was in agreement with previous studies ( gauffin et al . to investigate recovery of landing strategies , tip analysis was used to evaluate sagittal plane kinematics and kinetics . 2 ) showed that acld used a more pendular strategy than aclr and cont as they used reduced knee flexion / extension range of motion ( romknee ) throughout landing ( table 3 ) . this pendular strategy coincided with decreased knee extensor moments and increased hip flexion and plantar flexion moments . aclr also seemed to use a more telescopic strategy but performed the hop with a significantly smaller mknee(max ) than cont ( table 3 ) . this constraint could explain that they did not show a reduced tdec despite a reduced dhop . we demonstrated that sagittal plane control during landing was intermediate in aclr between acld and cont . ( 2000 ) observed that low functioning individuals used a smaller range of knee flexion , smaller knee extensor moments and a greater contribution from the hip to the total support moment . ( 2012 ) also found that acld performed a hop using higher moments at the ankle and hip compared to cont . in an earlier study van deursen and phillips ( 2006 ) showed that the landing technique during a run and stop task was adapted in acld compared to healthy subjects and that acld used a knee avoidance technique . their findings with reduced knee extension and increased hip flexion and plantar flexion moments agreed with our findings for a single leg hop . these findings collectively support our conclusion that acld used a more pendular strategy , which coincides with a smaller romknee and mknee(max ) and larger mhip(max ) and mankle(max ) than aclr and cont . we investigated medio - lateral control and coronal plane kinetics . in respect of medio - lateral control , knee movement was least fluent in acld , intermediate in aclr and most fluent in cont ( table 4 ) . peak displacement of the knee ( dknee(max ) ) showed a similar relationship , with the largest knee displacement medial relative to the ankle in acld and the smallest in cont . at the peak extensor moment the knee was however at a similar position relative to the ankle in all groups . the knee movement was less fluent in the acl patients and showed larger medio - lateral excursions , which is interpreted as a sign of lack of knee control . either they were unable to provide sufficient motor control for an external adductor moment of the same magnitude as in cont , or it could be interpreted as a protective strategy to avoid challenging adductor moments altogether . although the peak knee moments were reduced , the lack of control could arguably result in loading of the articular cartilage on locations that are normally not loaded in such a manner and this presumed altered loading could have implications for early development of osteoarthritis ( blagojevic et al . our study is the first to demonstrate that knee control was not fully recovered in acld and aclr . there is no previous literature evaluating coronal plane control of the knee during single leg hop in acl patients . the cont group had a larger proportion of females than the acld and aclr groups . it could be expected that if the groups were perfectly matched with a larger proportion of male subjects in the cont group their mean maximum hop distance would have been increased , which would have exaggerated our results and increased significance . another limitation was that the acl injured patients had other accompanying injuries besides acl rupture . the participants in this study also had a relatively wide range according to time since injury and surgery . this study demonstrated that to improve treatment outcome other aspects need to be evaluated , such as movement strategies and medio - lateral knee control . neuromuscular control and perturbation exercises are already recommended but need to be further developed . this study showed that acld and aclr patients had not recovered landing strategies and medio - lateral knee control . this altered control may result in altered stresses within the knee that could result in further damage and early onset osteoarthritis .

initiating mutations in colorectal cancer ( crc ) most commonly target the apc tumor suppressor gene ( kinzler and vogelstein , 1996 ) . apc is a negative regulator of wnt signaling that is required to target -catenin for proteosomal degradation . the loss of the apc gene results in the accumulation of -catenin in the nucleus , subsequent activation of wnt transcriptional targets , and ultimately adenoma formation ( korinek et al . , 1997 ; morin et al . , 1997 ; sansom et al . , normal intestinal homeostasis is maintained by a number of intestinal stem cells ( iscs ) . recent experiments have shown the presence of a cycling stem cell population marked by lgr5 along with a longer - lived population that can repopulate the crypt after injury ( barker et al . lgr5 expression marks crypt columnar stem cells that have a readily identifiable transcriptional profile termed the isc signature . genetic deletion of apc within lgr5 iscs leads to rapid adenoma formation , whereas deletion within more differentiated lineages leads to poorly proliferative lesions that fail to progress without additional oncogenic mutations ( schwitalla et al . , interestingly , although an established wnt target , lgr5 is only expressed in a subset of apc - deficient tumor cells . this may be due to lgr5 marking cells with the highest levels of wnt signaling or to co - operation of multiple pathways in conferring the lgr5 isc phenotype . our previous studies have shown that the wnt target gene myc is required for the phenotypes induced by apc loss and that reduced levels of myc slows intestinal tumorigenesis ( athineos and sansom , 2010 ; sansom et al . , 2007 ) . these studies set a precedent that targeting the downstream effectors of wnt signaling may be of therapeutic benefit in colorectal cancer . in the case of myc , although there is great interest in it as a therapeutic target ( soucek et al . , 2008 ) therefore identification of other pathways downstream of apc loss , in particular those highly active in lgr5 iscs may provide candidates to target apc - deficient cells . rac1 is a gtpase that acts as a key signaling node modulating a diverse set of cellular processes including proliferation , apoptosis , migration , and invasion . it influences a variety of signaling pathways including mtor , nf-b , jnk , and reactive oxygen species ( ros ) production ( ellenbroek and collard , 2007 ) . rac1 cycles between an inactive gdp and active gtp - bound state and is controlled by guanine nucleotide exchange factors ( gefs ) ( which activate rac1 ) and gtpase - activating proteins ( which inactivate rac1 ) . we previously identified myc - dependent upregulation of a number of racgefs after apc loss , and racgefs are commonly overexpressed during tumor progression ( lindsay et al . rac1-activating mutations have also been discovered in melanoma and a constitutively active rac1 isoform termed rac1b identified in colon and lung tumor samples can promote lung tumorigenesis ( hodis et al . , 2012 ; krauthammer et al . , 2012 ; zhou et al . , rac1 is also required for kras - mediated tumorigenesis in skin and the lung , but the mechanism of how rac1 loss suppresses tumorigenesis is still unclear ( kissil et al . , 2007 ; samuel et al . , 2011 two studies suggest that rac1 is required for nuclear localization of -catenin and wnt signaling ( phelps et al . , 2009 ; first , wu and colleagues demonstrated that rac1 is required for -catenin nuclear localization in the developing limb bud and the effects of its deletion phenocopy those of -catenin . as nuclear localization of -catenin is a key event during crc initiation if this dependency remains once apc is deleted , it would suggest that rac1 would be an excellent therapeutic target . additionally , phelps and colleagues demonstrated that in zebrafish , apc loss alone was insufficient to cause nuclear localization of -catenin ( phelps et al . , 2009 ) . here the consequence of apc loss was perturbed differentiation , which was dependent on the transcriptional repressor ctbp1 . additional activation of kras was required for nuclear accumulation of -catenin and hyperproliferation after apc loss . the authors proposed that rac1 activation downstream of constitutive kras activity was the mechanism that allowed the nuclear accumulation of -catenin . these results are controversial as a number of studies have shown that ( 1 ) -catenin can be nuclear localized in human adenomas without kras mutation ( obrador - hevia et al . , 2010 ) , ( 2 ) wnt target genes can be upregulated in human adenomas without kras mutation ( sabates - bellver et al . , 2007 ) , and ( 3 ) murine studies have shown nuclear -catenin and wnt target gene upregulation in adenomas from the apc mouse ( sansom et al . 2008 ) . therefore , given this evidence for a role of rac1 in wnt signaling , we investigated the importance of rac1 activation after apc deletion . we show that a number of racgefs and rac1b are upregulated after apc deletion , which leads to increased activity of rac1 . while rac1 deletion did not stop the nuclear accumulation of -catenin and activation of the majority of the tcf / lef targets , it attenuated hyperproliferation after apc loss and subsequent tumorigenesis . finally , we show that this is due to rac1-mediated control of ros production and nf-b activation . previous microarray analysis revealed upregulation of several racgefs after apc loss ( sansom et al . , 2007 ) . we found significant overexpression of vav3 and tiam1 transcripts after apc loss in a myc - dependent manner ( as synchronous deletion of apc and myc returned expression levels to wild - type ) ( figure 1a ) . chromatin immunoprecipitation ( chip ) revealed that myc was bound to the tiam1 promoter , demonstrating it is a direct myc target ( figure s1a available online ) . moreover , we observed increased expression of rac1b but not rac1 ( figure 1a ) . to investigate whether this led to increased rac1 activation , we performed immunohistochemistry ( ihc ) using an antibody raised against rac - gtp ( samuel et al . , 2011 ) . we observed increased , specific positivity for rac - gtp in apc - deficient tissue that was dependent on myc ( figure 1b ) . pull - downs for active rac1 in wild - type and apc - deficient intestinal extracts confirmed these results ( figure s1b ) . levels of rac1 protein and mrna remained unchanged ( figures 1a and s1c ) . to assess rac activation in human crc , we stained a tma containing 50 normal and 650 primary crc tumor cores for rac - gtp ( figures 1c and s1d ) ( duncan et al . , 2008 ; odwyer et al . , 2011 ) . we found a significant ( p 0.0001 ) increase in rac - gtp staining intensity at all tumor stages compared to normal tissue , indicating that rac activation is an early event in crc . we also stained a commercial tma for rac - gtp , tiam1 , vav3 , and myc and found a significant correlation between the expressions of these proteins ( figures 1d and s1e ) . these data are consistent with previous studies suggesting that rac1 expression ( and presumably activation ) is elevated in crc ( espina et al . , 2008 ) . therefore , our data suggest that in mammalian cells , activation of rac1 occurs downstream of wnt activation . to assess the functional significance of rac1 activation after apc loss , we generated vil - cre - er apc rac1 ( apc rac1 ) mice ( el marjou et al . four days after cre induction , we observed loss of both rac1 and rac1b mrna and rac1 protein ( figures 1a and s2a ) . in contrast to induced vil - cre - er apc mice ( apc ) , the intestines from apc rac1 mice had smaller hyperproliferative crypts with significantly reduced incorporation of brdu ( figures 2a and s2b ) . acute deletion of rac1 alone ( vil - cre - er rac1 ) did not affect crypt size , proliferation , or isc lineage - tracing capacity ( figures 2a and s2c ) , although increased apoptosis of villi enterocytes and intestinal barrier breakdown was observed at later time points ( data not shown ) . this prevented later time point analysis and ruling out of potential subtle homeostatic effects over the longer term . transgene also induces recombination in the colonic epithelium and the rac1-dependent intestinal proliferation phenotype was recapitulated in this tissue ( figure s2d ) . thus , rac1 is a critical component in permitting crypt progenitor hyperproliferation after apc loss in the intestinal and colonic epithelia . given previous studies reporting that rac1 is required for accumulation of nuclear -catenin ( phelps et al . , 2009 ; 2008 ) , we predicted that this and downstream target gene activation should be impaired in apc rac1 intestines . however , ihc analysis showed nuclear -catenin in apc rac1- ( and apc- ) deleted intestines ( figure 2b ) . moreover , quantitative rt - pcr ( qrt - pcr ) of selected tcf / lef targets that are deregulated after apc loss were unchanged in apc rac1 intestines ( figure s2e ) ( sansom et al . , 2007 ) . thus , rac1 is not required for -catenin nuclear localization and/or its functional activity in the absence of apc . to delineate the mechanism , we compared global gene expression changes between apc and apc rac1 intestines by microarray . gene set enrichment analysis ( gsea ) identified a significant overlap between our data set and the isc signatures associated with lgr5 + or ephb2 high iscs ( chi - square test with yates s correction , lgr5 p < 0.0001 , ephb2 p = 0.001 ) ( barker et al . , 2007 ; merlos - surez et al . , 2011 ; van der flier et al . , we confirmed that the isc signature was upregulated after apc loss and reverted back to wild - type levels in apc rac1 intestines using qrt - pcr ( figure 2c ) . to visualize changes in the lgr5-expressing population , we generated inducible vil - cre - er wt , rac1 , apc , and apc rac1 mice carrying the lgr5 transgene ( barker et al . lgr5-gfp expression is restricted to the base of the crypt in wild - type and rac1-deficient mice by ihc ( figure s2f ) . after apc loss , there is a significant increase in the number of lgr5-gfp - positive cells that are suppressed in apc rac1 intestines ( figure 2d ) . moreover , scoring the location of lgr5-expressing cells revealed a striking expansion of the stem cell zone that codeletion of rac1 prevented ( figure 2e ) . this was confirmed using multiphoton microscopy on intestines ( figures s2 g and s2h ) . to investigate whether rac1 deletion suppressed the proliferative potential of all isc populations , we purified crypts from apc and apc rac1 intestines and cultured them ex vivo . remarkably , while apc crypts formed colonies , apc rac1 crypts did not , suggesting that rac1 is required for the clonogenic capacity of all intestinal cell types ( figure s2i ) therefore , rac1 is required for two critical constituents of apc loss , progenitor hyperproliferation and lgr5 isc expansion . as rac1 integrates many pathways , a plethora of downstream effectors could account for the phenotype that we observed . rac1 is a member of the superoxide - generating nadph oxidase complex , and rac1b overexpression has been shown to lead to ros production ( bromberg et al . , 1994 ; radisky et al . , the generation of ros by nadph oxidase involves the conversion of nadph to nadp+ so its activity can be evaluated by the ratio of these molecules . we observed a shift in the nadp+/nadph ratio in apc intestinal extracts compared to wt , indicating increased complex activity that was rac1 dependent ( figure s3a ) . to analyze ros levels within the intestinal epithelium , we stained wt , rac1 , apc , and apc rac1 intestines with the ros - responsive dye dihydroethidium ( dhe ) . we observed a significant increase in staining intensity in apc but not apc rac1 intestines ( figures 3a and 3b ) . thus , apc loss leads to increased ros generation in the intestinal epithelium via activation of the rac1-containing nadph oxidase complex . moreover , dhe staining indicated that in wt tissue ros generation was particularly high at the crypt base ( figure 3a ) . scoring dhe fluorescence intensity based on crypt cell position showed that ros generation was highest at cell positions 13 ( where lgr5 + cells reside ) and was absent in rac1-deficient intestines ( figure s3b ) . to confirm that lgr5 + cells have high levels of ros , we isolated intestinal epithelial cells from mice carrying the lgr5 transgene ( barker et al . , 2007 ) , stained them with the ros - responsive dye cellrox deep red , and analyzed them by flow cytometry . we found that lgr5-gfp - expressing cells contained around 2-fold more ros than the cell population as a whole ( figures 3c , 3d , and s3c ) . as ros generation at the crypt base was rac1 dependent ( figures 3a and s3b ) , we investigated the expression of tiam1 , vav3 , and rac1b in lgr5 + cells sorted by qrt - pcr and found that all were significantly enriched , though rac1 levels were unchanged ( figure 3e ) . we hypothesized increased rac1-driven ros generation may be a critical process in conferring the isc / progenitor phenotypes associated with apc loss . first , we assessed whether reduction of intracellular ros levels through nac treatment could recapitulate the phenotypes associated with rac1 deletion . remarkably , treatment of apc - deficient mice with nac strongly suppressed overexpression of the isc signature genes ( lgr5 , olfm4 , and rgmb ) ( figure 4a ) . this suppression did not extend to non - isc wnt target genes , indicating that wnt signaling was not perturbed by this treatment ( figure s4a ) . nac treatment also significantly attenuated proliferation in apc intestines ( figures 4b and s4b ) . importantly , nac treatment had no effect on proliferation , number of lgr5-gfp cells , or gross villus histology in wt mice ( 4 day and 8 week time points ) ( figure 4b and data not shown ) . second , we asked whether induction of ros could rescue the reduced isc signature in apc rac1 intestines . systemic treatment with the ros - inducing compound paraquat led to increased expression of isc marker genes in apc rac1 intestines ( figures 4c and s4c ) . in situ microscopy of apc rac1 mice carrying the lgr5 transgene demonstrated a significant expansion of the isc zone upon paraquat treatment ( figure s4d ) . in wt and apc - deficient mice treated with we also observed a significant increase in proliferation in the intestines of the three cohorts of paraquat - treated mice ( figures 4d and s4e ) . thus , induction of ros can induce proliferation and , in the absence of rac1 , can partially compensate for its loss . we have recently demonstrated a requirement for the nf-b transcription factor p65 in isc expansion after -catenin activation ( schwitalla et al . , 2013 ) . given the remarkable overlap with this study , we addressed whether nf-b signaling is an important mediator of lgr5 isc / progenitor proliferation after apc loss . we observed a significant reduction of nf-b signaling components p65 , ikk , and acetyl - p65 in apc rac1 compared to apc - deficient intestines ( figures 5a , s5a , and s5b ) . also , nf-b binding activity was perturbed in apc rac1 compared to apc intestinal extracts ( figure s5c ) . we also observed an increase in p65 binding to the promoters of lgr5 , olfm4 , and rgmb after apc loss that was lost upon codeletion of rac1 ( figures 5b and 5c ) . we next sought to functionally determine whether constitutive nf-b signaling can compensate for rac1 deletion . we crossed apc rac1 mice to mice carrying the r26stopikk2ca allele ( an inducible , constitutively active ikk2 allele from now on referred to as ikk ) to generate apc rac1 ikk mice ( sasaki et al . , 2006 ) this was sufficient to almost completely rescue the proliferation defect and partially rescue isc marker expression in apc rac1 mice ( figures 5e , 5f , and s5d ) . thus , nf-b signaling is an important downstream effector of rac1 signaling in promoting the phenotypes of apc loss . we noted that neither increased ros nor nf-b activation were able to completely rescue the apc rac1 deletion phenotype . this is perhaps unsurprising given that we also observed effects on a number of other pathways downstream of rac1 signaling , such as stat3 and mtor , which could impinge on the phenotypes of apc loss ( figure s5e ) . our previous studies have shown that apc deletion within iscs using lgr5 leads to rapid tumorigenesis ( barker et al . , 2009 ) . given that rac1 deletion prevents increased lgr5 expression within apc - deficient crypts , we hypothesized that this would reduce the tumor - forming capacity of iscs . to test this , we generated control lgr5 apc ( lgr5 apc ) and experimental lgr5 apc rac1 ( lgr5 apc rac1 ) mice . to induce recombination , we gave mice a daily injection of tamoxifen for 4 days ( a total of four injections ) . most control lgr5 apc mice developed a lethal intestinal adenoma burden by 20 days ; however , none of the lgr5 apc rac1 did until much later time points ( figure 6a ) . when they did succumb to adenoma formation , all adenomas expressed rac1 , indicating that they most likely arose from cells in which rac1 deletion had not occurred ( figure s6a ) . histological analysis revealed that in contrast to the adenomas found in controls , lgr5 apc rac1 mice developed numerous cystic structures and micro adenomas ( figures 6b and 6e ) . these lesions phenotypically resemble those that arise via apc deletion outside the stem cell zone ( barker et al . , 2009 ) and had a very low proliferative index ( figure 6e ) . despite high levels of nuclear -catenin , they had reduced levels of lgr5-gfp , suggesting that rac1 activity is essential for tumor growth and the efficient expansion of lgr5 cells after apc loss ( figures 6f , 6 g , and s6b ) . lgr5 also marks colonic stem cells , and deletion of apc with lgr5 leads to formation of colonic adenomas ( barker et al . , , few colonic adenomas were macroscopically visible in either lgr5 apc or lgr5 apc rac1 mice . however , microscopic analysis uncovered numerous aberrant crypt foci and micro adenomas in the colons of lgr5 apc mice that stained positive for nuclear -catenin ( figure s6c and data not shown ) . we observed a significant reduction in the number of these lesions in the colons of lgr5 apc rac1 mice ( figure s6d ) , with the majority of these mice ( 6/8 ) being tumor free . thus , rac1 is required for intestinal and colonic adenoma formation after apc loss . to test whether ros production by rac1 was also important this led to a significant extension of tumor - free lifespan , further emphasizing the role ros plays in intestinal transformation ( figure 6h ) . as crc progresses , tumors accumulate additional mutations , of which the activation of kras ( kras ) is one of the most common ( cancer genome atlas network , 2012 ) . therefore , we generated vil - cre - er apc kras ( apc kras ) and vil - cre - er apc kras rac1 ( apc kras rac1 ) mice . three days after induction , we purified crypts from these mice and assessed their tumor formation capacity by injecting them into nude mice . strikingly , crypts from apc kras intestines were able to efficiently form tumors , whereas those from apc kras rac1 animals did not ( figure 6i ) . in the two out of six cases in which delayed tumor formation occurred from apc kras rac1 crypts , they were found to express rac1 , indicating that these grew from rare crypts that had not lost rac1 ( data not shown ) . together , these data strongly indicate an essential role for rac1 in intestinal adenoma formation . a recent study has demonstrated that lgr5 marks a population of cancer stem cells ( cscs ) within mouse intestinal adenomas ( schepers et al . , 2012 ) . as rac1 permits the proliferation of lgr5 iscs after apc loss , we asked whether rac1 is important for maintaining this population in established tumors . specifically , we wanted to assess the impact of deletion of rac1 from the lgr5 + population within adenomas . for this , we intercrossed mice carrying the apc allele ( pollard et al . , we aged lgr5 apc carrying the rosa26-rfp reporter ( figure s6e ) to 60 days , when mice have small tumors and induced recombination with tamoxifen . in agreement with previously published work , we observed lineage tracing within both wild - type epithelium and in adenomas , with marked rfp - positive clones 3 weeks after induction in both ( figures s6f and s6 g ) ( schepers et al . , 2012 ) . given that we were able to induce recombination within adenomas , we aged lgr5 apc rac1 mice until they developed signs of intestinal tumor burden ( weight loss and anemia ) and induced recombination . rac1 deletion significantly reduced the number of lgr5-gfp - positive cells within the tumors ( figure 6j ) . moreover , the reduction in the number of lgr5-gfp - positive cells was partially ablated by concurrent treatment of mice with paraquat ( figure s6j ) . here , we demonstrate a critical requirement for rac1 in intestinal transformation after apc loss ( figure 7 ) . in contrast to previous studies suggesting that rac1 is required for nuclear localization of -catenin ( wu et al . , 2008 ) , we find that rac1 acts downstream of constitutive wnt signaling to promote progenitor cell proliferation and expansion of the lgr5 isc population . this may reflect a difference between the effects of wnt3a stimulation and apc loss or may indicate a tissue - specific role for rac1 in -catenin activation . we find that the dependence on rac1 for crypt hyperproliferation is translated to a tumor model driven by apc loss in lgr5 iscs . these findings are striking as other oncogenic mediators overexpressed after apc loss , including cyclin d1/d2 , cd44 , and mbd2 , show much less dramatic effects on crypt hyperproliferation or intestinal tumorigenesis ( cole et al . 2008 ) . as rac1 also suppresses proliferation of apc - deficient cells after kras activation , we propose that it is essential for intestinal transformation . the role of ros in stem cell maintenance , cellular transformation , and csc survival appears to be context and tissue specific . in proliferative neural stem cells , high levels of ros regulate self - renewal by driving pi3k / akt signaling ( le belle et al . , 2011 ) . conversely , hematopoietic stem cells are sensitive to ros levels and higher levels limit their lifespan ( ito et al . , 2006 ) . additionally , although production of ros via nox1 upregulation is important for tumorigenesis ( mitsushita et al . , 2004 ) , it has been reported that low levels of ros in cscs help protect against radiotherapy - induced dna damage ( diehn et al . , 2009 ) . our data suggest that after apc loss , lgr5 isc / progenitor cell expansion is a critical process during tumor initiation and is dependent on rac1-driven ros production . given that lgr5 iscs maintain high levels of ros but are not dependent on it , we would hypothesize that increased rac1-ros is critical for proliferation outside the normal niche ( figure 7 ) . rac1 is also required for efficient nf-b signaling after apc loss and , importantly , constitutive activation of nf-b in the absence of rac1 partially rescued the attenuated proliferation and isc expansion phenotypes . we also found that loss of rac1 prevented p65 recruitment to the promoters of isc genes after apc deletion . our findings are similar to those observed with p65 deletion after -catenin activation and suggest that after apc loss , lgr5 isc marker genes require both -catenin and p65 for full transcriptional activation ( schwitalla et al . , 2013 ) . the finding that ros and nf-b both play important roles in intestinal tumor initiation indicates a role for inflammation in this process . interestingly , a recent report outlines a role for inflammatory pathways in progenitor cell transformation in the foregut ( liu et al . , 2013 ) . thus , it is tempting to speculate that inflammatory pathways are common mediators of stem / progenitor transformation in multiple tissues . one is highly proliferative , located at the crypt base , and marked by lgr5 and the other is relatively quiescent , label retaining , and expresses bmi1 . the focus of our study was on the proliferative lgr5 population and we have not directly addressed whether rac1 is required for the transformation of bmi1 iscs . a level of hierarchy between isc populations has been proposed . under normal homeostatic conditions , lgr5 iscs populate the intestine , but this population can be depleted during times of intestinal stress . when this occurs , bmi1 + cells can produce lgr5 iscs and subsequently repopulate the intestine ( tian et al . , 2011 ) . interestingly , lgr5 + cells are also capable of generating the + 4 population ( takeda et al . , 2011 ) . this would argue against a strict hierarchical relationship between these populations and instead indicate that , depending on various contexts , these isc populations are interchangeable . the location , function , and plasticity of isc populations are still subject to keen debate . recent data has argued that lgr5 + cells express markers of alternative isc populations and that immediate progenitor cells can regain stemness after crypt damage ( muoz et al . , 2012 ; van es et al . , perhaps the simplest explanation is that lgr5 + iscs are defined by their crypt location and progenitor cells can regain isc properties if they enter the lgr5 + niche . interestingly , we observed lgr5 + cells throughout the crypt after apc loss , and this is dependent on rac1 . perhaps this is why rac1 deletion and ros inhibition do not affect normal lgr5 isc function , i.e. , these factors are critical for unrestrained proliferation outside the conventional niche . it should be noted that despite being expanded after apc loss , the lgr5 + zone does not encompass all apc - deficient cells and apc deletion alone in lgr5 cells does not lead to tumorigenesis . thus , other mutations are probably required to permit tumor formation from non - iscs . we have recently shown that activation of kras in concert with apc loss can induce dedifferentiation of villus enterocytes , leading to tumor formation ( schwitalla et al . , 2013 ) . it would be interesting to determine whether this process is rac1 dependent . given the plasticity of the normal intestine if multiple csc populations exist and are interconvertible , then so - called stem cell therapies , which specifically target a particular subpopulation of cells would be unlikely to prove beneficial . as specific csc populations are depleted , they may be rapidly repopulated from alternative reserve , it would be important to target both the cscs and the proliferative capacity of their descendants . in this regard , our data demonstrate that rac1 would be a particularly attractive candidate to target these populations . rac1 deletion suppressed both lgr5 isc and progenitor hyperproliferation after deletion of apc and prevented tumor formation from lgr5 + cells . importantly , deleting rac1 also suppressed proliferation of cells with activated kras , thus expanding the potential range of tumors likely to respond to rac1 inhibition . lgr5 marks a csc population with tumor lineage - tracing properties in murine adenomas and lgr5 + apc - deficient cells have enhanced in vitro clonogenicity over lgr5 cells , but no in vivo clonogenicity data are available ( schepers et al . , 2012 ) . we have shown that lgr5 isc expansion , in vitro clonogenicity of apc crypts , and adenoma initiation are rac1 dependent . however , as sorted cells from adenomas do not transplant , we were not able to determine their clonogenicity in vivo . thus , we can not state categorically that rac1 is required for apc - deficient isc function . two recent studies have suggested very different outcomes for crc patients with high levels of lgr5 isc markers . merlos - suarez and colleagues have shown that expression of ephb receptors is closely correlated with lgr5 and other isc markers in crc , and those cancers with high levels of these markers had a greater chance of relapse ( merlos - surez et al . , 2011 ) . in contrast , de sousa e melo and colleagues showed that while high levels of wnt signaling marks csc populations , those cancers in which the promoters of lgr5 isc markers were methylated were more likely to relapse ( de sousa e melo et al . , 2011 ) . thus , it is not clear how well lgr5 isc marker expression defines cscs in human tumors and how this is linked to disease progression . we previously demonstrated that deletion of myc was sufficient to rescue all of the phenotypes of apc loss . although rac1 deletion did not completely recapitulate this , the comprehensive suppression of tumor formation suggests that rac1 is required for transformation after apc loss . as the majority of crcs contain apc mutations , this has been the focus of our study . however , it would be interesting to determine whether rac1 deletion also suppresses tumorigenesis in apc - independent murine crc models ( heid et al . thus , rac1 may be a worthwhile therapeutic target during the early stages of crc . interestingly , our data indicate that ros and nf-b are two parallel pathways involved in promoting isc / progenitor proliferation downstream of rac1 . it should be noted that activation of ros or nf-b was not sufficient to completely rescue the phenotypes associated with rac1 loss . first , it demonstrates how robust the requirement for rac1 is to intestinal tumor formation . as a key signaling node that integrates numerous downstream signaling pathways , the potential therapeutic benefits of targeting it should be high also , it would likely increase the range of tumors that would be sensitive to its inhibition and reduce the scope for drug resistance to develop . indeed , activation of two other known target pathways of rac1 , mtor ( saci et al . , both of these pathways have been shown to reduce though not prevent polyposis in the apc mouse ( fujishita et al . this may also explain why our results do not perfectly phenocopy studies on any of the rac1-modified factors that we have examined . for example , ikk deletion in an aom / dss - driven model does not completely prevent tumorigenesis , whereas in our model , rac1 deletion does ( greten et al . , 2004 ) . thus , the combined suppression of a number of downstream effectors appears to have a much more profound effect than deleting them individually . thus , if efficient rac1 inhibitors remain elusive , it may be worth combining inhibition of these downstream pathways . it is interesting to note that in the case of mtor inhibition , additional kras mutation strongly suppresses the efficacy of rapalogs in mouse models ( hung et al . , 2010 ) . we have demonstrated that rac1 is essential for the transformation of kras - mutated crypts . given previous reports that the transformation activity of kras is dependent on rac1 ( qiu et al . , 1995 ) , it seems likely that rac1 would remain an efficacious target even in the setting of a kras mutation . in summary , we have elucidated an important axis downstream of wnt / myc signaling after apc loss that is crucial for lgr5 isc / progenitor hyperproliferation and hence tumorigenesis . all experiments were performed under the uk home office guidelines . the background of mice were as follows : ahcre experiments were at least ten generations c57bl6j , apc experiments were at least five generations c57bl6j , vilcre - er experiments were performed on a mixed background ( 50% c57bl6j , 50% s129 ) . the alleles used for this study were as follows : c - myc ( sansom et al . , 2007 ) , ahcre ( ireland et al . , 2004 ) , apc ( sansom et al . , 2007 ) , rac1 ( walmsley et al . , 2003 ) , vilcre - er ( el marjou et al . , 2004 ) , lgr5-creer ( barker et al . , 2007 ) , rosa - tdrfp ( luche et al . , 2007 ) , and r26stopikk2ca ( sasaki et al . , 2006 ) . injection of 80 mg / kg -napthoflavone and analyzing gfp expression 14 days later . this protocol was also used for inducing recombination within preformed adenomas . for ros inhibition , 0.5% n - acetyl cysteine was added to drinking water for 1 week prior to cre induction by tamoxifen treatment . primary antibodies and concentrations can be found in the supplemental experimental procedures . for each antibody , staining was performed on at least three mice of each genotype and average staining intensity over the entire tissue area was scored . we reverse transcribed 1 g of total rna isolated from intestinal tissue was to cdna and hybridized to affymetrix mouse genome 430 2.0 microarrays . rma normalization and log2 transformation of the data was followed by the differential gene expression analysis using anova and post hoc linear contrasts between all pairs of experimental conditions . enrichment analysis was performed with chi - square test with yates s correction ( gold et al . , 2007 ) .

summarythe adenomatous polyposis coli ( apc ) gene is mutated in the majority of colorectal cancers ( crcs ) . loss of apc leads to constitutively active wnt signaling , hyperproliferation , and tumorigenesis . identification of pathways that facilitate tumorigenesis after apc loss is important for therapeutic development . here , we show that rac1 is a critical mediator of tumorigenesis after apc loss . we find that rac1 is required for expansion of the lgr5 intestinal stem cell ( isc ) signature , progenitor hyperproliferation , and transformation . mechanistically , rac1-driven ros and nf-b signaling mediate these processes . together , these data highlight that ros production and nf-b activation triggered by rac1 are critical events in crc initiation .

Introduction Results Discussion Experimental Procedures

apc is a negative regulator of wnt signaling that is required to target -catenin for proteosomal degradation . the loss of the apc gene results in the accumulation of -catenin in the nucleus , subsequent activation of wnt transcriptional targets , and ultimately adenoma formation ( korinek et al . , normal intestinal homeostasis is maintained by a number of intestinal stem cells ( iscs ) . recent experiments have shown the presence of a cycling stem cell population marked by lgr5 along with a longer - lived population that can repopulate the crypt after injury ( barker et al . genetic deletion of apc within lgr5 iscs leads to rapid adenoma formation , whereas deletion within more differentiated lineages leads to poorly proliferative lesions that fail to progress without additional oncogenic mutations ( schwitalla et al . , interestingly , although an established wnt target , lgr5 is only expressed in a subset of apc - deficient tumor cells . this may be due to lgr5 marking cells with the highest levels of wnt signaling or to co - operation of multiple pathways in conferring the lgr5 isc phenotype . our previous studies have shown that the wnt target gene myc is required for the phenotypes induced by apc loss and that reduced levels of myc slows intestinal tumorigenesis ( athineos and sansom , 2010 ; sansom et al . these studies set a precedent that targeting the downstream effectors of wnt signaling may be of therapeutic benefit in colorectal cancer . , 2008 ) therefore identification of other pathways downstream of apc loss , in particular those highly active in lgr5 iscs may provide candidates to target apc - deficient cells . rac1 is a gtpase that acts as a key signaling node modulating a diverse set of cellular processes including proliferation , apoptosis , migration , and invasion . we previously identified myc - dependent upregulation of a number of racgefs after apc loss , and racgefs are commonly overexpressed during tumor progression ( lindsay et al . , rac1 is also required for kras - mediated tumorigenesis in skin and the lung , but the mechanism of how rac1 loss suppresses tumorigenesis is still unclear ( kissil et al . , 2011 two studies suggest that rac1 is required for nuclear localization of -catenin and wnt signaling ( phelps et al . , 2009 ; first , wu and colleagues demonstrated that rac1 is required for -catenin nuclear localization in the developing limb bud and the effects of its deletion phenocopy those of -catenin . as nuclear localization of -catenin is a key event during crc initiation if this dependency remains once apc is deleted , it would suggest that rac1 would be an excellent therapeutic target . here the consequence of apc loss was perturbed differentiation , which was dependent on the transcriptional repressor ctbp1 . additional activation of kras was required for nuclear accumulation of -catenin and hyperproliferation after apc loss . therefore , given this evidence for a role of rac1 in wnt signaling , we investigated the importance of rac1 activation after apc deletion . we show that a number of racgefs and rac1b are upregulated after apc deletion , which leads to increased activity of rac1 . while rac1 deletion did not stop the nuclear accumulation of -catenin and activation of the majority of the tcf / lef targets , it attenuated hyperproliferation after apc loss and subsequent tumorigenesis . finally , we show that this is due to rac1-mediated control of ros production and nf-b activation . previous microarray analysis revealed upregulation of several racgefs after apc loss ( sansom et al . we found significant overexpression of vav3 and tiam1 transcripts after apc loss in a myc - dependent manner ( as synchronous deletion of apc and myc returned expression levels to wild - type ) ( figure 1a ) . to investigate whether this led to increased rac1 activation , we performed immunohistochemistry ( ihc ) using an antibody raised against rac - gtp ( samuel et al . these data are consistent with previous studies suggesting that rac1 expression ( and presumably activation ) is elevated in crc ( espina et al . to assess the functional significance of rac1 activation after apc loss , we generated vil - cre - er apc rac1 ( apc rac1 ) mice ( el marjou et al . four days after cre induction , we observed loss of both rac1 and rac1b mrna and rac1 protein ( figures 1a and s2a ) . in contrast to induced vil - cre - er apc mice ( apc ) , the intestines from apc rac1 mice had smaller hyperproliferative crypts with significantly reduced incorporation of brdu ( figures 2a and s2b ) . transgene also induces recombination in the colonic epithelium and the rac1-dependent intestinal proliferation phenotype was recapitulated in this tissue ( figure s2d ) . thus , rac1 is a critical component in permitting crypt progenitor hyperproliferation after apc loss in the intestinal and colonic epithelia . given previous studies reporting that rac1 is required for accumulation of nuclear -catenin ( phelps et al . moreover , quantitative rt - pcr ( qrt - pcr ) of selected tcf / lef targets that are deregulated after apc loss were unchanged in apc rac1 intestines ( figure s2e ) ( sansom et al . thus , rac1 is not required for -catenin nuclear localization and/or its functional activity in the absence of apc . to delineate the mechanism , we compared global gene expression changes between apc and apc rac1 intestines by microarray . , we confirmed that the isc signature was upregulated after apc loss and reverted back to wild - type levels in apc rac1 intestines using qrt - pcr ( figure 2c ) . to visualize changes in the lgr5-expressing population , we generated inducible vil - cre - er wt , rac1 , apc , and apc rac1 mice carrying the lgr5 transgene ( barker et al . after apc loss , there is a significant increase in the number of lgr5-gfp - positive cells that are suppressed in apc rac1 intestines ( figure 2d ) . moreover , scoring the location of lgr5-expressing cells revealed a striking expansion of the stem cell zone that codeletion of rac1 prevented ( figure 2e ) . to investigate whether rac1 deletion suppressed the proliferative potential of all isc populations , we purified crypts from apc and apc rac1 intestines and cultured them ex vivo . remarkably , while apc crypts formed colonies , apc rac1 crypts did not , suggesting that rac1 is required for the clonogenic capacity of all intestinal cell types ( figure s2i ) therefore , rac1 is required for two critical constituents of apc loss , progenitor hyperproliferation and lgr5 isc expansion . rac1 is a member of the superoxide - generating nadph oxidase complex , and rac1b overexpression has been shown to lead to ros production ( bromberg et al . we observed a shift in the nadp+/nadph ratio in apc intestinal extracts compared to wt , indicating increased complex activity that was rac1 dependent ( figure s3a ) . to analyze ros levels within the intestinal epithelium , we stained wt , rac1 , apc , and apc rac1 intestines with the ros - responsive dye dihydroethidium ( dhe ) . thus , apc loss leads to increased ros generation in the intestinal epithelium via activation of the rac1-containing nadph oxidase complex . to confirm that lgr5 + cells have high levels of ros , we isolated intestinal epithelial cells from mice carrying the lgr5 transgene ( barker et al . as ros generation at the crypt base was rac1 dependent ( figures 3a and s3b ) , we investigated the expression of tiam1 , vav3 , and rac1b in lgr5 + cells sorted by qrt - pcr and found that all were significantly enriched , though rac1 levels were unchanged ( figure 3e ) . we hypothesized increased rac1-driven ros generation may be a critical process in conferring the isc / progenitor phenotypes associated with apc loss . first , we assessed whether reduction of intracellular ros levels through nac treatment could recapitulate the phenotypes associated with rac1 deletion . remarkably , treatment of apc - deficient mice with nac strongly suppressed overexpression of the isc signature genes ( lgr5 , olfm4 , and rgmb ) ( figure 4a ) . in situ microscopy of apc rac1 mice carrying the lgr5 transgene demonstrated a significant expansion of the isc zone upon paraquat treatment ( figure s4d ) . in wt and apc - deficient mice treated with we also observed a significant increase in proliferation in the intestines of the three cohorts of paraquat - treated mice ( figures 4d and s4e ) . thus , induction of ros can induce proliferation and , in the absence of rac1 , can partially compensate for its loss . given the remarkable overlap with this study , we addressed whether nf-b signaling is an important mediator of lgr5 isc / progenitor proliferation after apc loss . we observed a significant reduction of nf-b signaling components p65 , ikk , and acetyl - p65 in apc rac1 compared to apc - deficient intestines ( figures 5a , s5a , and s5b ) . we also observed an increase in p65 binding to the promoters of lgr5 , olfm4 , and rgmb after apc loss that was lost upon codeletion of rac1 ( figures 5b and 5c ) . we crossed apc rac1 mice to mice carrying the r26stopikk2ca allele ( an inducible , constitutively active ikk2 allele from now on referred to as ikk ) to generate apc rac1 ikk mice ( sasaki et al . , 2006 ) this was sufficient to almost completely rescue the proliferation defect and partially rescue isc marker expression in apc rac1 mice ( figures 5e , 5f , and s5d ) . thus , nf-b signaling is an important downstream effector of rac1 signaling in promoting the phenotypes of apc loss . we noted that neither increased ros nor nf-b activation were able to completely rescue the apc rac1 deletion phenotype . this is perhaps unsurprising given that we also observed effects on a number of other pathways downstream of rac1 signaling , such as stat3 and mtor , which could impinge on the phenotypes of apc loss ( figure s5e ) . given that rac1 deletion prevents increased lgr5 expression within apc - deficient crypts , we hypothesized that this would reduce the tumor - forming capacity of iscs . to test this , we generated control lgr5 apc ( lgr5 apc ) and experimental lgr5 apc rac1 ( lgr5 apc rac1 ) mice . most control lgr5 apc mice developed a lethal intestinal adenoma burden by 20 days ; however , none of the lgr5 apc rac1 did until much later time points ( figure 6a ) . despite high levels of nuclear -catenin , they had reduced levels of lgr5-gfp , suggesting that rac1 activity is essential for tumor growth and the efficient expansion of lgr5 cells after apc loss ( figures 6f , 6 g , and s6b ) . lgr5 also marks colonic stem cells , and deletion of apc with lgr5 leads to formation of colonic adenomas ( barker et al . we observed a significant reduction in the number of these lesions in the colons of lgr5 apc rac1 mice ( figure s6d ) , with the majority of these mice ( 6/8 ) being tumor free . thus , rac1 is required for intestinal and colonic adenoma formation after apc loss . to test whether ros production by rac1 was also important this led to a significant extension of tumor - free lifespan , further emphasizing the role ros plays in intestinal transformation ( figure 6h ) . therefore , we generated vil - cre - er apc kras ( apc kras ) and vil - cre - er apc kras rac1 ( apc kras rac1 ) mice . together , these data strongly indicate an essential role for rac1 in intestinal adenoma formation . as rac1 permits the proliferation of lgr5 iscs after apc loss , we asked whether rac1 is important for maintaining this population in established tumors . specifically , we wanted to assess the impact of deletion of rac1 from the lgr5 + population within adenomas . for this , we intercrossed mice carrying the apc allele ( pollard et al . , we aged lgr5 apc carrying the rosa26-rfp reporter ( figure s6e ) to 60 days , when mice have small tumors and induced recombination with tamoxifen . in agreement with previously published work , we observed lineage tracing within both wild - type epithelium and in adenomas , with marked rfp - positive clones 3 weeks after induction in both ( figures s6f and s6 g ) ( schepers et al . here , we demonstrate a critical requirement for rac1 in intestinal transformation after apc loss ( figure 7 ) . in contrast to previous studies suggesting that rac1 is required for nuclear localization of -catenin ( wu et al . , 2008 ) , we find that rac1 acts downstream of constitutive wnt signaling to promote progenitor cell proliferation and expansion of the lgr5 isc population . we find that the dependence on rac1 for crypt hyperproliferation is translated to a tumor model driven by apc loss in lgr5 iscs . these findings are striking as other oncogenic mediators overexpressed after apc loss , including cyclin d1/d2 , cd44 , and mbd2 , show much less dramatic effects on crypt hyperproliferation or intestinal tumorigenesis ( cole et al . as rac1 also suppresses proliferation of apc - deficient cells after kras activation , we propose that it is essential for intestinal transformation . the role of ros in stem cell maintenance , cellular transformation , and csc survival appears to be context and tissue specific . additionally , although production of ros via nox1 upregulation is important for tumorigenesis ( mitsushita et al . our data suggest that after apc loss , lgr5 isc / progenitor cell expansion is a critical process during tumor initiation and is dependent on rac1-driven ros production . rac1 is also required for efficient nf-b signaling after apc loss and , importantly , constitutive activation of nf-b in the absence of rac1 partially rescued the attenuated proliferation and isc expansion phenotypes . we also found that loss of rac1 prevented p65 recruitment to the promoters of isc genes after apc deletion . our findings are similar to those observed with p65 deletion after -catenin activation and suggest that after apc loss , lgr5 isc marker genes require both -catenin and p65 for full transcriptional activation ( schwitalla et al . the finding that ros and nf-b both play important roles in intestinal tumor initiation indicates a role for inflammation in this process . the focus of our study was on the proliferative lgr5 population and we have not directly addressed whether rac1 is required for the transformation of bmi1 iscs . this would argue against a strict hierarchical relationship between these populations and instead indicate that , depending on various contexts , these isc populations are interchangeable . the location , function , and plasticity of isc populations are still subject to keen debate . interestingly , we observed lgr5 + cells throughout the crypt after apc loss , and this is dependent on rac1 . , these factors are critical for unrestrained proliferation outside the conventional niche . it should be noted that despite being expanded after apc loss , the lgr5 + zone does not encompass all apc - deficient cells and apc deletion alone in lgr5 cells does not lead to tumorigenesis . given the plasticity of the normal intestine if multiple csc populations exist and are interconvertible , then so - called stem cell therapies , which specifically target a particular subpopulation of cells would be unlikely to prove beneficial . rac1 deletion suppressed both lgr5 isc and progenitor hyperproliferation after deletion of apc and prevented tumor formation from lgr5 + cells . we have shown that lgr5 isc expansion , in vitro clonogenicity of apc crypts , and adenoma initiation are rac1 dependent . however , as sorted cells from adenomas do not transplant , we were not able to determine their clonogenicity in vivo . thus , we can not state categorically that rac1 is required for apc - deficient isc function . merlos - suarez and colleagues have shown that expression of ephb receptors is closely correlated with lgr5 and other isc markers in crc , and those cancers with high levels of these markers had a greater chance of relapse ( merlos - surez et al . we previously demonstrated that deletion of myc was sufficient to rescue all of the phenotypes of apc loss . although rac1 deletion did not completely recapitulate this , the comprehensive suppression of tumor formation suggests that rac1 is required for transformation after apc loss . as the majority of crcs contain apc mutations , this has been the focus of our study . interestingly , our data indicate that ros and nf-b are two parallel pathways involved in promoting isc / progenitor proliferation downstream of rac1 . first , it demonstrates how robust the requirement for rac1 is to intestinal tumor formation . , both of these pathways have been shown to reduce though not prevent polyposis in the apc mouse ( fujishita et al . this may also explain why our results do not perfectly phenocopy studies on any of the rac1-modified factors that we have examined . we have demonstrated that rac1 is essential for the transformation of kras - mutated crypts . , 1995 ) , it seems likely that rac1 would remain an efficacious target even in the setting of a kras mutation . in summary , we have elucidated an important axis downstream of wnt / myc signaling after apc loss that is crucial for lgr5 isc / progenitor hyperproliferation and hence tumorigenesis . , 2007 ) , and r26stopikk2ca ( sasaki et al . primary antibodies and concentrations can be found in the supplemental experimental procedures .

rheumatoid arthritis ( ra ) is an inflammatory disease characterized by infiltrating leukocytes , monocytes , and macrophages in the joint synovial fluid ( sf ) and in the surrounding synovial tissue . these cells play a critical role in the pathogenesis of ra , by secreting proinflammatory cytokines , which , in turn , perpetuate joint destruction [ 2 , 3 ] . fibroblast - like synoviocytes ( fls ) , in the synovial intimal lining , also contribute to inflammation of the joint and destruction of the cartilage and bone , through cell - cell contact as well as elaboration of soluble products , such as cytokines , chemokines , growth factors , prostaglandins , and leukotrienes . sf thus condenses a mix of factors , secreted by all the cells located within the joint , with prosurvival and proliferative effects that are important in the development of expanded fibroblast network and in the maintaining the inflammatory response [ 4 , 5 ] . synovial fluid from ra patients ( ra sf ) contains higher levels of il-1 , il-6 , and tnf than synovial fluid from osteoarthritis ( oa ) patients [ 68 ] . the effects of these cytokines include the induction of cytokine synthesis , the upregulation of adhesion molecules , the activation of osteoclasts , the induction of other inflammatory mediators such as prostaglandins , nitric oxide , and matrix metalloproteinases , the induction of the acute phase response , and the activation of b cells ( il-6 ) . the ra joint microenvironment also contains high levels of il-15 , it stimulates migration of neutrophils and t lymphocytes into the joint [ 9 , 10 ] , it protects these cells from apoptosis , and it induces triggering and proliferation of t cells [ 12 , 13 ] . in addition , il15 indirectly induces the expression of proinflammatory cytokines tnf- , il-1 , il-17 , and il-8 , as well as inflammation - inciting free radicals [ 1418 ] . il-17 is higher in ra sf , compared with the concentrations found in oa patients . this cytokine has pleiotropic effects on leukocytes and stromal cells , and it induces il-6 and il-8 production by fibroblasts and stimulates macrophage il-1 and tnf- production . il-17 is thought to play a crucial role in the bone reabsorption process during ra . other cytokines increased in ra sf are as follows : il-8 involved in cellular recruitment , gm - csf involved in macrophage development , and il-23 involved in increasing th17 lymphocytes differentiation . in ra sf , there are also soluble mediators of inflammation , such as prostaglandins , leukotrienes , and matrix metalloproteinases , which may diffuse from blood and/or be formed locally within the joint cavity . elevated il-18 levels have been detected in synovial tissues and synovial fluids of ra patients , in significantly higher levels with respect to oa controls . ra synovial cells are characterized by a resistance to apoptosis , and this contributes to the accumulation of cells in the rheumatoid lesion . in the inflamed rheumatoid joint , synovial fluid factors tend to inhibit the apoptosis of cd4 t cells , neutrophils [ 25 , 26 ] , and fls . in addition , this impaired apoptosis may contribute to the development of the microenvironment and to maintain the inflammatory response that characterizes established ra [ 4 , 27 ] . sf factors that contribute to cells survival are the high levels of common -chain cytokines ( il-2 , il-4 , and il-15 ) , g - csf , gm - csf , and macrophage derived ifn-. nitric oxide ( no ) levels are also increased in ra synovial fluid . no mediates many different cell functions at the site of synovial inflammation , including cytokine production , signal transduction , mitochondrial functions , and apoptosis [ 28 , 29 ] . multiple mechanisms by which no may regulate apoptosis have been identified , one of them might be the suppression by no donor of the proteolytic processing and activation of caspase-3 in fas - treated synovial cells . like no , thioredoxin 1 ( trx1 ) levels are elevated in the synovial tissue , fluid , and serum of ra patients [ 32 , 33 ] . trx1 has multiple biological activities in regulation of apoptosis , albeit the mechanisms responsible for these activities have not been completely established . the composition of sf is very complex and strongly influences the microenvironment of joints , thus representing an inseparable element of the disease . for these reasons studies about the ra pathology should be performed in sf conditioned medium , rather than recombinant cytokines conditioned medium or medium alone , in order to recreate the correct physiopathological microenvironment , typical of ra . in our study we compared the effect of sf , tnf , and the medium alone on in vitro ra fls , which play pivotal roles both in the initiation and the perpetuation of ra . we observed a greater response of the synovial fibroblasts to sf to confirm our hypothesis . the study was approved by the hospital ethics committee of the gaetano pini hospital of milan ( italy ) . the tissues were minced and treated for 4 h with 2.5 mg / ml of type i collagenase ( sigma aldrich , saint louis , mo , usa ) in dulbecco 's modified eagle 's medium ( dmem ) ( euroclone , italy ) at 37c in 5% co2 . dissociated cells were then centrifuged at 1000 g , resuspended in dmem supplemented with 10% fetal calf serum ( fcs ) ( fetalclone1 hyclone logan , ut , usa ) , 2 mm l - glutamine , 100 units / ml penicillin , and 100 mg / ml streptomycin ( euroclone , italy ) , and plated in 75 cm flasks ( primo cell culture flask , euroclone , italy ) . after overnight culture , nonadherent cells were removed , and adherent cells were cultivated in dmem supplemented with 10% fcs . the cultures were kept at 37c in 5% co2 , and the medium was replaced every 3 days . the purity of the cells was tested by flow - cytometric analysis using phycoerythrin - conjugated anti - cd14 ( pharmingen , san diego , ca , usa ) and fluorescein isothiocyanate phycoerythrin - conjugated anti - cd3 , anti - cd19 , anti - cd14 , or anti - thy-1 ( cd90 ) monoclonal antibodies ( r&d systems minneapolis , mn ) . a facs calibur flow cytometer ( 488ex/620em ) ( becton dickinson , san jos , ca , usa ) was used for the analysis . at passage 3 , the cells were morphologically homogeneous and exhibited the appearance of fls , with typical bipolar configuration under inverse microscopy . most cells ( > 98% ) expressed the surface markers for fibroblasts ( thy-1 ) and were negative for the expression of cd3 , cd19 , and cd14 . sf was directly aspirated from the joints of ra patients , and the fluid was collected into heparinized tubes and spun at 1000 g for 10 min . fls were cultured in complete medium alone or in the presence of recombinant human tumor necrosis factor - alpha ( tnf ) ( 25 ng / ml ) ( immunological sciences , italy ) or sf , at the dilution of 1 : 8 in culture medium . we used three pools of sf , each from 10 ra patients ' fluid , in order to minimize the variability in responses among different pools . at indicated time points the evaluation of newly synthesized dna was carried out using the click - it edu hcs assay ( life technologies italia ) in accordance with the kit instructions . 5-ethynyl-2-deoxyuridine ( edu ) ( 10 m ) was added to the cultures 24 h before the detection , cells were then fixed and permeabilized , and edu incorporated into newly synthesized dna was detected using the fluorescent alexa fluor 488 azide . the detection of apoptosis was performed by the annexin v - fluorescein isothiocyanate ( fitc)/propidium iodide ( pi ) kit ( biolegend , san diego , ca ) according to the manufacturer 's instructions . briefly , after treatments cells were washed and resuspended in annexin v binding buffer , and then annexin v - fitc and propidium iodide ( pi ) solution were added . after 15 min at room temperature in the dark , annexin v binding buffer was added . the resulting fluorescence was detected by flow cytometry ( facscan - becton dickinson , mountain view , ca ) with cellquest analysis software . the percentage of apoptosis was calculated as apoptotic index considering cells both in early and late apoptosis . fls were incubated in absence or presence of tnf ( 25 ng / ml ) or sf ( 1 : 8 dilution in culture medium ) . after 7 days cells were processed in triplicate with the taqman gene expression cells - to - ct kit ( life technologies italia ) according to the manufacturer 's instructions . briefly , cells were washed with cold pbs and resuspended in lysis solution + dnase i and incubated for 5 min at room temperature . stop solution was then added and after 2 min of incubation at room temperature the samples were transferred at 80c . taqman gene expression master mix and taqman gene expression assays ( life technologies italia ) were used to perform real - time quantitative polymerase chain reaction . data were analysed according to the comparative ct method and were normalized by gapdh expression in each sample . fls were cultured on coverslips to a 10% to 20% confluence and then incubated in absence or presence of tnf ( 25 ng / ml ) or sf ( 1 : 8 dilution in culture medium ) . after 7 days cells were fixed with 4% formaldehyde for 15 min at room temperature followed by permeabilization of the cells with pbs plus 0.1% triton x-100 for 5 min . cells were incubated with alexa fluor 594 phalloidin ( actin filament staining ; invitrogen ) for 15 min . a zeiss axiovert 200 m fluorescent microscope was used for visualization with the appropriate filters , with zeiss axioversion 4.7 software ( jena , germany ) . cultures were loaded for 3540 min at 37c with 2 m fura-2-am in krebs - ringer solution buffered with hepes , 125 mm nacl , 5 mm kcl , 1.2 mm mgso4 , 2 mm cacl2 , 10 mm glucose , and 25 mm hepes ( ph 7.4 ) and were washed twice with prewarmed krebs - ringer solution before recordings were made . the recording setting comprised an inverted microscope ( leica , dmi600b ) equipped with a ca imaging unit . fura-2 fluorescence images were collected with a andor ccd camera ( axon instruments , ca , usa ) and analyzed with imaging workbench 6 ( indec biosystem , santa clara usa ) . single - cell 340/380 nm fluorescence ratios were analysed with origin 6.0 ( microcal software inc . , ma , usa ) . differences with a confidence level of > 95% were considered statistically significant ( p < 0.05 ) . to compare the effect of sf , tnf , and culture medium alone , we first evaluated the proliferation of synovial fibroblasts , established from rheumatoid synovial tissue . cells were cultured in medium additioned with 10% fcs in the presence / absence of either tnf or sf from ra patients . we utilized tnf at the optimal concentration of 25 ng / ml as our reference standard , since this concentration is known to induce maximal cell proliferation of synovial fibroblasts . we used three sf pools ; at a final dilution of 1 : 8 in culture medium , the lower dilution is able to induce maximal cell proliferation of synovial fibroblasts ( data not shown ) . exposure to sf provided a significant growth advantage for fls , compared with exposure to tnf or medium alone , with an increase in dna synthesis that was evident already after 48 h of incubation , and it was also maintained at later times ( figure 1 ) . comparing the three different pools , we did not observed a significant difference in cellular responses . we also evaluated the influence of sf on apoptosis , given that it is known that ra fls are more resistant to apoptosis , and one of the possible mechanisms responsible for this resistance is the presence of antiapoptotic factors in the sf . we assessed the spontaneous apoptosis of fls cultured in medium alone , in medium with sf or with tnf. our results indicate that the presence of sf in the culture medium significantly decreased apoptosis , compared to the medium alone , and this protective effect on the cells increased when we extended the treatment beyond 48 h ( i.e. , 7 days ) . also in this condition , no differences between the three sf pools were observed . in the presence of tnf , a protective effect on apoptosis was observed , but this effect was less than the one induced by sf ( figure 2 ) . then , we explored the modulation of the expression of four inflammatory cytokines known to be involved in ra pathology , namely , il1 , il6 , il15 , and tnf. fls were exposed to sf or tnf and selected gene expression was then quantified by real - time pcr . as shown in figure 3 , unstimulated fls constitutively produced il1 , il6 , il15 , and tnf , as expected , and the chronic exposure to sf or tnf increased the expression of these cytokines . the mrna expression was strongest in cells exposed to the three sf pools , and its maximal stimulatory effect was detected after 7 days of exposure . given that we did not evidence significant differences of the responses interpool , neither in cell proliferation and apoptosis nor in the expression of cytokines , we used a single sf pool for the subsequent experiments . we concentrated our attention on the effect of sf and tnf on cytoskeletal reorganization in ra fls ( figure 4 ) . indeed , it is known that these cells exhibit rearranged cytoskeleton that may lead to morphological changes , ultimately resulting in the transformed phenotype of the cells , characterized by increased migration , adhesion , and proliferation properties . treatments with sf and tnf induced the reorganization of the actin cytoskeleton , but sf effects on cytoskeleton and morphological changes were more pronounced , with an observable cell spreading effect . subsequently , a functional characterization of fls , exposed to either tnf or sf , was carried out by quantitative evaluation of intracellular calcium dynamics . as a first step , ra fibroblasts were exposed to tnf or sf for either 2 or 7 days prior to experimental recording . a quantitative evaluation of basal intracellular calcium levels did not show particular changes in the resting status of fls exposed to the different conditions ( figure 5(a ) ) . however , when the same cells were exposed to inflammatory challenge ( such as 1 mm atp resembling exacerbated extracellular inflammatory conditions , such as the ones which characterize ra pathological contexts ) , significant differences were observed , both in terms of percentage of responding cells as well as peak intensity of response . in particular , already after 48 h of exposure , fls primed with tnf showed a significantly higher intensity of response with respect to sf ( figure 5(b ) ) , even though this was not coupled to a concomitant change in percentage of responding cells ( figure 5(c ) ) , thus suggesting an acute effect of tnf on the presence of p2 purinergic receptors at the plasma membrane . at 7 days , the situation was somewhat even more drastic but different , with a significantly higher increase both in the percentage of responsive cells as well as in the mean peak amplitude of response in sf - primed fls , as compared with tnf samples ( figures 5(b ) and 5(c ) ) . overall , tnf exposure seemed to have an acute effect at shorter times of exposure on the quantity of functional p2 receptor at the plasma membrane of fls , which is somewhat maintained at longer times of exposure ; on the contrary , sf - exposed fls showed an important change in responsiveness at longer times of exposure . it is very interesting to observe that after 7 days , all ra fls exposed chronically to sf responded to atp exposure with a rapid kinetic , typical of ionotropic channel activation . in order to confirm this hypothesis , the experiment was replicated by administering 1 mm atp in the absence of extracellular calcium . these data suggest that the atp - mediated response of fls is due to the ionotropic component of the purinergic receptor family ( p2x ) , rather than the metabotropic component ( p2y receptor ) . the composition of sf in ra pathology is very complex and strongly influences the microenvironment of joints . in fact , it is an inseparable element of the disease and for these reasons we think that in vitro ra studies should be performed in the presence of sf in the culture medium , to recreate the physiopathological microenvironment of ra . in this study , we compared the effect of tnf , a proinflammatory cytokine typically used for in vitro ra studies , with respect to sf and the culture medium alone on ra fibroblast synovial cells , to verify our hypothesis . our observations were performed using three different pools of sf , each from 10 ra patients ' fluid . we used the pools of ra synovial fluids to minimize the variability in responses between the different individual synovial fluid samples and to ensure the reproducibility of the results . in addition , we decided to use pools composed of 10 ra patients ' fluid to reduce the variability in responses between the different pools . we used fls because their involvement and critical contribution to the initiation and perpetuation of the disease are well known and are characterized , along with their ability to modulate both joint destruction and propagation of inflammation . challenged fls show alterations in morphology and behaviour , including apoptotic responses , inappropriate production of chemokines , adhesion molecules , and matrix - degrading components . for their active role in ra , fls represent today an important target for novel therapeutic approaches aimed at inhibiting joint destruction . it is well known that the significantly increased proliferation and insufficient apoptosis of fls are implicated in the pathogenesis and progression of ra [ 34 , 35 ] . our observations indicate that the presence of sf in the culture medium can strongly influence these aspects of fls , in a more efficient way than that observed in the presence of tnf and culture medium alone ( figures 1 and 2 ) . indeed , all the three sf pools provided a significant growth advantage for fls , their effect on dna synthesis was more evident , compared with tnf or medium alone , and it was also maintained for longer time in culture ( data not shown ) . along the same line , the spontaneous apoptosis of fls observed was decreased in the presence of the sf pools . this effect was detected starting from 48 h following treatment , with a more pronounced effect at 72 h , and the persistence of sf for longer times in the culture medium stabilized apoptosis . after 7 days , an increase in spontaneous apoptosis in cells cultured with medium alone was detected , probably due to nutrient limitations in the culture supernatant and/or the accumulation of metabolites , while the cells cultured in the presence of sf maintained a very low level of apoptosis , significantly lower than tnf-cultured cells . here we did not want to analyse the mechanisms which promote resistance to apoptosis in sf - maintained synovial cells , given that this aspect is documented and many factors contribute to this mechanism [ 2729 , 31 ] , but our observations highlight the important role of sf in conferring this resistance to ra fls in vitro . for this reason , we think that sf should be used as a more physiologically relevant tool in in vitro studies , in particular when new therapeutic molecules involved in apoptotic pathways are evaluated . when we compared the effects of sf and tnf on the expression of cytokines known to be involved in ra pathology , such as il1 , il6 , il15 , and tnf , we observed that chronic exposure to sf ( 7 days ) induced more pronounced effects on the cytokines synthesis in synovial fibroblasts ( figure 3 ) . sf influences the expression of genes involved in modulation of synovial cells and these cells have specific characteristics due to their high inflammation state . in respect to the three different sf pools , we did not evidence significant differences of the responses interpool , neither in cell proliferation and apoptosis nor in the expression of cytokines ( figures 1 , 2 , and 3 ) , to confirm that the elevated number of synovial fluid samples , 10 , used in each pool reduces the variability in responses between the different pools this is of particular importance when considering the possibility of using such a pool for drug profiling purposes . cytoskeleton plays a critical role in the regulation of various cellular processes linked to cell transforming and tumorigenesis , such as contact inhibition and anchorage - independent cell growth . ra fls exhibit rearranged cytoskeleton , and this reorganization may lead to morphological changes in the fls , ultimately resulting in the transformed phenotype of these cells , characterized by increased migration , adhesion , and proliferation properties . previously reports found that recombinant tnf induced morphological changes as well as the reorganization of the actin cytoskeleton . in addition , it was demonstrated that pharmacological inhibition of actin cytoskeleton dynamics alters potential pathogenic properties of the synovial fibroblast , such as proliferation , migration , and resistance to apoptosis . here we compared the effect of tnf and sf on cytoskeleton reorganization ( figure 4 ) and observed that although both in vitro treatments induced the reorganization of the actin cytoskeleton , fls cultured with sf exhibited more pronounced stress fibres and more significant effects on cytoskeletal and overall morphological changes . when we focused our attention on the functional responses of fls subjected to the different experimental scenarios , in terms of quantitative evaluation of intracellular calcium dynamics , we confirmed a clear distinct effect of tnf and sf on fls responsiveness . in particular , tnf exerts a very rapid effect , with an increase in the peak of the intensity response to 1 mm atp exposure . in contrast , fls exposed to sf show a significant change at longer times of stimulation ( i.e. , 7 days ) with a striking increase in the percentage of responding cells as well as in the mean peak amplitude of response , thus suggesting a more chronic effect on the purinergic receptor portfolio at the plasma membrane surface of challenged fls . we tried to give hints on the purinergic receptor components influencing such differences and observed that fls response was completely abolished when cells were challenged with atp in the absence of extracellular calcium , thus suggesting a role for the p2x rather than the p2y component of the purinergic receptor family in the observed effects . a complete characterization of the purinergic receptor components on the surface of fls , challenged with either tnf or sf , would be worthy of future investigation in order to give insights on the molecular reasons behind this clearly observed effect . these results emphasize the importance of using the ra sf in in vitro studies of ra synovial fibroblasts rather than typically used tnf challenge , in order to ensure culture conditions as similar as possible to those typical of the physiopathological environmental in rheumatoid arthritis joints . this should ensure the possibility to have more accurate biological information , both to clarify the pathogenesis of rheumatoid arthritis and to evaluate the therapeutic effectiveness of new molecules .

the composition of synovial fluid in rheumatoid arthritis ( ra ) is complex and strongly influences the microenvironment of joints and it is an inseparable element of the disease . currently , in vitro studies are performed on ra cells cultured in the presence of either recombinant proinflammatory cytokines - conditioned medium or medium alone . in this study , we evaluated the use of synovial fluid , derived from ra patients , as optimal culture condition to perform in vitro studies on ra synovial fibroblasts . we observed that synovial fluid is more effective in inducing cell proliferation with respect to tnf - alpha or culture medium alone . spontaneous apoptosis in fibroblasts was also decreased in response to synovial fluid . the expression of proinflammatory cytokines in the presence of synovial fluid was significantly elevated with respect to cells cultured with tnf - alpha or medium , and the overall morphology of cells was also modified . in addition , modulation of intracellular calcium dynamics elicited in response to synovial fluid or tnf - alpha exposure is different and suggests a role for the purinergic signalling in the modulation of the effects . these results emphasize the importance of using ra synovial fluid in in vitro studies involving ra cells , in order to reproduce faithfully the physiopathological environmental characteristic of ra joints .

1. Introduction 2. Material and Methods 3. Results 4. Discussion 5. Conclusion

rheumatoid arthritis ( ra ) is an inflammatory disease characterized by infiltrating leukocytes , monocytes , and macrophages in the joint synovial fluid ( sf ) and in the surrounding synovial tissue . these cells play a critical role in the pathogenesis of ra , by secreting proinflammatory cytokines , which , in turn , perpetuate joint destruction [ 2 , 3 ] . fibroblast - like synoviocytes ( fls ) , in the synovial intimal lining , also contribute to inflammation of the joint and destruction of the cartilage and bone , through cell - cell contact as well as elaboration of soluble products , such as cytokines , chemokines , growth factors , prostaglandins , and leukotrienes . synovial fluid from ra patients ( ra sf ) contains higher levels of il-1 , il-6 , and tnf than synovial fluid from osteoarthritis ( oa ) patients [ 68 ] . the effects of these cytokines include the induction of cytokine synthesis , the upregulation of adhesion molecules , the activation of osteoclasts , the induction of other inflammatory mediators such as prostaglandins , nitric oxide , and matrix metalloproteinases , the induction of the acute phase response , and the activation of b cells ( il-6 ) . the ra joint microenvironment also contains high levels of il-15 , it stimulates migration of neutrophils and t lymphocytes into the joint [ 9 , 10 ] , it protects these cells from apoptosis , and it induces triggering and proliferation of t cells [ 12 , 13 ] . in addition , il15 indirectly induces the expression of proinflammatory cytokines tnf- , il-1 , il-17 , and il-8 , as well as inflammation - inciting free radicals [ 1418 ] . this cytokine has pleiotropic effects on leukocytes and stromal cells , and it induces il-6 and il-8 production by fibroblasts and stimulates macrophage il-1 and tnf- production . il-17 is thought to play a crucial role in the bone reabsorption process during ra . in ra sf , there are also soluble mediators of inflammation , such as prostaglandins , leukotrienes , and matrix metalloproteinases , which may diffuse from blood and/or be formed locally within the joint cavity . elevated il-18 levels have been detected in synovial tissues and synovial fluids of ra patients , in significantly higher levels with respect to oa controls . ra synovial cells are characterized by a resistance to apoptosis , and this contributes to the accumulation of cells in the rheumatoid lesion . in the inflamed rheumatoid joint , synovial fluid factors tend to inhibit the apoptosis of cd4 t cells , neutrophils [ 25 , 26 ] , and fls . in addition , this impaired apoptosis may contribute to the development of the microenvironment and to maintain the inflammatory response that characterizes established ra [ 4 , 27 ] . sf factors that contribute to cells survival are the high levels of common -chain cytokines ( il-2 , il-4 , and il-15 ) , g - csf , gm - csf , and macrophage derived ifn-. nitric oxide ( no ) levels are also increased in ra synovial fluid . no mediates many different cell functions at the site of synovial inflammation , including cytokine production , signal transduction , mitochondrial functions , and apoptosis [ 28 , 29 ] . multiple mechanisms by which no may regulate apoptosis have been identified , one of them might be the suppression by no donor of the proteolytic processing and activation of caspase-3 in fas - treated synovial cells . like no , thioredoxin 1 ( trx1 ) levels are elevated in the synovial tissue , fluid , and serum of ra patients [ 32 , 33 ] . the composition of sf is very complex and strongly influences the microenvironment of joints , thus representing an inseparable element of the disease . for these reasons studies about the ra pathology should be performed in sf conditioned medium , rather than recombinant cytokines conditioned medium or medium alone , in order to recreate the correct physiopathological microenvironment , typical of ra . in our study we compared the effect of sf , tnf , and the medium alone on in vitro ra fls , which play pivotal roles both in the initiation and the perpetuation of ra . we observed a greater response of the synovial fibroblasts to sf to confirm our hypothesis . the study was approved by the hospital ethics committee of the gaetano pini hospital of milan ( italy ) . dissociated cells were then centrifuged at 1000 g , resuspended in dmem supplemented with 10% fetal calf serum ( fcs ) ( fetalclone1 hyclone logan , ut , usa ) , 2 mm l - glutamine , 100 units / ml penicillin , and 100 mg / ml streptomycin ( euroclone , italy ) , and plated in 75 cm flasks ( primo cell culture flask , euroclone , italy ) . after overnight culture , nonadherent cells were removed , and adherent cells were cultivated in dmem supplemented with 10% fcs . the cultures were kept at 37c in 5% co2 , and the medium was replaced every 3 days . the purity of the cells was tested by flow - cytometric analysis using phycoerythrin - conjugated anti - cd14 ( pharmingen , san diego , ca , usa ) and fluorescein isothiocyanate phycoerythrin - conjugated anti - cd3 , anti - cd19 , anti - cd14 , or anti - thy-1 ( cd90 ) monoclonal antibodies ( r&d systems minneapolis , mn ) . a facs calibur flow cytometer ( 488ex/620em ) ( becton dickinson , san jos , ca , usa ) was used for the analysis . most cells ( > 98% ) expressed the surface markers for fibroblasts ( thy-1 ) and were negative for the expression of cd3 , cd19 , and cd14 . sf was directly aspirated from the joints of ra patients , and the fluid was collected into heparinized tubes and spun at 1000 g for 10 min . fls were cultured in complete medium alone or in the presence of recombinant human tumor necrosis factor - alpha ( tnf ) ( 25 ng / ml ) ( immunological sciences , italy ) or sf , at the dilution of 1 : 8 in culture medium . we used three pools of sf , each from 10 ra patients ' fluid , in order to minimize the variability in responses among different pools . briefly , after treatments cells were washed and resuspended in annexin v binding buffer , and then annexin v - fitc and propidium iodide ( pi ) solution were added . after 15 min at room temperature in the dark , annexin v binding buffer was added . fls were incubated in absence or presence of tnf ( 25 ng / ml ) or sf ( 1 : 8 dilution in culture medium ) . taqman gene expression master mix and taqman gene expression assays ( life technologies italia ) were used to perform real - time quantitative polymerase chain reaction . fls were cultured on coverslips to a 10% to 20% confluence and then incubated in absence or presence of tnf ( 25 ng / ml ) or sf ( 1 : 8 dilution in culture medium ) . after 7 days cells were fixed with 4% formaldehyde for 15 min at room temperature followed by permeabilization of the cells with pbs plus 0.1% triton x-100 for 5 min . cultures were loaded for 3540 min at 37c with 2 m fura-2-am in krebs - ringer solution buffered with hepes , 125 mm nacl , 5 mm kcl , 1.2 mm mgso4 , 2 mm cacl2 , 10 mm glucose , and 25 mm hepes ( ph 7.4 ) and were washed twice with prewarmed krebs - ringer solution before recordings were made . to compare the effect of sf , tnf , and culture medium alone , we first evaluated the proliferation of synovial fibroblasts , established from rheumatoid synovial tissue . cells were cultured in medium additioned with 10% fcs in the presence / absence of either tnf or sf from ra patients . we utilized tnf at the optimal concentration of 25 ng / ml as our reference standard , since this concentration is known to induce maximal cell proliferation of synovial fibroblasts . we used three sf pools ; at a final dilution of 1 : 8 in culture medium , the lower dilution is able to induce maximal cell proliferation of synovial fibroblasts ( data not shown ) . exposure to sf provided a significant growth advantage for fls , compared with exposure to tnf or medium alone , with an increase in dna synthesis that was evident already after 48 h of incubation , and it was also maintained at later times ( figure 1 ) . comparing the three different pools , we did not observed a significant difference in cellular responses . we also evaluated the influence of sf on apoptosis , given that it is known that ra fls are more resistant to apoptosis , and one of the possible mechanisms responsible for this resistance is the presence of antiapoptotic factors in the sf . we assessed the spontaneous apoptosis of fls cultured in medium alone , in medium with sf or with tnf. our results indicate that the presence of sf in the culture medium significantly decreased apoptosis , compared to the medium alone , and this protective effect on the cells increased when we extended the treatment beyond 48 h ( i.e. in the presence of tnf , a protective effect on apoptosis was observed , but this effect was less than the one induced by sf ( figure 2 ) . then , we explored the modulation of the expression of four inflammatory cytokines known to be involved in ra pathology , namely , il1 , il6 , il15 , and tnf. fls were exposed to sf or tnf and selected gene expression was then quantified by real - time pcr . as shown in figure 3 , unstimulated fls constitutively produced il1 , il6 , il15 , and tnf , as expected , and the chronic exposure to sf or tnf increased the expression of these cytokines . the mrna expression was strongest in cells exposed to the three sf pools , and its maximal stimulatory effect was detected after 7 days of exposure . given that we did not evidence significant differences of the responses interpool , neither in cell proliferation and apoptosis nor in the expression of cytokines , we used a single sf pool for the subsequent experiments . indeed , it is known that these cells exhibit rearranged cytoskeleton that may lead to morphological changes , ultimately resulting in the transformed phenotype of the cells , characterized by increased migration , adhesion , and proliferation properties . subsequently , a functional characterization of fls , exposed to either tnf or sf , was carried out by quantitative evaluation of intracellular calcium dynamics . as a first step , ra fibroblasts were exposed to tnf or sf for either 2 or 7 days prior to experimental recording . a quantitative evaluation of basal intracellular calcium levels did not show particular changes in the resting status of fls exposed to the different conditions ( figure 5(a ) ) . in particular , already after 48 h of exposure , fls primed with tnf showed a significantly higher intensity of response with respect to sf ( figure 5(b ) ) , even though this was not coupled to a concomitant change in percentage of responding cells ( figure 5(c ) ) , thus suggesting an acute effect of tnf on the presence of p2 purinergic receptors at the plasma membrane . at 7 days , the situation was somewhat even more drastic but different , with a significantly higher increase both in the percentage of responsive cells as well as in the mean peak amplitude of response in sf - primed fls , as compared with tnf samples ( figures 5(b ) and 5(c ) ) . in order to confirm this hypothesis , the experiment was replicated by administering 1 mm atp in the absence of extracellular calcium . these data suggest that the atp - mediated response of fls is due to the ionotropic component of the purinergic receptor family ( p2x ) , rather than the metabotropic component ( p2y receptor ) . the composition of sf in ra pathology is very complex and strongly influences the microenvironment of joints . in fact , it is an inseparable element of the disease and for these reasons we think that in vitro ra studies should be performed in the presence of sf in the culture medium , to recreate the physiopathological microenvironment of ra . in this study , we compared the effect of tnf , a proinflammatory cytokine typically used for in vitro ra studies , with respect to sf and the culture medium alone on ra fibroblast synovial cells , to verify our hypothesis . our observations were performed using three different pools of sf , each from 10 ra patients ' fluid . we used the pools of ra synovial fluids to minimize the variability in responses between the different individual synovial fluid samples and to ensure the reproducibility of the results . in addition , we decided to use pools composed of 10 ra patients ' fluid to reduce the variability in responses between the different pools . we used fls because their involvement and critical contribution to the initiation and perpetuation of the disease are well known and are characterized , along with their ability to modulate both joint destruction and propagation of inflammation . challenged fls show alterations in morphology and behaviour , including apoptotic responses , inappropriate production of chemokines , adhesion molecules , and matrix - degrading components . it is well known that the significantly increased proliferation and insufficient apoptosis of fls are implicated in the pathogenesis and progression of ra [ 34 , 35 ] . our observations indicate that the presence of sf in the culture medium can strongly influence these aspects of fls , in a more efficient way than that observed in the presence of tnf and culture medium alone ( figures 1 and 2 ) . indeed , all the three sf pools provided a significant growth advantage for fls , their effect on dna synthesis was more evident , compared with tnf or medium alone , and it was also maintained for longer time in culture ( data not shown ) . along the same line , the spontaneous apoptosis of fls observed was decreased in the presence of the sf pools . this effect was detected starting from 48 h following treatment , with a more pronounced effect at 72 h , and the persistence of sf for longer times in the culture medium stabilized apoptosis . after 7 days , an increase in spontaneous apoptosis in cells cultured with medium alone was detected , probably due to nutrient limitations in the culture supernatant and/or the accumulation of metabolites , while the cells cultured in the presence of sf maintained a very low level of apoptosis , significantly lower than tnf-cultured cells . here we did not want to analyse the mechanisms which promote resistance to apoptosis in sf - maintained synovial cells , given that this aspect is documented and many factors contribute to this mechanism [ 2729 , 31 ] , but our observations highlight the important role of sf in conferring this resistance to ra fls in vitro . for this reason , we think that sf should be used as a more physiologically relevant tool in in vitro studies , in particular when new therapeutic molecules involved in apoptotic pathways are evaluated . when we compared the effects of sf and tnf on the expression of cytokines known to be involved in ra pathology , such as il1 , il6 , il15 , and tnf , we observed that chronic exposure to sf ( 7 days ) induced more pronounced effects on the cytokines synthesis in synovial fibroblasts ( figure 3 ) . sf influences the expression of genes involved in modulation of synovial cells and these cells have specific characteristics due to their high inflammation state . in respect to the three different sf pools , we did not evidence significant differences of the responses interpool , neither in cell proliferation and apoptosis nor in the expression of cytokines ( figures 1 , 2 , and 3 ) , to confirm that the elevated number of synovial fluid samples , 10 , used in each pool reduces the variability in responses between the different pools this is of particular importance when considering the possibility of using such a pool for drug profiling purposes . ra fls exhibit rearranged cytoskeleton , and this reorganization may lead to morphological changes in the fls , ultimately resulting in the transformed phenotype of these cells , characterized by increased migration , adhesion , and proliferation properties . previously reports found that recombinant tnf induced morphological changes as well as the reorganization of the actin cytoskeleton . in addition , it was demonstrated that pharmacological inhibition of actin cytoskeleton dynamics alters potential pathogenic properties of the synovial fibroblast , such as proliferation , migration , and resistance to apoptosis . here we compared the effect of tnf and sf on cytoskeleton reorganization ( figure 4 ) and observed that although both in vitro treatments induced the reorganization of the actin cytoskeleton , fls cultured with sf exhibited more pronounced stress fibres and more significant effects on cytoskeletal and overall morphological changes . when we focused our attention on the functional responses of fls subjected to the different experimental scenarios , in terms of quantitative evaluation of intracellular calcium dynamics , we confirmed a clear distinct effect of tnf and sf on fls responsiveness . in particular , tnf exerts a very rapid effect , with an increase in the peak of the intensity response to 1 mm atp exposure . , 7 days ) with a striking increase in the percentage of responding cells as well as in the mean peak amplitude of response , thus suggesting a more chronic effect on the purinergic receptor portfolio at the plasma membrane surface of challenged fls . we tried to give hints on the purinergic receptor components influencing such differences and observed that fls response was completely abolished when cells were challenged with atp in the absence of extracellular calcium , thus suggesting a role for the p2x rather than the p2y component of the purinergic receptor family in the observed effects . a complete characterization of the purinergic receptor components on the surface of fls , challenged with either tnf or sf , would be worthy of future investigation in order to give insights on the molecular reasons behind this clearly observed effect . these results emphasize the importance of using the ra sf in in vitro studies of ra synovial fibroblasts rather than typically used tnf challenge , in order to ensure culture conditions as similar as possible to those typical of the physiopathological environmental in rheumatoid arthritis joints . this should ensure the possibility to have more accurate biological information , both to clarify the pathogenesis of rheumatoid arthritis and to evaluate the therapeutic effectiveness of new molecules .

the authors have attempted to measure the activity concentration of cs-137 in the natural environment of the gorce mts . the distribution of contamination by radioactive cs-137 in poland ( south poland in particular ) ( cf . section deposition of cs-137 and [ 1 , 1013 ] ) and in its immediate vicinity ( e.g. [ 2 , 3 ] ) is well known , but there is a lack of detailed data pertaining to the level and distribution of this contamination in smaller areas , covering only several hundred square kilometres . such investigations provide , on the one hand , information on the current distribution of the activity concentration , and on the other hand , may establish a point of reference in the future when dealing with the changes of this distribution in the course of time . moreover , baseline studies of this type are necessary because of the location of nuclear power plants in the immediate vicinity of poland and the anticipated construction of such a plant in poland around the year 2025 . the gorce mts . situated in southern poland ( fig . , approximately 44 km in length , a width of approximately 15 km and an area of approximately 530 km . turbacz ( 1,310 m asl ) . in 1981 , the central and north - eastern parts of the gorce mts . were declared the gorce national park , which presently has an area of around 70 km . the geological structure of the gorce mts . is made of folded and thrusted flysch formations : mainly the magura quartz sandstones cemented with silica ( silicon oxide ) or calcite ( calcium carbonate ) , minor conglomerates and shales . the soils of the study area belong mainly to various brown soil sub - types , in the lower subalpine zone and various podzoilic soil sub - types in the upper subalpine zone . the cs-137 deposition values have been presented in numerical and graphic form and rounded up to 0.5 kbq / m . the border shown on the map ( solid bold line ) separates two areas of different cs-137 deposition deposition of cs-137 in the selected sites of the gorce mts . the cs-137 deposition values have been presented in numerical and graphic form and rounded up to 0.5 kbq / m . the border shown on the map ( solid bold line ) separates two areas of different cs-137 deposition the gorce mts . have been selected for the study as it is an area which has been practically unaffected by human economic activity , it is reflected , among others , in the presence of an intact surface soil layer and low level of contamination of the natural environment . additionally , as a large part of the study area is located within the gorce national park , it is most probable that this part of the gorce mts . will not be significantly affected by human economic activity in the future . therefore , it is a very good testing ground for long - term measurements of the dynamics of radioactive contamination . the study included : soil;lichens , because this species is commonly used as bioindicators of air pollution ( cf . ) . the lichen hypogymnia physodes , often used in environmental studies , was chosen in our measurements . lichens , because this species is commonly used as bioindicators of air pollution ( cf . ) . the lichen hypogymnia physodes , often used in environmental studies , was chosen in our measurements . sampling was carried out in the years 20012005 over the whole area of the gorce mts . , in sites of dense forest complexes to minimise interference by human activity . in particular , the selected sampling sites differed in their absolute altitude ( between 530 and 1,250 m asl . ) , slope inclination , morphology ( valleys , slopes , ridges , etc . ) , and so on . they had to be representative of a study area and to cover it with possible regularity . a total of 74 samples of soil ( 49 samples ) and lichen ( 25 samples ) were collected . soil samples were taken from the top 10-cm layer in the form of cores with a diameter of 10 cm and a length of 10.0 cm ( uncertainty 0.5 cm ) , using a pvc cutter . the core was next divided into three layers , according to the soil horizon types distinguished ( organic , mineral ) . the samples were further prepared for radiometric measurements in accordance with iaea recommendations , applicable to environmental samples . larger rock fragments , exceeding a diameter of 3 mm , were discarded , the remaining material was dried to a constant weight at 105 c and ground to a grain size below 1 mm . the ground material was placed in cylindrical measuring vessels with a volume of 121 cm , the activity concentration of cs-137 ( bq / kg ) in the soil samples ( layers ) distinguished was measured , and the cs-137 deposition per 1 m ( its activity concentration in the top 10-cm soil layer ; kbq / m ) calculated . the lichen samples were collected from the bark of european beech and norwegian spruce trees at a height of between 0.5 and 1.5 m above the soil surface . this procedure should minimise the effect of dependence of the cs-137 concentration upon the height of the sampling site ( cf . ) . the area in which samples were taken did not exceed 1 ha , while the mass of lichen samples ranged between single grams and several tens of grams . in the laboratory the next stage of cleaning was carried out under a magnifying glass and involved final removal of other lichen species and splitting the thallus of the lichen from the tree bark . for lichens the material collected was dried to a constant mass at around 70 c , and next , depending on the amount of the material , either ground to a grain size below 1 mm , or broken up by hand into fragments below 5 mm . the material was placed in cylindrical measuring vessels of a volume of 48 or 121 cm . the activity concentration was determined using a semiconductor hpge detector ( canberra gx4020 ) with 42 % relative efficiency and resolution of 1.9 kev , placed in lead housing with walls 10 cm thick . the standard solution of gamma emitting radionuclides , was used as a calibration source ; the uncertainty of radionuclide activities should not exceed 2 % . the duration of measurements was generally chosen so that the relative uncertainty of the net count for 662 kev line ( cs-137 ) was less than 3 % . in further calculation a detailed description of the methodology is set out elsewhere [ 7 , 8 ] . the activity concentrations of cs-137 originating from global fallout and the chernobyl fallout were decay corrected to 1st july 2005 . results for all sampling sites are presented in table 1 ; comprehensive results are contained in the final report of the study .table description of sampling sites , cs-137 deposition , cs-137 activity concentration in the soil and lichen h. physodes site nosite location coordinates altitude ( m asl)gorce mts . part cs-137 deposition ( kbq / m ) cs-137 activity concentration ( bq / kg d.m.)soil layer lichen 1st2nd3rd10 cm1maciejowa ( f)493455n 200035e830nw10.537556910131443.01amaciejowa ( g)493458n 200007e830nw5.11858690872rdzawka ponice ( f)493312n 195921e770nw9.41259259821423stare wierchy ( f)493344n 200256e1,030nw9.675323882721041.25redni wierch ( f)493244n 200420e1,110nw15.83708484125116koninki / valley ( f)493442n 200522e700nw7.04198118521048kocurka glade ( f)493323n 200517e1,080nw11.02516761143069turbacz ( f)493236n 200712e1,240nw13.33939472435399turbacz ( f)493232n 200717e1,180nw7.6623226814919752.69ahala duga glade ( g)493247n 200755e1,200se3.44697461679bhala duga glade ( g)493242n 200746e1,200se4.5216038166321turbaczyk ( f)493444n 200655e1,050nw7.1420113299687.722konina valley ( f)493449n 200801e680nw17.137233220828632.923mostownica ( f)493352n 200755e950se3.1613325134624lubomierz ( f)493531n 201008e840nw8.6534360610525kosarzysko glade ( f)493445n 201013e1,020se3.641655675126kudo ( f)493418n 201035e1,250se6.0729425011719527podskale ( f)493504n 201133e970nw11.914069531233327.729kiczora kamien . ( f)493252n 200948e1,250se5.2834723514720543kamienica valley ( f)493344n 201115e970se2.474238333650.745jaszcze due ( f)493211n 201201e800se2.25872363047skaka ( f)493323n 201343e1,050se5.944282031410948gorc ( f)493353n 201453e1,110se3.10314106278173.149gorcowe ( f)493245n 201614e750se7.27349833311361bukowina obidow . ( f)493125n 200556e1,050se4.203336256865may kowaniec ( f)493038n 200347e780se3.08161107123541.466jankwki ( f)493133n 200814e1,050se7.392873945020445.868opuszna / valley ( f)493026n 200750e730se2.18721569910627.269fordwki ( f)493105n 201136e870se2.788430315154.980knurowska pass ( f)492950n 201046e800se6.173791923512481studzionki ( f)492941n 201260e890se3.5811120227720082szlembark ( f)492919n 201248e780se4.61268119127464.683runek ( f)492952n 201549e980se6.211994187331.784morgi glade ( f)492931n 201800e1,000se2.344952324285mizerna / valley ( f)492843n 201746e760se4.31187100439113.487lubanskie ( f)493035n 202014e640se4.5118745157088luba ( f)492922n 202029e1,180se7.593612889519845.488aluba ( g)492918n 202029e1,190se4.493384836889kotelnica ( f)492747n 202337e660se3.154939353969.891ochotnica stasichy ( f)493014n 201431e730se6.402851846013092ochotnica kudows . ( f)492858n 202340e530se4.93236781876 ( f)forest , ( g)glade ; kamien.kamieniecka , obidow.obidowska , waksm.waksmundzka , ochotn . ochotnickie , kudows.kudowskie , ziem.ziemianki uncertainty of the site location identification amounts to 80 m two contaminated gorce mts . parts distinguished by the authors : nw north - western part , se south - eastern part relative uncertainty of less than 10 % ; uncertainty of core height was included activity concentration in 1st , 2nd , 3rd soil layer and in a 10 cm soil layer ( whole core ) respectively ; relative uncertainty less than 5 % relative uncertainty less than 10 % results of the cs-137 analysis in the gorce mts . : description of sampling sites , cs-137 deposition , cs-137 activity concentration in the soil and lichen h. physodes ( f)forest , ( g)glade ; kamien.kamieniecka , obidow.obidowska , waksm.waksmundzka , ochotn . ochotnickie , kudows.kudowskie , ziem.ziemianki uncertainty of the site location identification amounts to 80 m two contaminated gorce mts . parts distinguished by the authors : nw north - western part , se south - eastern part relative uncertainty of less than 10 % ; uncertainty of core height was included activity concentration in 1st , 2nd , 3rd soil layer and in a 10 cm soil layer ( whole core ) respectively ; relative uncertainty less than 5 % relative uncertainty less than 10 % as the result of measurements , cs-137 activity concentration was determined in individual soil layers , and then the vertical distribution of cs-137 ( depth profile ) . statistical parameters for each of the three layers are presented in table 2 ; due to the different cs-137 deposition in the gorce mts . next chapter ) , the data is presented separately for the two distinguished parts of the study area . the values presented in table 2 only provide information on the mean concentrations in individual layers , thus they should not be the used as a basis for estimation of concentration variability in soil profile.table 2cs-137 activity concentration in soil layerslayerrangemean / medianstandard deviationcvgorce mts.north-western part ( bq / kg)(bq / kg)(bq / kg)(%)1 . layer 6412154/11113386mean 87540253/21014859gorce mts.south-eastern part ( bq / kg)(bq / kg)(bq / kg)(%)1 layer 327743/3153125mean 3020591/755358gorce mts.whole area ( % ) ( % ) ( % ) ( % ) 1 layer 167738/3314373 . layer 26423/181670 number of samples : nw gorce mts . 34 , gorce mts.whole area 49 cs-137 activity concentration in a successive soil layer mean activity concentration in a 10 cm soil layer percentage of a successive soil layer in cs-137 total activity in 10 cm soil layer cs-137 activity concentration in soil layers number of samples : nw gorce mts . 34 , gorce mts.whole area 49 cs-137 activity concentration in a successive soil layer mean activity concentration in a 10 cm soil layer percentage of a successive soil layer in cs-137 total activity in 10 cm soil layer the change of cs-137 activity concentration in soil profile is not uniform . in the entire gorce mts . , the profiles , whose concentration rapidly falls with depth ( 69 % ) dominate , there are profiles in which the maximum concentration appears in the second layer ( 16 % ) , and also profiles in which concentration is more or less constant in all the layers ( 10 % ) . the total activity of cs-137 ( bq ) in individual layers was calculated based on the cs-137 activity concentrations in individual soil layers ( bq / kg ) ; results show that , on average , in the gorce : in the first layer there is approx . 39 % of cs-137 deposited per 10 cm soil layer , in the second 38 % , and in the third 23 % . based on the analysis of deposition of cs-137 in the selected sampling points , the authors distinguished two areas of the gorce mts . ( cf . fig . 1 ; tables 1 , 3 ) : south - eastern and north - western ; one has double the caesium contamination of the other . the deposition in the north - western part , which has a higher level of contamination , is in the range 4.5 - 17.1 kbq / m with an average of 9.9 kbq / m , while in the less contaminated south - eastern part deposition ranges between 2.2 and 7.6 kbq / m with an average of 4.4 kbq / m . the border of the two parts runs approximately along the line nowy targ mt . 1).table 3deposition of cs-137 and cs-137 activity concentration in h. physodes in the gorce mts.arearangeaverage/medianstandard deviationcvcs-137 deposition(kbq / m)(kbq / m)(kbq / m)(%)gorce mts.north-western part 4.517.19.9/9.73.636gorce mts.south-eastern part 2.27.64.4/4.41.535gorce mts.whole area 2.217.16.1/4.93.557mt . mostownica local variability 2.28.24.8/3.91.940 hypogymnia physodes ( bq / kg)(bq / kg)(bq mostownica 19 number of samples : 25 deposition of cs-137 and cs-137 activity concentration in h. physodes in the gorce mts . mostownica 19 number of samples : 25 it seems that the reason for such significant differences in contamination in the two distinguished parts may be possibly due to the various meteorological conditions when radioactive contamination ( radioactive cloud ) from the chernobyl disaster spread in the atmosphere . however , the polish institute of meteorology and water management ( imgw ) data indicates that the impact of rainfall differences should probably be excluded . it was found that in the period when the chernobyl cloud was spreading over southern poland , i.e. , between 28th april and mid - may 1986 , the rainfall in the gorce mts . area was minimal ( up to fifteen or so mm daily ; mainly on 30th april and between 8th may and 11th may ) and no differences in the rainfall level between the two parts of the gorce mts . 2 . as data is scarce , it is difficult to determine the nature of these distributions . nevertheless , it is characteristic that in each of the two distinguished parts of the gorce mts . se part has been presented twice : once at the bin width being the same as in the nw part ( for comparison of the two parts ) , and again at the bin width being half of that in the nw part statistical distribution of cs-137 deposition in the gorce mts . se part has been presented twice : once at the bin width being the same as in the nw part ( for comparison of the two parts ) , and again at the bin width being half of that in the nw part the relationship between the cs-137 deposition values and the absolute altitude of the sampling sites is shown for the south - eastern part of the study area ( fig . 3 ) as more data was available just for this part of the gorce mts . the analysis of the graphs indicates that there is no basis to assume any dependence between cs-137 deposition and the absolute altitude.fig . each entry shows : the mean value and its uncertainty ( longer horizontal line ) and variability range ( shorter horizontal line ) dependence between the cs-137 deposition and the altitude of the sampling site . each entry shows : the mean value and its uncertainty ( longer horizontal line ) and variability range ( shorter horizontal line ) in order to compare the results obtained with those of other authors , the literature data has been decay - corrected to 1st july 2005 . according to mietelski et al . , the gorce mts . are situated on the border between the deposition areas 1.54.4 and 4.47.3 kbq / m . the radioecological map of poland elaborated using the in situ method assigns the gorce mts . to the area with a deposition of 05.9 kbq / m , while the average deposition in the then nowy scz voivodeship that encompassed the area of the gorce mts . the central laboratory for radiological protection ( clor ) carried out measurements in five villages situated in the gorce mts . . the deposition of cs-137 ranged between 0.7 and 2.0 kbq / m , while the average deposition in the maopolska voivodeship encompassing at that time the gorce mts . 30 km from gorce mts . ) and obtained results between 0.2 and 17.5 kbq / m with an average of 5.6 kbq / m . comparison of the results obtained with the previous data is justified because the cs-137 deposition decreases only slightly faster than cs-137 decays ( cs-137 t1/2 = 30 years ) . the average deposition of 9.9 kbq / m measured by the present authors in the north - western part of the gorce mts . is significantly higher than the values presented on the maps of cs-137 deposition in poland [ 1012 ] . this difference is probably mainly due to averaging the local values on these maps as the maps were constructed to reflect regional variations . therefore , they do not show small areas with a higher deposition of cs-137 . when interpreting the cs-137 deposition data , its significant local variability should be taken into account . it is defined as the coefficient of variation ( cv ) of statistical distribution of the deposition in the case of multiple sampling points around one site . the author determined local variability of cs-137 deposition in 19 points on the 400 m slope located in the mt . ( cf . fig . 4 and table 3 ) . the deposition value varied in the interval 2.59.2 kbq / m , the mean value was 5.3 kbq / m , and cv 40 % . the cv value is slightly above the variability interval of the literature data oscillating ( for an area of approximately 0.11 km ) in the range 1030 % ( cf . the cv value slightly higher than quoted by other authors , can be explained by the undulations of the gorce mts . area and the possibility of cs-137 accumulation in surface irregularities of the area ; as a result , there are points of significantly higher deposition and the cv value increases . an analysis of the data presented in table 3 indicates that the variability in the north - western and south - eastern parts of the gorce mts . 35 % ) is comparable with the local variability ( 40 % ) . therefore , it may be assumed that each of the two parts is contaminated evenly , and the spreads of deposition values obtained in these parts for various sampling points ( cf . 4cs-137 deposition value in 19 sampling points located on 400 m long slope profile of mt . values on the x - axis represent the distance of a sampling point from the centre of the investigated area ; the + and symbols , indicate that the point is located accordingly above or below the centre of the area cs-137 deposition value in 19 sampling points located on 400 m long slope profile of mt . values on the x - axis represent the distance of a sampling point from the centre of the investigated area ; the + and symbols , indicate that the point is located accordingly above or below the centre of the area the results of measurements of cs-137 activity concentration in h. physodes from selected sites in the gorce mts . the cs-137 activity concentration in lichens is in the range 1388 bq / kg d.m . the cs-137 activity concentration values measured are comparable to the literature data of around 100 bq / kg d.m . for samples of pseudevernia furfuracea and parmeliaceae family from south and south - eastern poland [ 17 , 18 ] . the local variability ( cf . turbacz ( site no 9 ) , where lichen samples were collected from ten trees ( five beech and five spruce specimens ) . 50 % being comparable to the variability established over the whole of the gorce mts . area therefore , it may be accepted that the spreads of cs-137 activity concentrations in lichens , obtained for various sampling points in the gorce mts . , one of the reasons for the high local variability of the cs-137 activity concentration in the lichen is the growth of the lichen resulting in two co - occurring types of the thallus in a single sample : the thallus highly contaminated shortly after the chernobyl disaster in 1986 ( direct contamination ) or the thallus contaminated by the former due to cs-137 leaching ( indirect contamination),the thallus formed much later , i.e. , in the period when the activity concentration of cs-137 in the air was lower by several orders of magnitude . the thallus highly contaminated shortly after the chernobyl disaster in 1986 ( direct contamination ) or the thallus contaminated by the former due to cs-137 leaching ( indirect contamination ) , the thallus formed much later , i.e. , in the period when the activity concentration of cs-137 in the air was lower by several orders of magnitude . to sum up , the cs-137 activity concentration in the lichen is currently due first of all to the contribution of the thallus contaminated shortly after the chernobyl disaster , to the total thallus volume sampled . as the result of measurements , cs-137 activity concentration was determined in individual soil layers , and then the vertical distribution of cs-137 ( depth profile ) . statistical parameters for each of the three layers are presented in table 2 ; due to the different cs-137 deposition in the gorce mts . next chapter ) , the data is presented separately for the two distinguished parts of the study area . the values presented in table 2 only provide information on the mean concentrations in individual layers , thus they should not be the used as a basis for estimation of concentration variability in soil profile.table 2cs-137 activity concentration in soil layerslayerrangemean / medianstandard deviationcvgorce mts.north-western part ( bq / kg)(bq / kg)(bq / kg)(%)1 . layer 85533286/259122432 . layer 60947405/332283703 . layer 6412154/11113386mean 87540253/21014859gorce mts.south-eastern part ( bq / kg)(bq / kg)(bq / kg)(%)1 . layer 42428207/187113542 . layer 327743/3153125mean 3020591/755358gorce mts.whole area ( % ) ( % ) ( % ) ( % ) 1 . 34 , gorce mts.whole area 49 cs-137 activity concentration in a successive soil layer mean activity concentration in a 10 cm soil layer percentage of a successive soil layer in cs-137 total activity in 10 cm soil layer cs-137 activity concentration in soil layers number of samples : nw gorce mts . 34 , gorce mts.whole area 49 cs-137 activity concentration in a successive soil layer mean activity concentration in a 10 cm soil layer percentage of a successive soil layer in cs-137 total activity in 10 cm soil layer the change of cs-137 activity concentration in soil profile is not uniform . in the entire gorce mts . , the profiles , whose concentration rapidly falls with depth ( 69 % ) dominate , there are profiles in which the maximum concentration appears in the second layer ( 16 % ) , and also profiles in which concentration is more or less constant in all the layers ( 10 % ) . the total activity of cs-137 ( bq ) in individual layers was calculated based on the cs-137 activity concentrations in individual soil layers ( bq / kg ) ; results show that , on average , in the gorce : in the first layer there is approx . 39 % of cs-137 deposited per 10 cm soil layer , in the second 38 % , and in the third 23 % . based on the analysis of deposition of cs-137 in the selected sampling points , the authors distinguished two areas of the gorce mts . ( cf . fig . 1 ; tables 1 , 3 ) : south - eastern and north - western ; one has double the caesium contamination of the other . the deposition in the north - western part , which has a higher level of contamination , is in the range 4.5 - 17.1 kbq / m with an average of 9.9 kbq / m , while in the less contaminated south - eastern part deposition ranges between 2.2 and 7.6 kbq / m with an average of 4.4 kbq / m . the border of the two parts runs approximately along the line nowy targ mt . turbacz 1).table 3deposition of cs-137 and cs-137 activity concentration in h. physodes in the gorce mts.arearangeaverage/medianstandard deviationcvcs-137 deposition(kbq / m)(kbq / m)(kbq / m)(%)gorce mts.north-western part 4.517.19.9/9.73.636gorce mts.south-eastern part 2.27.64.4/4.41.535gorce mts.whole area 2.217.16.1/4.93.557mt . mostownica 19 number of samples : 25 deposition of cs-137 and cs-137 activity concentration in h. physodes in the gorce mts . mostownica 19 number of samples : 25 it seems that the reason for such significant differences in contamination in the two distinguished parts may be possibly due to the various meteorological conditions when radioactive contamination ( radioactive cloud ) from the chernobyl disaster spread in the atmosphere . however , the polish institute of meteorology and water management ( imgw ) data indicates that the impact of rainfall differences should probably be excluded . it was found that in the period when the chernobyl cloud was spreading over southern poland , i.e. , between 28th april and mid - may 1986 , the rainfall in the gorce mts . area was minimal ( up to fifteen or so mm daily ; mainly on 30th april and between 8th may and 11th may ) and no differences in the rainfall level between the two parts of the gorce mts . were identified . the statistical distribution of the cs-137 deposition in the whole gorce mts . and 2 . as data is scarce , it is difficult to determine the nature of these distributions . nevertheless , it is characteristic that in each of the two distinguished parts of the gorce mts . se part has been presented twice : once at the bin width being the same as in the nw part ( for comparison of the two parts ) , and again at the bin width being half of that in the nw part statistical distribution of cs-137 deposition in the gorce mts . se part has been presented twice : once at the bin width being the same as in the nw part ( for comparison of the two parts ) , and again at the bin width being half of that in the nw part the relationship between the cs-137 deposition values and the absolute altitude of the sampling sites is shown for the south - eastern part of the study area ( fig . 3 ) as more data was available just for this part of the gorce mts . the analysis of the graphs indicates that there is no basis to assume any dependence between cs-137 deposition and the absolute altitude.fig . each entry shows : the mean value and its uncertainty ( longer horizontal line ) and variability range ( shorter horizontal line ) dependence between the cs-137 deposition and the altitude of the sampling site . each entry shows : the mean value and its uncertainty ( longer horizontal line ) and variability range ( shorter horizontal line ) in order to compare the results obtained with those of other authors , the literature data has been decay - corrected to 1st july 2005 . according to mietelski et al . , the gorce mts . are situated on the border between the deposition areas 1.54.4 and 4.47.3 kbq / m . the radioecological map of poland elaborated using the in situ method assigns the gorce mts . to the area with a deposition of 05.9 kbq / m , while the average deposition in the then nowy scz voivodeship that encompassed the area of the gorce mts . the central laboratory for radiological protection ( clor ) carried out measurements in five villages situated in the gorce mts . . the deposition of cs-137 ranged between 0.7 and 2.0 kbq / m , while the average deposition in the maopolska voivodeship encompassing at that time the gorce mts . 30 km from gorce mts . ) and obtained results between 0.2 and 17.5 kbq / m with an average of 5.6 kbq / m . comparison of the results obtained with the previous data is justified because the cs-137 deposition decreases only slightly faster than cs-137 decays ( cs-137 t1/2 = 30 years ) . the average deposition of 9.9 kbq / m measured by the present authors in the north - western part of the gorce mts . is significantly higher than the values presented on the maps of cs-137 deposition in poland [ 1012 ] . this difference is probably mainly due to averaging the local values on these maps as the maps were constructed to reflect regional variations . therefore , they do not show small areas with a higher deposition of cs-137 . when interpreting the cs-137 deposition data , its significant local variability should be taken into account it is defined as the coefficient of variation ( cv ) of statistical distribution of the deposition in the case of multiple sampling points around one site . the author determined local variability of cs-137 deposition in 19 points on the 400 m slope located in the mt . ( cf . fig . 4 and table 3 ) . the deposition value varied in the interval 2.59.2 kbq / m , the mean value was 5.3 kbq / m , and cv 40 % . the cv value is slightly above the variability interval of the literature data oscillating ( for an area of approximately 0.11 km ) in the range 1030 % ( cf . the cv value slightly higher than quoted by other authors , can be explained by the undulations of the gorce mts . area and the possibility of cs-137 accumulation in surface irregularities of the area ; as a result , there are points of significantly higher deposition and the cv value increases . an analysis of the data presented in table 3 indicates that the variability in the north - western and south - eastern parts of the gorce mts . 35 % ) is comparable with the local variability ( 40 % ) . therefore , it may be assumed that each of the two parts is contaminated evenly , and the spreads of deposition values obtained in these parts for various sampling points ( cf . fig . 1 ) , are associated with the local variability of the deposition.fig . 4cs-137 deposition value in 19 sampling points located on 400 m long slope profile of mt . values on the x - axis represent the distance of a sampling point from the centre of the investigated area ; the + and symbols , indicate that the point is located accordingly above or below the centre of the area cs-137 deposition value in 19 sampling points located on 400 m long slope profile of mt . values on the x - axis represent the distance of a sampling point from the centre of the investigated area ; the + and symbols , indicate that the point is located accordingly above or below the centre of the area the results of measurements of cs-137 activity concentration in h. physodes from selected sites in the gorce mts . are presented in table 3 . the cs-137 activity concentration in lichens is in the range 1388 bq / kg d.m . the cs-137 activity concentration values measured are comparable to the literature data of around 100 bq / kg d.m . for samples of pseudevernia furfuracea and parmeliaceae family from south and south - eastern poland [ 17 , 18 ] . the local variability ( cf . ) has been estimated in the sampling site ( an area of approx turbacz ( site no 9 ) , where lichen samples were collected from ten trees ( five beech and five spruce specimens ) . 50 % being comparable to the variability established over the whole of the gorce mts . area ( cv = 38 % ) . therefore , it may be accepted that the spreads of cs-137 activity concentrations in lichens , obtained for various sampling points in the gorce mts . , are associated with the local variability . in the opinion of the present authors , one of the reasons for the high local variability of the cs-137 activity concentration in the lichen is the growth of the lichen resulting in two co - occurring types of the thallus in a single sample : the thallus highly contaminated shortly after the chernobyl disaster in 1986 ( direct contamination ) or the thallus contaminated by the former due to cs-137 leaching ( indirect contamination),the thallus formed much later , i.e. , in the period when the activity concentration of cs-137 in the air was lower by several orders of magnitude . the thallus highly contaminated shortly after the chernobyl disaster in 1986 ( direct contamination ) or the thallus contaminated by the former due to cs-137 leaching ( indirect contamination ) , the thallus formed much later , i.e. , in the period when the activity concentration of cs-137 in the air was lower by several orders of magnitude . to sum up , the cs-137 activity concentration in the lichen is currently due first of all to the contribution of the thallus contaminated shortly after the chernobyl disaster , to the total thallus volume sampled . ( several hundred km ) , the authors established in detail the activity concentration of cs-137 and its spatial distribution in soils and lichen . such investigations provide , on the one hand , information on the current distribution of the caesium contamination , and on the other hand , may establish a point of reference in future changes of this distribution in the course of time . two parts of the gorce mts . have been distinguished on the basis of different deposition of cs-137 . the respective average values are 4.4 and 9.9 kbq / m as at 1st july 2005 , providing a relative difference of around 2 . the value 9.9 kbq / m is much higher than the values given in the maps of cs-137 deposition in poland . the average cs-137 activity concentration in the h. physodes from the gorce mts . is 47 bq / kg d.m . when interpreting this data , significant local variability of measured quantities ( cs-137 deposition , cs-137 activity concentration in lichen ) should be taken into account . this local variability amounts to several dozen percent and is comparable with the variability calculated for the whole area of the gorce mts . thus , there are no grounds to draw isolines of measured quantities on the map of the gorce mts .

concentration of activity of cs-137 and its spatial distribution in soils and lichen hypogymnia physodes were determined in the gorce mts . ( several hundred km2 ) in s poland . the authors distinguished two areas of the gorce mts . on the basis of markedly different cs-137 depositions , whose respective average values are 4.4 and 9.9 kbq / m2 as at 1st july 2005 . the average cs-137 activity concentration in the lichen h. physodes from the gorce is 47 bq / kg d.m . a significant local variability of quantities measured amounts to a few dozen percent was found .

Introduction Materials and methods Results and discussion Vertical distribution of Cs-137 Deposition of Cs-137 Lichens Conclusions

the authors have attempted to measure the activity concentration of cs-137 in the natural environment of the gorce mts . such investigations provide , on the one hand , information on the current distribution of the activity concentration , and on the other hand , may establish a point of reference in the future when dealing with the changes of this distribution in the course of time . the geological structure of the gorce mts . the border shown on the map ( solid bold line ) separates two areas of different cs-137 deposition deposition of cs-137 in the selected sites of the gorce mts . the border shown on the map ( solid bold line ) separates two areas of different cs-137 deposition the gorce mts . additionally , as a large part of the study area is located within the gorce national park , it is most probable that this part of the gorce mts . the lichen hypogymnia physodes , often used in environmental studies , was chosen in our measurements . sampling was carried out in the years 20012005 over the whole area of the gorce mts . the ground material was placed in cylindrical measuring vessels with a volume of 121 cm , the activity concentration of cs-137 ( bq / kg ) in the soil samples ( layers ) distinguished was measured , and the cs-137 deposition per 1 m ( its activity concentration in the top 10-cm soil layer ; kbq / m ) calculated . in the laboratory the next stage of cleaning was carried out under a magnifying glass and involved final removal of other lichen species and splitting the thallus of the lichen from the tree bark . for lichens the material collected was dried to a constant mass at around 70 c , and next , depending on the amount of the material , either ground to a grain size below 1 mm , or broken up by hand into fragments below 5 mm . results for all sampling sites are presented in table 1 ; comprehensive results are contained in the final report of the study .table description of sampling sites , cs-137 deposition , cs-137 activity concentration in the soil and lichen h. physodes site nosite location coordinates altitude ( m asl)gorce mts . part cs-137 deposition ( kbq / m ) cs-137 activity concentration ( bq / kg d.m. ochotnickie , kudows.kudowskie , ziem.ziemianki uncertainty of the site location identification amounts to 80 m two contaminated gorce mts . parts distinguished by the authors : nw north - western part , se south - eastern part relative uncertainty of less than 10 % ; uncertainty of core height was included activity concentration in 1st , 2nd , 3rd soil layer and in a 10 cm soil layer ( whole core ) respectively ; relative uncertainty less than 5 % relative uncertainty less than 10 % results of the cs-137 analysis in the gorce mts . : description of sampling sites , cs-137 deposition , cs-137 activity concentration in the soil and lichen h. physodes ( f)forest , ( g)glade ; kamien.kamieniecka , obidow.obidowska , waksm.waksmundzka , ochotn . ochotnickie , kudows.kudowskie , ziem.ziemianki uncertainty of the site location identification amounts to 80 m two contaminated gorce mts . parts distinguished by the authors : nw north - western part , se south - eastern part relative uncertainty of less than 10 % ; uncertainty of core height was included activity concentration in 1st , 2nd , 3rd soil layer and in a 10 cm soil layer ( whole core ) respectively ; relative uncertainty less than 5 % relative uncertainty less than 10 % as the result of measurements , cs-137 activity concentration was determined in individual soil layers , and then the vertical distribution of cs-137 ( depth profile ) . statistical parameters for each of the three layers are presented in table 2 ; due to the different cs-137 deposition in the gorce mts . the values presented in table 2 only provide information on the mean concentrations in individual layers , thus they should not be the used as a basis for estimation of concentration variability in soil profile.table 2cs-137 activity concentration in soil layerslayerrangemean / medianstandard deviationcvgorce mts.north-western part ( bq / kg)(bq / kg)(bq / kg)(%)1 . 34 , gorce mts.whole area 49 cs-137 activity concentration in a successive soil layer mean activity concentration in a 10 cm soil layer percentage of a successive soil layer in cs-137 total activity in 10 cm soil layer cs-137 activity concentration in soil layers number of samples : nw gorce mts . 34 , gorce mts.whole area 49 cs-137 activity concentration in a successive soil layer mean activity concentration in a 10 cm soil layer percentage of a successive soil layer in cs-137 total activity in 10 cm soil layer the change of cs-137 activity concentration in soil profile is not uniform . the total activity of cs-137 ( bq ) in individual layers was calculated based on the cs-137 activity concentrations in individual soil layers ( bq / kg ) ; results show that , on average , in the gorce : in the first layer there is approx . based on the analysis of deposition of cs-137 in the selected sampling points , the authors distinguished two areas of the gorce mts . the deposition in the north - western part , which has a higher level of contamination , is in the range 4.5 - 17.1 kbq / m with an average of 9.9 kbq / m , while in the less contaminated south - eastern part deposition ranges between 2.2 and 7.6 kbq / m with an average of 4.4 kbq / m . 1).table 3deposition of cs-137 and cs-137 activity concentration in h. physodes in the gorce mts.arearangeaverage/medianstandard deviationcvcs-137 deposition(kbq / m)(kbq / m)(kbq / m)(%)gorce mts.north-western part 4.517.19.9/9.73.636gorce mts.south-eastern part 2.27.64.4/4.41.535gorce mts.whole area 2.217.16.1/4.93.557mt . mostownica local variability 2.28.24.8/3.91.940 hypogymnia physodes ( bq / kg)(bq / kg)(bq mostownica 19 number of samples : 25 deposition of cs-137 and cs-137 activity concentration in h. physodes in the gorce mts . area was minimal ( up to fifteen or so mm daily ; mainly on 30th april and between 8th may and 11th may ) and no differences in the rainfall level between the two parts of the gorce mts . nevertheless , it is characteristic that in each of the two distinguished parts of the gorce mts . se part has been presented twice : once at the bin width being the same as in the nw part ( for comparison of the two parts ) , and again at the bin width being half of that in the nw part statistical distribution of cs-137 deposition in the gorce mts . each entry shows : the mean value and its uncertainty ( longer horizontal line ) and variability range ( shorter horizontal line ) in order to compare the results obtained with those of other authors , the literature data has been decay - corrected to 1st july 2005 . are situated on the border between the deposition areas 1.54.4 and 4.47.3 kbq / m . to the area with a deposition of 05.9 kbq / m , while the average deposition in the then nowy scz voivodeship that encompassed the area of the gorce mts . the central laboratory for radiological protection ( clor ) carried out measurements in five villages situated in the gorce mts . the deposition of cs-137 ranged between 0.7 and 2.0 kbq / m , while the average deposition in the maopolska voivodeship encompassing at that time the gorce mts . the average deposition of 9.9 kbq / m measured by the present authors in the north - western part of the gorce mts . the author determined local variability of cs-137 deposition in 19 points on the 400 m slope located in the mt . the cv value is slightly above the variability interval of the literature data oscillating ( for an area of approximately 0.11 km ) in the range 1030 % ( cf . the cv value slightly higher than quoted by other authors , can be explained by the undulations of the gorce mts . an analysis of the data presented in table 3 indicates that the variability in the north - western and south - eastern parts of the gorce mts . values on the x - axis represent the distance of a sampling point from the centre of the investigated area ; the + and symbols , indicate that the point is located accordingly above or below the centre of the area cs-137 deposition value in 19 sampling points located on 400 m long slope profile of mt . values on the x - axis represent the distance of a sampling point from the centre of the investigated area ; the + and symbols , indicate that the point is located accordingly above or below the centre of the area the results of measurements of cs-137 activity concentration in h. physodes from selected sites in the gorce mts . the cs-137 activity concentration in lichens is in the range 1388 bq / kg d.m . the cs-137 activity concentration values measured are comparable to the literature data of around 100 bq / kg d.m . 50 % being comparable to the variability established over the whole of the gorce mts . area therefore , it may be accepted that the spreads of cs-137 activity concentrations in lichens , obtained for various sampling points in the gorce mts . , one of the reasons for the high local variability of the cs-137 activity concentration in the lichen is the growth of the lichen resulting in two co - occurring types of the thallus in a single sample : the thallus highly contaminated shortly after the chernobyl disaster in 1986 ( direct contamination ) or the thallus contaminated by the former due to cs-137 leaching ( indirect contamination),the thallus formed much later , i.e. , in the period when the activity concentration of cs-137 in the air was lower by several orders of magnitude . , in the period when the activity concentration of cs-137 in the air was lower by several orders of magnitude . to sum up , the cs-137 activity concentration in the lichen is currently due first of all to the contribution of the thallus contaminated shortly after the chernobyl disaster , to the total thallus volume sampled . as the result of measurements , cs-137 activity concentration was determined in individual soil layers , and then the vertical distribution of cs-137 ( depth profile ) . statistical parameters for each of the three layers are presented in table 2 ; due to the different cs-137 deposition in the gorce mts . the values presented in table 2 only provide information on the mean concentrations in individual layers , thus they should not be the used as a basis for estimation of concentration variability in soil profile.table 2cs-137 activity concentration in soil layerslayerrangemean / medianstandard deviationcvgorce mts.north-western part ( bq / kg)(bq / kg)(bq / kg)(%)1 . 34 , gorce mts.whole area 49 cs-137 activity concentration in a successive soil layer mean activity concentration in a 10 cm soil layer percentage of a successive soil layer in cs-137 total activity in 10 cm soil layer cs-137 activity concentration in soil layers number of samples : nw gorce mts . 34 , gorce mts.whole area 49 cs-137 activity concentration in a successive soil layer mean activity concentration in a 10 cm soil layer percentage of a successive soil layer in cs-137 total activity in 10 cm soil layer the change of cs-137 activity concentration in soil profile is not uniform . the total activity of cs-137 ( bq ) in individual layers was calculated based on the cs-137 activity concentrations in individual soil layers ( bq / kg ) ; results show that , on average , in the gorce : in the first layer there is approx . based on the analysis of deposition of cs-137 in the selected sampling points , the authors distinguished two areas of the gorce mts . the deposition in the north - western part , which has a higher level of contamination , is in the range 4.5 - 17.1 kbq / m with an average of 9.9 kbq / m , while in the less contaminated south - eastern part deposition ranges between 2.2 and 7.6 kbq / m with an average of 4.4 kbq / m . turbacz 1).table 3deposition of cs-137 and cs-137 activity concentration in h. physodes in the gorce mts.arearangeaverage/medianstandard deviationcvcs-137 deposition(kbq / m)(kbq / m)(kbq / m)(%)gorce mts.north-western part 4.517.19.9/9.73.636gorce mts.south-eastern part 2.27.64.4/4.41.535gorce mts.whole area 2.217.16.1/4.93.557mt . mostownica 19 number of samples : 25 deposition of cs-137 and cs-137 activity concentration in h. physodes in the gorce mts . area was minimal ( up to fifteen or so mm daily ; mainly on 30th april and between 8th may and 11th may ) and no differences in the rainfall level between the two parts of the gorce mts . the statistical distribution of the cs-137 deposition in the whole gorce mts . nevertheless , it is characteristic that in each of the two distinguished parts of the gorce mts . se part has been presented twice : once at the bin width being the same as in the nw part ( for comparison of the two parts ) , and again at the bin width being half of that in the nw part statistical distribution of cs-137 deposition in the gorce mts . each entry shows : the mean value and its uncertainty ( longer horizontal line ) and variability range ( shorter horizontal line ) in order to compare the results obtained with those of other authors , the literature data has been decay - corrected to 1st july 2005 . to the area with a deposition of 05.9 kbq / m , while the average deposition in the then nowy scz voivodeship that encompassed the area of the gorce mts . the deposition of cs-137 ranged between 0.7 and 2.0 kbq / m , while the average deposition in the maopolska voivodeship encompassing at that time the gorce mts . the average deposition of 9.9 kbq / m measured by the present authors in the north - western part of the gorce mts . when interpreting the cs-137 deposition data , its significant local variability should be taken into account it is defined as the coefficient of variation ( cv ) of statistical distribution of the deposition in the case of multiple sampling points around one site . the author determined local variability of cs-137 deposition in 19 points on the 400 m slope located in the mt . the cv value slightly higher than quoted by other authors , can be explained by the undulations of the gorce mts . an analysis of the data presented in table 3 indicates that the variability in the north - western and south - eastern parts of the gorce mts . values on the x - axis represent the distance of a sampling point from the centre of the investigated area ; the + and symbols , indicate that the point is located accordingly above or below the centre of the area the results of measurements of cs-137 activity concentration in h. physodes from selected sites in the gorce mts . the cs-137 activity concentration in lichens is in the range 1388 bq / kg d.m . the cs-137 activity concentration values measured are comparable to the literature data of around 100 bq / kg d.m . 50 % being comparable to the variability established over the whole of the gorce mts . therefore , it may be accepted that the spreads of cs-137 activity concentrations in lichens , obtained for various sampling points in the gorce mts . in the opinion of the present authors , one of the reasons for the high local variability of the cs-137 activity concentration in the lichen is the growth of the lichen resulting in two co - occurring types of the thallus in a single sample : the thallus highly contaminated shortly after the chernobyl disaster in 1986 ( direct contamination ) or the thallus contaminated by the former due to cs-137 leaching ( indirect contamination),the thallus formed much later , i.e. , in the period when the activity concentration of cs-137 in the air was lower by several orders of magnitude . , in the period when the activity concentration of cs-137 in the air was lower by several orders of magnitude . to sum up , the cs-137 activity concentration in the lichen is currently due first of all to the contribution of the thallus contaminated shortly after the chernobyl disaster , to the total thallus volume sampled . ( several hundred km ) , the authors established in detail the activity concentration of cs-137 and its spatial distribution in soils and lichen . such investigations provide , on the one hand , information on the current distribution of the caesium contamination , and on the other hand , may establish a point of reference in future changes of this distribution in the course of time . have been distinguished on the basis of different deposition of cs-137 . the respective average values are 4.4 and 9.9 kbq / m as at 1st july 2005 , providing a relative difference of around 2 . the value 9.9 kbq / m is much higher than the values given in the maps of cs-137 deposition in poland . the average cs-137 activity concentration in the h. physodes from the gorce mts . is 47 bq / kg d.m . when interpreting this data , significant local variability of measured quantities ( cs-137 deposition , cs-137 activity concentration in lichen ) should be taken into account . this local variability amounts to several dozen percent and is comparable with the variability calculated for the whole area of the gorce mts . thus , there are no grounds to draw isolines of measured quantities on the map of the gorce mts .

low molecular weight heparins ( lmwhs ) are fragments of commercial grade heparin produced by either chemical or enzymatic depolymerization ( 1 ) . both heparin and lwmh are high molecular weight hydrophilic polyanions ( 2 ) with poor oral bioavailability and are being administered parenterally . poor oral absorption is a result of ionic repulsion from negatively charged mucus and epithelial tissue ( 3 ) , destruction by gastrointestinal bacteria ( 4 ) and , to a lesser extent , by the acidic conditions of the stomach ( 5 ) . lmwhs are potentially more advantageous than heparin due to their reduced hemorrhagic to antithrombotic ratio , reduced risk of bleeding , greater bioavailability at low doses , longer half - life and more predictable anticoagulant response at fixed doses ( 6 , 7 ) . they have proven their role as potent anticoagulants in the prevention and treatment of deep vein thrombosis ( 8) and pulmonary embolism ( 9 ) and are the preferred agents for primary prophylaxis in medical and surgical patients in hospital . two large trails have demonstrated the safety and efficacy of outpatient treatment with lmwhs ( 10 , 11 ) . the studies have shown that isl has wide pharmacological effects including anti - flammatory and analgesic activity ( 12 ) , cytoprotective action ( 13 ) , antitumor ( 14 , 15 ) , antioxidant ( 16 , 17 ) , anti - allergic ( 18 ) and anti - platelet coagulation effects ( 19 ) , radical - scavenging ( 20 ) and antiviral activities ( 21 ) . however , isl is poorly water - soluble . the elimination half - life of isl is short , which is about 4.4 - 4.8 h following oral administration and about 3.13 - 5.63 h after intravenous injection in rat ( 22 - 24 ) . intravenous administration of frequent and high doses may be needed , which possibly leads to severe and acute side effects . therefore , extended drug delivery systems for isl such as isoliquiritigenin - loaded nanostructured lipid carrier ( 22 ) and isoliquiritigenin - loaded solid lipid nanoparticle ( to be published ) were developed by our laboratory . it is interesting that acute toxicity of low molecular weight heparin - modified isoliquiritigenin - loaded solid lipid nanoparticle ( lmwh - isl - sln ) reduced . the objective of the present work was studied the formulation and characteristers of low molecular weight heparin - modified isoliquiritigenin - loaded solid lipid nanoparticle ( lmwh - isl - sln ) systematically . particle diameter , zeta potential , entrapment efficiency ( ee ) , drug loading ( dl ) , the in - vitro release models and stabilities of lmwh - isl - sln were investigated . the hemocompatibility , dose - related toxic effects and the in vitro cytotoxicity of lmwh - isl - sln were evaluated . the pharmacokinetics and biodistribution of lmwh - isl - sln in mice following single intravenious injection at doses of 50 , 100 and 200 mg / kg were also studied . isoliquiritigenin ( isl , 99.0% purity ) was purchased from shanghai bangcheng chemical co. ( shanghai , china ) . low molecular weight heparin ( lmwh , average mw 5000 kda ) was purchased from hebei changshan biochemical pharmaceutical limited by share ltd . ( hebei , china ) . stearic acid ( sa ) , hexadecanol , and soya lecithin were purchased from sinopharm chemical reagent co. , ltd . , medium - chain triglyceride ( mct ) and lutrol f68 ( poloxamer 188 , basf , germany ) was obtained from shanghai chemical reagent co. , ltd . , the human heparin carcinoma cell line ( hep - g2 ) was purchased from chinese academy of sciences shanghai cell library ( shanghai , china ) . rpmi 1640 , 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoli - um bromide ( mtt ) , dimethyl sulfoxide ( dmso ) , cona ( concanavalin agglutinin ) and penicillin - streptomycin sulphate were obtained from sigma chemical co. ( shanghai , china ) . hplc grade methanol and acetonitrile were obtained from shandong yuwang ( shandong , china ) . sodium chloride ( analytical grade ) and mannitol were purchased from beijing beihua fine chemicals co. , ltd . preparation of lmwh - isl - sln low molecular weight heparin - modified isoliquiritigenin - loaded solid lipid nanoparticle ( lmwh - isl - sln ) was prepared by our previously reported method ( 25 ) . the preparation process was optimized by a single factor experiment and the optimal process was detailed as follows . isl , stearic acid , hexadecanol , medium - chain triglyceride and soya lecithin were dissolved into 5 ml of mixed organic solvent of ethanol and acetone ( 1:1 , v / v ) in a water bath at 60 c . the resultant organic solution was quickly injected into 5 ml of aqueous solution of poloxamer188 ( 1% , w / v ) containing low molecular weight heparin at the same temperature ( 60 c ) under mechanical agitation ( dc-40 , hangzhou electrical engineering instruments , china ) with 3000 rpm and the resulting mixture was kept at a certain temperature with the same agitation speed for 30 min to remove the organic solvent and formed emulsion . the resulted emulsion was cooled in aqueous phase under mechanical agitation at 1000 rpm at 0 c for 1 h in order to form solid nanoparticle . the lmwh - isl - sln obtained was stored at 4 c.the drug - free nanoparticle was prepared with exactly the same procedures except the drug . the obtained slns were ultra - centrifuged for 1 h at 80,000 g ( 4 c ) using a super - speed refrigerated centrifuge ( mikr022 , heettich , germany ) . the bottom pellet after centrifugation was resuspended in double distilled water containing 8% ( w / v ) mannitol . the slns suspensions were fast frozen in an aqueous mannitol solution under -80 c in a ult 2586 - 5-a14 freezer ( revco scientific , asheville nc , usa ) for 12 h and then the samples were moved to the freeze - drier ( lgj0.5-ii , beijing , china ) and lyophilized at -50 c for 48 h. the slns dried powders were collected and stored at 4 c for further experiments . size and zeta potential analysis of lmwh - isl - sln size and zeta potential analysis of lmwh - isl - sln is similar as described previously ( 22 ) . particle size analysis was performed by dynamic light scattering ( dls ) with a malvern zetasizer 3000 hsa ( malvern instruments , uk ) . transmission electron microscopy ( tem ) examination the morphological observation of lmwh - isl - sln nanoparticle was performed by transmission electron microscopy ( tem ) ( jem 1200 ex , japan ) , using a negative - staining method . a drop of dispersion was spread on a 200-mesh copper grid coating and the excess droplets were removed with filter paper . after 5 min , a drop of 4% ( w / v ) phosphotungstic acid solution was then dropped onto the grids . after being negatively stained and air - dried under room temperature , the samples were completed for the tem investigation . drug encapsulation efficiency ( ee ) and drug loading ( dl ) percentage the obtained lmwh - isl - sln was ultra - centrifuged for 1 h at 80,000 g ( 4 c ) using a super - speed refrigerated centrifuge ( mikr022 , heettich , germany ) . the drug content in the supernatant after centrifugation was measured by hplc method reported previously ( 23 , 24 ) . briefly , analysis was performed by hplc using an agilent 1200 hplc ( bblingen , germany ) system consisting of g1322a vacuum degasser , g1311a quat gradient pump , g1316a thermostatted column compartment , g1329a autosampler , g1315b diode array detector and lc 3d instrument chem station for liquid chromatography systems . chromatographic separation was achieved on a ymc - packed ods - a c18 column , 150 4.6 mm , 5 m ( ymc co. ltd . , kyoto , japan ) preceded by a guard column ( c18 , 10 mm 4.6 mm ) . the ph of the mobile phase was adjusted to 2 with 85% phosphoric acid . prior to use , the mobile phase was filtered through a 0.45 m hydrophilic membrane filter . detection was performed at a wavelength of 242 nm while reference wavelength was set at 360 nm during 0 to 5 min , and from 5 to 9 min detection and reference wavelengths were set at 360 nm and 700 nm , respectively . the drug encapsulation efficiency ( ee ) and drug loading ( dl ) percentage of lmwh - isl - sln were then calculated from formulas ( 1 ) and ( 2 ) : ee = wtotal - wfreewtotal100% dl = wtotal - wfreewlipids100% where wtotal , wfree and wlipids are the weight of drug added , the drug weight in supernatant and weight of lipid added , respectively . storage stabilities of lmwh - isl - sln the storage stabilities of lmwh - isl - sln were determined as follows . lyophilized lmwh - isl - sln was stored at 4 c for 3 months . before the measurement of particle size , zeta potential , ee and dl , the nanoparticle powders were redispersed in distilled water by vortexing ( xw-80a , instruments factory of shanghai medical university ) for 3 min . the particle sizes , zeta potential values , ee and dl of the nanoparticle were determined by the method described above . the in - vitro release study of lmwh - isl - sln the release experiments of lmwh - isl - sln were performed by previously reported dialysis technique ( 25 ) . phosphate butter solution ( pbs , ph 7.4 ) , 200 ml , was poured into a well - closed glass vessel as the dissolution medium for the in - vitro release test . lmwh - isl - sln ( 2 ml ) was transferred to a dialysis bag ( molecular weight cut - off 5000 - 10,000 ) and then the dialysis bag was placed in the glass vessel . the vessels were placed in a shaker and shaken horizontally ( incubator shaker zhwy-200b , shanghai zhicheng analysis instrument company , china ) at 37 c and 100 strokes per min . the sample ( 1 ml ) was withdrawn at predetermined time intervals and filtered through a 0.45 m hydrophilic filter membrane . at the same time , the same volume of fresh buffer was added . a profile showing the cumulative amount of drug release as a function of time was plotted . to describe the drug releasing mechanism from nanoparticle , release profiles were analyzed applying four different mathematical models , which were exponential kinetic model , logarithmic kinetic model , higuchi equation and weibull s distribution law . the exponential kinetic model is 100 - q = a*e , where q is the amount of drug dissolved in time t , a is a constant while t is zero , and k is the release rate constant . logarithmic kinetic model is q = k*ln(t ) + a , where q is the cumulative percentage of drug released at time t , a is a constant while t is 1 h , and k is the release rate constant . higuchi equation is q = kh * t , which describes the release of drug as the square root of time based on the fickian diffusion , and kh is higuchi coefficient . weibull s distribution equation is ln [ ln [ 1/(1 - q ) ] ] = k*ln ( t ) + a , where q is the amount of drug dissolved in time t , a is a constant while t is 1 h , and k is the weibull s distribution rate constant . in - vitro cytotoxicity assay the cytotoxic effects of lmwh - isl - sln and isl solution ( isl - sol ) on hep - g2 cell lines were determined using mtt assay . 100 l of tumor cells ( 5 * 10/ml ) were seeded in 96-well plates and incubated at 37 c for 24 h in a humidified environment with 5% co2 . cells were then treated with lmwh - isl - sln and isl - sol ( at concentrations of 2 , 4 , 6 , 8 , and 10 g / ml ) for 24 , 48 , and 72 h , respectively . then , 50 l of 3-(4,5-dimethylthiazol-2-yl)-2 , 5-diphenyl - tetrazolium bromide ( mtt , sigma , shanghai , china ) solution ( 2.5 mg / ml in pbs ) was added to each well and incubated at 37 c for 4 h. microplates were then centrifuged at 275 g for 5 min and the culture medium carefully aspirated and replaced with 100 l of 100% dimethylsulfoxide ( dmso ) . complete and homogeneous solubilisation of formazan crystals was achieved after 20 min of incubation and a slight plate shaking . the absorbance was measured on a 400 atc microculture plate reader ( slt lab instruments , austria ) at 490 nm . ic50s were calculated by the analysis of single dose response curves , each final value being the mean of 8 - 9 independent experiments ( 26 ) . safety tests of lmwh - isl - sln hemolytic test in - vitro erythrocytes were isolated from fresh whole rabbit blood . 0.1 , 0.2 , 0.3 , 0.4 and 0.5 ml of lmwh - isl - sln ( 15 mg / ml ) were added to five tubes along with 2.5 ml 2% rabbit erythrocyte suspension , respectively . then , normal saline was added to every tube to obtain a final volume of 5 ml . positive and negative controls were prepared by the addition of water ( 2.5 ml ) and normal saline ( 2.5 ml ) to 2.5 ml samples of 2% erythrocyte dispersion . following incubation at 37 c for 4 h , the samples were centrifuged at 2000 rpm for 10 min and the color of the supernatant was compared with controls . if the supernatant solution was absolute achromatic , it implied that there was no hemolysis . dose - related toxic effects kunming mice ( equal numbers of males and females , 18 - 22 g ) were housed under normal conditions with free access to food and water . six male and six female mice per dosing group ( n = 12 ) were used . lmwh - isl - sln and isl - sol were injected via the tail vein at doses of 190 - 610 mg / kg . dose - related toxic effects were observed immediately , 4 h post - injection and then daily for 2 weeks in all groups , and the number of mice surviving was recorded . the median lethal dose ( ld50 ) in - vivo studies of lmwh - isl - sln kunming mice ( 18 - 22 g ) used for this study were supplied by the laboratory animal center of lanzhou university . the procedures used in this experiment were conducted according to the approved protocols of the institutional animal care and use committee of the lanzhou university . pharmacokinetic and tissue distribution study mice were randomly assigned to six different groups ( n = 72 in each group ) . lmwh - isl - sln and isl - sol were injected in mice via a tail vein injection at a dose of 50 , 100 and 200 mg / kg to different groups , respectively . at predetermined time intervals ( 0.08 , 0.17 , 0.25 , 0.33 , 0.5 , 0.75 , 1 , 1.5 , 2 , 2.5 , 3 and 6 h ) , six mice at each time point from each group were given anesthesia and the blood samples were collected by removing the eyeball , placed into heparinized test tubes and centrifuged ( 4500 g , 10 min ) to get corresponding plasma samples . thereafter , tissue samples ( heart , liver , spleen , kidneys , lung and brain ) were immediately collected after cervical dislocation washed with physiological saline and dried with filter paper . plasma and tissue sample analysis a previously validated hplc method ( 23 , 24 ) was used to analyze the drug in the samples . briefly , a 100 l of each plasma sample was transferred into a 1.5 ml polyethylene centrifuge tube . 500 l of acetonitrile containing 20 l of acetanilide ( internal standard , is ) solution ( 100 g / ml , dissolved in methanol ) were added to each plasma sample and vortex - mixed ( sw-80a vortex shaker , shanghai medical university instrument plant , shanghai , china ) for 3 min . thereafter , 300 mg of sodium chloride were added to the mixture and vortex - mixed for 1 min . after placing for 1 min the mixture was centrifuged for 5 min at 12,000 g to separate precipitated proteins . the supernatant ( acetonitrile layer ) was transferred into a new 1.5 ml eppendorf tube and evaporated to dryness at 40 c under a gentle stream of nitrogen . the residue was then reconstituted with 100 l of a mixed solution of methanol and water in the ratio of 50 : 50 ( v / v ) , and ultrasonitated for 1 min and vortex - mixed for 1 min again , then centrifuged at 12,000 g for 5 min , and an aliquot ( 50 l ) of the clear supernatant was injected into the lc system . tissue samples were weighed accurately and homogenized with a two fold aliquot of saline in a tissue homogenizer ( fsh-2a , jintan medical instrument factory , china ) in an ice bath . non - compartmental analysis of the pharmacokinetic data was performed by the statistical moment method using the das 2.1 pharmacokinetic program ( chinese pharmacological society , china ) . the area under the total concentration time curve from time zero to infinity was calculated by : auc0 = auc0t + ct / k , where ct is the last observed isl concentration , and k is the apparent elimination rate constant obtained from the terminal slope of the individual concentration - time curves after logarithmic transformation of the concentration values and application of linear regression ( 27 , 28 ) . the mean residence time ( mrt ) was calculated as follows : mrt = aumc / auc ( aumc is the area under the first moment - time curve ) . the peak concentrations ( cmax ) were derived directly from the original measured values . formulation of lmwh - isl - sln models used in the in - vitro release ic50s of lmwh - isl - sln and isl solution on the growth of hep - g2 cell lines after 24 , 48 and 72 h exposure . statistical difference from isl solution group ( p < 0.05 ) . pharmacokinetic parameters of lmwh - isl - sln and isl - sol after intravenous administration in mice ( mean value sd , n = 6 ) . schematic picture of lmwh - isl - sln structure tem photo of lmwh - isl - sln particle size distribution of lmwh - isl - sln zeta potential value of lmwh - isl - sln the particle size , zeta potential ( a ) and drug encapsulation efficiency ( ee ) , drug loading ( dl ) ( b ) of lmwh - isl - sln lyophilized powder against storage time at 4 c the in vitro release curve of isl from lmwh - isl - sln in pbs at 37c time - dependent inhibition rates of lmwh - isl - sln ( a ) and isl - sol ( b ) in the hep - g2 cell line in vitro the plasma concentration - time profiles of isl in mice after intravenous administration of lmwh - isl - sln ( a ) and isl solution ( b ) at doses of 50 , 100 and 200 mg / kg . ( n = 6 concentrations of isl ( g / g tissue ) in mice after intravenous administration of lmwh - isl - sln at different tissues ( n = 6 concentrations of isl ( g / g tissue ) in mice after intravenous administration of isl - sol at 100 mg / kg dose ( n = 6 ) . characteristics of lmwh - isl - sln the morphology and size of the lmwh - isl - sln were shown in figure 2 . the mean particle size of lmwh - isl - sln was ( 217.53 4.86 ) nm ( figure 3 . ) with a narrow polydispersity index ( pi = 0.16 0.03 ) , and its zeta potential was ( 18.24 2.47 ) mv ( figure 4 . ) . drug loading was ( 18.68 1.51 ) % , and the entrapment efficiency was ( 99.80 3.27)% . storage stability of lmwh - isl - sln due to the instabilities of the sln suspensions , the freeze - dried lmwh - isl - sln was employed to improve the storage stability . mannitol ( 8% , w / v ) was chosen as the cryoprotectants to prevent the aggregation between nanoparticles during the freeze drying process . figure 5a showed the particle size and zeta potential of lmwh - isl - sln against storage time . even though the lyophilized powders of lmwh - isl - sln were stored for 3 months , the particle sizes only slightly increased , but zeta potential values of the lyophilized nanoparticles only slightly decreased . the in - vitro release of lmwh - isl - sln the in - vitro release curve of the lmwh - isl - sln was shown in figure 6 . nearly 60% drug was released from the lmwh - isl - sln during the first 8 h , which was caused by the different melting points between solid lipid and liquid lipid . firstly , the solid lipid which has a higher melting point could crystallize forming a core without liquid lipid or with little liquid lipid . next , most of the liquid lipid located at the outer shell of the nanoparticles led to drug enriched shell . meanwhile , the medium - chain triglyceride - enriched outer layers possessed a soft and considerably higher solubility for lipophilic drug , in which the drug was easily loaded with higher amount and could be easily released by the drug diffusion or the matrix erosion manners . therefore , lmwh - isl - sln showed the burst release at the initial stage and sustained release subsequently . the results in table 2 . demonstrated that release mechanism of lmwh - isl - sln was in line with weibull s distribution law . antiproliferative activity figure 7 . showed the inhibition rates of lmwh - isl - sln and isl - sol in hep - g2 cell lines in - vitro after 24 , 48 and 72 h incubation period . lmwh - isl - sln and isl - sol showed a similar time - dependent and concentration dependent behavior in the hep - g2 cell line . the inhibition rates of lmwh - isl - sln , at a concentration of 10 g / ml , caused a strong inhibition of hep - g2 cell growth after 24 , 48 and 72 h exposure , were 65.03 1.81% , 77.21 2.53% , and 87.16 2.12% , in case of isl - sol , the inhibition rates were 59.33 2.72% , 66.14 1.93% and 78.08 2.06 % , respectively . the half maximal inhibitory concentration ( ic50 ) was calculated in order to compare the nanoparticles cytotoxicity ( 29 ) . the safety of lmwh - isl - sln can be evaluated by the red cell haemolysis assay . the hemolytic potential of lmwh - isl - sln was evaluated to ensure their hemocompatibility . in the present study , lmwh - isl - sln at the concentration of 1.5 mg / ml did not cause any hemolysis on rabbit erythrocyte comparing with the negative control . the ld50 of lmwh - isl - sln and isl - sol were determined to compare their dose - related toxic effects . the ld50 and 95% confidence limits of lmwh - isl - sln was 525.23 mg / kg and 504.08 - 548.12 mg / kg respectively . the ld50 and 95% confidence limits of isl - sol was 415.13 mg / kg and the ld50 of lmwh - isl - sln was 1.27 fold higher than that of isl - sol . no mouse died after being injected with lmwh - isl - sln though the dose went up to 430 mg / kg . there were no marked toxic effects evident in the experimental animals injected with lmwh - isl - sln at a dose of 430 mg / kg . pharmacokinetics study after a single intravenous administration of lmwh - isl - sln and isl - sol in mice at doses of 50 , 100 and 200 mg / kg , the plasma drug concentration - time curves ( mean sd ) of isl are presented in figure 8 and the corresponding pharmacokinetic parameters are listed in table 4 . respectively . as shown in table 4 . there were no statistically significant differences in several pharmacokinetic parameters including mrt , t1/2z , clz and vss of isl after an intravenous administration of lmwh - isl - sln at the three doses of 50 , 100 and 200 mg / kg . however , these pharmacokinetic parameters showed statistically significant differences between lmwh - isl - sln and isl - sol at same dose . compared to isl - sol , the parameters of auc0 and vss of isl were significantly increased ( p < 0.05 ) , and the mrt and t1/2z was prolonged ( p < 0.05 ) after an intravenous administration of lmwh - isl - sln at the three doses of 50 , 100 and 200 mg / kg . these differences indicated that lmwh - isl - sln could postpone the elimination of isl and lead to a long blood circulating effect in mice plasma . in table 4 , the cmax and auc(0 ) of lmwh - isl - sln increased in proportion to the test doses , respectively , suggesting the linear pharmacokinetic behavior of isl after intravenous administration of lmwh - isl - sln over the above dose range , respectively , which was consistent with that previously reported by us ( 24 ) . for lmwh - isl - sln , the auc ( 0 ) of isl was higher 1.32 , 1.67 and 1.58 times than that of isl - sol at the three doses of 50 , 100 and 200 mg / kg , respectively . the results of the pharmacokinetics studies showed that the pharmacokinetic behaviors of isl after administration of the lmwn - isl - sln were significantly different from that of isl - sol . tissue distribution the organ concentrations of isl after intravenous administration of lmwh - isl - sln in mice were shown in figure 9 . biodistribution of isl - sol was shown in figure 10 . at 0.08 h after administration of lmwh - isl - sln , the concentrations of isl in tissues all reached a maximum and then decreased rapidly . at 2 h after intravenous administration , the isl concentrations from isl - sol were low in all collected tissues , that is , there was no long - term accumulation following intravenous injection of isl - sol . while the concentrations of isl from lmwh - isl - sln were higher than that from isl - sol in all collected tissues at 6 h after intravenous administration , that is , there was to a certain degree long - term accumulation following intravenous injection of lmwh - isl - sln . the total concentrations of isl from lmwh - isl - sln were highest in liver , followed by kidney , lung , spleen , brain and heart . it was noteworthy that at each time point the concentrations of isl in heart were higher for isl - sol than for lmwh - isl - sln . the concentrations of isl in brain were lower for isl - sol than for lmwh - isl - sln . this indicated a possible reduction of the cardiac toxicity and enhancement of the therapeutic index of brain when administered lmwh - isl - sln . this is the first report describing the in - vitro antiproliferative activity , safety , pharmacokinetics and biodistribution of lmwh - isl - sln after intravenous administration . antiproliferative activity testing in hep - g2 cell lines of lmwh - isl - sln exhibited superior proliferation inhibition effects . incorporation of isl in low molecular weight heparin - modified sln possessed good blood compatibility . the in - vitro release study revealed that the encapsulated isl in lmwh - isl - sln prolonged the release behavior of isl . work of lmwh - isl - sln on hematology and cytotoxic effects are in progress . our results suggest that the low molecular weight heparin - modified sln system is a promising approach for the intravenous delivery of isl .

low molecular weight heparin - modified isoliquiritigenin - loaded solid lipid nanoparticle ( lmwh - isl - sln ) was developed for injective application . the morphological observation , particle diameter and zeta potential of lmwh - isl - sln were characterized using transmission electron microscopy ( tem ) and a malvern zetasizer . its entrapment efficiency ( ee ) and drug loading ( dl ) were determined by ultracentrifuge . the in - vitro release experiments were performed by dialysis technique . the cytotoxic effects of lmwh - isl - sln on hep - g2 cell lines were determined using an mtt assay . pharmacokinetic and tissue distribution studies were conducted in kunming mice after intravenous administration of lmwh - isl - sln . the average drug entrapment efficiency for lmwh - isl - sln was ( 99.80 3.27)% , drug loading was ( 18.68 1.51)% , mean particle size was ( 217.53 4.86 ) nm and zeta potential was ( 18.24 2.47 ) mv . the in - vitro release experiments demonstrated isoliquiritigenin release from lmwh - isl - sln was in line with weibull s distribution law . hemolysis test and dose - related toxic effects proved that lmwh - isl - sln was a safe and non toxic product when given by intravenous injection . the pharmacokinetics results of lmwh - isl - sln showed that the area under the concentration - time curve ( auc0)of lmwh - isl - sln was greater than that for the isoliquiritigenin solution in plasma . tissue distribution study indicated that isl were mainly distributed in the liver and lung . in conclusion , low molecular weight heparin - modified sln system is a promising carrier for the intravenous delivery of isl .

Introduction Experimental Results and discussions Conclusions Conflict of interest statement

the studies have shown that isl has wide pharmacological effects including anti - flammatory and analgesic activity ( 12 ) , cytoprotective action ( 13 ) , antitumor ( 14 , 15 ) , antioxidant ( 16 , 17 ) , anti - allergic ( 18 ) and anti - platelet coagulation effects ( 19 ) , radical - scavenging ( 20 ) and antiviral activities ( 21 ) . the elimination half - life of isl is short , which is about 4.4 - 4.8 h following oral administration and about 3.13 - 5.63 h after intravenous injection in rat ( 22 - 24 ) . therefore , extended drug delivery systems for isl such as isoliquiritigenin - loaded nanostructured lipid carrier ( 22 ) and isoliquiritigenin - loaded solid lipid nanoparticle ( to be published ) were developed by our laboratory . it is interesting that acute toxicity of low molecular weight heparin - modified isoliquiritigenin - loaded solid lipid nanoparticle ( lmwh - isl - sln ) reduced . the objective of the present work was studied the formulation and characteristers of low molecular weight heparin - modified isoliquiritigenin - loaded solid lipid nanoparticle ( lmwh - isl - sln ) systematically . particle diameter , zeta potential , entrapment efficiency ( ee ) , drug loading ( dl ) , the in - vitro release models and stabilities of lmwh - isl - sln were investigated . the hemocompatibility , dose - related toxic effects and the in vitro cytotoxicity of lmwh - isl - sln were evaluated . the pharmacokinetics and biodistribution of lmwh - isl - sln in mice following single intravenious injection at doses of 50 , 100 and 200 mg / kg were also studied . low molecular weight heparin ( lmwh , average mw 5000 kda ) was purchased from hebei changshan biochemical pharmaceutical limited by share ltd . , medium - chain triglyceride ( mct ) and lutrol f68 ( poloxamer 188 , basf , germany ) was obtained from shanghai chemical reagent co. , ltd . , the human heparin carcinoma cell line ( hep - g2 ) was purchased from chinese academy of sciences shanghai cell library ( shanghai , china ) . preparation of lmwh - isl - sln low molecular weight heparin - modified isoliquiritigenin - loaded solid lipid nanoparticle ( lmwh - isl - sln ) was prepared by our previously reported method ( 25 ) . the resultant organic solution was quickly injected into 5 ml of aqueous solution of poloxamer188 ( 1% , w / v ) containing low molecular weight heparin at the same temperature ( 60 c ) under mechanical agitation ( dc-40 , hangzhou electrical engineering instruments , china ) with 3000 rpm and the resulting mixture was kept at a certain temperature with the same agitation speed for 30 min to remove the organic solvent and formed emulsion . the lmwh - isl - sln obtained was stored at 4 c.the drug - free nanoparticle was prepared with exactly the same procedures except the drug . size and zeta potential analysis of lmwh - isl - sln size and zeta potential analysis of lmwh - isl - sln is similar as described previously ( 22 ) . particle size analysis was performed by dynamic light scattering ( dls ) with a malvern zetasizer 3000 hsa ( malvern instruments , uk ) . transmission electron microscopy ( tem ) examination the morphological observation of lmwh - isl - sln nanoparticle was performed by transmission electron microscopy ( tem ) ( jem 1200 ex , japan ) , using a negative - staining method . drug encapsulation efficiency ( ee ) and drug loading ( dl ) percentage the obtained lmwh - isl - sln was ultra - centrifuged for 1 h at 80,000 g ( 4 c ) using a super - speed refrigerated centrifuge ( mikr022 , heettich , germany ) . the drug encapsulation efficiency ( ee ) and drug loading ( dl ) percentage of lmwh - isl - sln were then calculated from formulas ( 1 ) and ( 2 ) : ee = wtotal - wfreewtotal100% dl = wtotal - wfreewlipids100% where wtotal , wfree and wlipids are the weight of drug added , the drug weight in supernatant and weight of lipid added , respectively . storage stabilities of lmwh - isl - sln the storage stabilities of lmwh - isl - sln were determined as follows . lyophilized lmwh - isl - sln was stored at 4 c for 3 months . before the measurement of particle size , zeta potential , ee and dl , the nanoparticle powders were redispersed in distilled water by vortexing ( xw-80a , instruments factory of shanghai medical university ) for 3 min . the particle sizes , zeta potential values , ee and dl of the nanoparticle were determined by the method described above . the in - vitro release study of lmwh - isl - sln the release experiments of lmwh - isl - sln were performed by previously reported dialysis technique ( 25 ) . phosphate butter solution ( pbs , ph 7.4 ) , 200 ml , was poured into a well - closed glass vessel as the dissolution medium for the in - vitro release test . lmwh - isl - sln ( 2 ml ) was transferred to a dialysis bag ( molecular weight cut - off 5000 - 10,000 ) and then the dialysis bag was placed in the glass vessel . to describe the drug releasing mechanism from nanoparticle , release profiles were analyzed applying four different mathematical models , which were exponential kinetic model , logarithmic kinetic model , higuchi equation and weibull s distribution law . weibull s distribution equation is ln [ ln [ 1/(1 - q ) ] ] = k*ln ( t ) + a , where q is the amount of drug dissolved in time t , a is a constant while t is 1 h , and k is the weibull s distribution rate constant . in - vitro cytotoxicity assay the cytotoxic effects of lmwh - isl - sln and isl solution ( isl - sol ) on hep - g2 cell lines were determined using mtt assay . cells were then treated with lmwh - isl - sln and isl - sol ( at concentrations of 2 , 4 , 6 , 8 , and 10 g / ml ) for 24 , 48 , and 72 h , respectively . safety tests of lmwh - isl - sln hemolytic test in - vitro erythrocytes were isolated from fresh whole rabbit blood . 0.1 , 0.2 , 0.3 , 0.4 and 0.5 ml of lmwh - isl - sln ( 15 mg / ml ) were added to five tubes along with 2.5 ml 2% rabbit erythrocyte suspension , respectively . dose - related toxic effects kunming mice ( equal numbers of males and females , 18 - 22 g ) were housed under normal conditions with free access to food and water . lmwh - isl - sln and isl - sol were injected via the tail vein at doses of 190 - 610 mg / kg . dose - related toxic effects were observed immediately , 4 h post - injection and then daily for 2 weeks in all groups , and the number of mice surviving was recorded . the median lethal dose ( ld50 ) in - vivo studies of lmwh - isl - sln kunming mice ( 18 - 22 g ) used for this study were supplied by the laboratory animal center of lanzhou university . pharmacokinetic and tissue distribution study mice were randomly assigned to six different groups ( n = 72 in each group ) . lmwh - isl - sln and isl - sol were injected in mice via a tail vein injection at a dose of 50 , 100 and 200 mg / kg to different groups , respectively . plasma and tissue sample analysis a previously validated hplc method ( 23 , 24 ) was used to analyze the drug in the samples . the area under the total concentration time curve from time zero to infinity was calculated by : auc0 = auc0t + ct / k , where ct is the last observed isl concentration , and k is the apparent elimination rate constant obtained from the terminal slope of the individual concentration - time curves after logarithmic transformation of the concentration values and application of linear regression ( 27 , 28 ) . the mean residence time ( mrt ) was calculated as follows : mrt = aumc / auc ( aumc is the area under the first moment - time curve ) . formulation of lmwh - isl - sln models used in the in - vitro release ic50s of lmwh - isl - sln and isl solution on the growth of hep - g2 cell lines after 24 , 48 and 72 h exposure . pharmacokinetic parameters of lmwh - isl - sln and isl - sol after intravenous administration in mice ( mean value sd , n = 6 ) . schematic picture of lmwh - isl - sln structure tem photo of lmwh - isl - sln particle size distribution of lmwh - isl - sln zeta potential value of lmwh - isl - sln the particle size , zeta potential ( a ) and drug encapsulation efficiency ( ee ) , drug loading ( dl ) ( b ) of lmwh - isl - sln lyophilized powder against storage time at 4 c the in vitro release curve of isl from lmwh - isl - sln in pbs at 37c time - dependent inhibition rates of lmwh - isl - sln ( a ) and isl - sol ( b ) in the hep - g2 cell line in vitro the plasma concentration - time profiles of isl in mice after intravenous administration of lmwh - isl - sln ( a ) and isl solution ( b ) at doses of 50 , 100 and 200 mg / kg . ( n = 6 concentrations of isl ( g / g tissue ) in mice after intravenous administration of lmwh - isl - sln at different tissues ( n = 6 concentrations of isl ( g / g tissue ) in mice after intravenous administration of isl - sol at 100 mg / kg dose ( n = 6 ) . characteristics of lmwh - isl - sln the morphology and size of the lmwh - isl - sln were shown in figure 2 . the mean particle size of lmwh - isl - sln was ( 217.53 4.86 ) nm ( figure 3 . ) with a narrow polydispersity index ( pi = 0.16 0.03 ) , and its zeta potential was ( 18.24 2.47 ) mv ( figure 4 . ) drug loading was ( 18.68 1.51 ) % , and the entrapment efficiency was ( 99.80 3.27)% . storage stability of lmwh - isl - sln due to the instabilities of the sln suspensions , the freeze - dried lmwh - isl - sln was employed to improve the storage stability . figure 5a showed the particle size and zeta potential of lmwh - isl - sln against storage time . even though the lyophilized powders of lmwh - isl - sln were stored for 3 months , the particle sizes only slightly increased , but zeta potential values of the lyophilized nanoparticles only slightly decreased . the in - vitro release of lmwh - isl - sln the in - vitro release curve of the lmwh - isl - sln was shown in figure 6 . nearly 60% drug was released from the lmwh - isl - sln during the first 8 h , which was caused by the different melting points between solid lipid and liquid lipid . therefore , lmwh - isl - sln showed the burst release at the initial stage and sustained release subsequently . demonstrated that release mechanism of lmwh - isl - sln was in line with weibull s distribution law . showed the inhibition rates of lmwh - isl - sln and isl - sol in hep - g2 cell lines in - vitro after 24 , 48 and 72 h incubation period . lmwh - isl - sln and isl - sol showed a similar time - dependent and concentration dependent behavior in the hep - g2 cell line . the inhibition rates of lmwh - isl - sln , at a concentration of 10 g / ml , caused a strong inhibition of hep - g2 cell growth after 24 , 48 and 72 h exposure , were 65.03 1.81% , 77.21 2.53% , and 87.16 2.12% , in case of isl - sol , the inhibition rates were 59.33 2.72% , 66.14 1.93% and 78.08 2.06 % , respectively . the safety of lmwh - isl - sln can be evaluated by the red cell haemolysis assay . the hemolytic potential of lmwh - isl - sln was evaluated to ensure their hemocompatibility . in the present study , lmwh - isl - sln at the concentration of 1.5 mg / ml did not cause any hemolysis on rabbit erythrocyte comparing with the negative control . the ld50 of lmwh - isl - sln and isl - sol were determined to compare their dose - related toxic effects . the ld50 and 95% confidence limits of lmwh - isl - sln was 525.23 mg / kg and 504.08 - 548.12 mg / kg respectively . the ld50 and 95% confidence limits of isl - sol was 415.13 mg / kg and the ld50 of lmwh - isl - sln was 1.27 fold higher than that of isl - sol . no mouse died after being injected with lmwh - isl - sln though the dose went up to 430 mg / kg . there were no marked toxic effects evident in the experimental animals injected with lmwh - isl - sln at a dose of 430 mg / kg . pharmacokinetics study after a single intravenous administration of lmwh - isl - sln and isl - sol in mice at doses of 50 , 100 and 200 mg / kg , the plasma drug concentration - time curves ( mean sd ) of isl are presented in figure 8 and the corresponding pharmacokinetic parameters are listed in table 4 . there were no statistically significant differences in several pharmacokinetic parameters including mrt , t1/2z , clz and vss of isl after an intravenous administration of lmwh - isl - sln at the three doses of 50 , 100 and 200 mg / kg . however , these pharmacokinetic parameters showed statistically significant differences between lmwh - isl - sln and isl - sol at same dose . compared to isl - sol , the parameters of auc0 and vss of isl were significantly increased ( p < 0.05 ) , and the mrt and t1/2z was prolonged ( p < 0.05 ) after an intravenous administration of lmwh - isl - sln at the three doses of 50 , 100 and 200 mg / kg . these differences indicated that lmwh - isl - sln could postpone the elimination of isl and lead to a long blood circulating effect in mice plasma . in table 4 , the cmax and auc(0 ) of lmwh - isl - sln increased in proportion to the test doses , respectively , suggesting the linear pharmacokinetic behavior of isl after intravenous administration of lmwh - isl - sln over the above dose range , respectively , which was consistent with that previously reported by us ( 24 ) . for lmwh - isl - sln , the auc ( 0 ) of isl was higher 1.32 , 1.67 and 1.58 times than that of isl - sol at the three doses of 50 , 100 and 200 mg / kg , respectively . the results of the pharmacokinetics studies showed that the pharmacokinetic behaviors of isl after administration of the lmwn - isl - sln were significantly different from that of isl - sol . tissue distribution the organ concentrations of isl after intravenous administration of lmwh - isl - sln in mice were shown in figure 9 . biodistribution of isl - sol was shown in figure 10 . at 0.08 h after administration of lmwh - isl - sln , the concentrations of isl in tissues all reached a maximum and then decreased rapidly . at 2 h after intravenous administration , the isl concentrations from isl - sol were low in all collected tissues , that is , there was no long - term accumulation following intravenous injection of isl - sol . while the concentrations of isl from lmwh - isl - sln were higher than that from isl - sol in all collected tissues at 6 h after intravenous administration , that is , there was to a certain degree long - term accumulation following intravenous injection of lmwh - isl - sln . the total concentrations of isl from lmwh - isl - sln were highest in liver , followed by kidney , lung , spleen , brain and heart . it was noteworthy that at each time point the concentrations of isl in heart were higher for isl - sol than for lmwh - isl - sln . the concentrations of isl in brain were lower for isl - sol than for lmwh - isl - sln . this indicated a possible reduction of the cardiac toxicity and enhancement of the therapeutic index of brain when administered lmwh - isl - sln . this is the first report describing the in - vitro antiproliferative activity , safety , pharmacokinetics and biodistribution of lmwh - isl - sln after intravenous administration . antiproliferative activity testing in hep - g2 cell lines of lmwh - isl - sln exhibited superior proliferation inhibition effects . incorporation of isl in low molecular weight heparin - modified sln possessed good blood compatibility . the in - vitro release study revealed that the encapsulated isl in lmwh - isl - sln prolonged the release behavior of isl . work of lmwh - isl - sln on hematology and cytotoxic effects are in progress . our results suggest that the low molecular weight heparin - modified sln system is a promising approach for the intravenous delivery of isl .

this study was conducted according to the guidelines laid down in the declaration of helsinki . the institutional review board at the instituto nacional de perinatologa isidro espinosa de los reyes this study was conducted according to the guidelines laid down in the declaration of helsinki . the institutional review board at the instituto nacional de perinatologa isidro espinosa de los reyes postmenopausal women were included if they were overweight or obese ( body mass index 25 kg / m ) and had mets ( three or more adult treatment panel iii risk criteria).17 women were excluded if they had type 2 diabetes mellitus ( t2 dm ) or a fasting glucose concentration higher than 126 mg / dl ( 7.0 mmol / l ) , had thyroid problems , or were taking medications that may affect nutrient metabolism or absorption . between 2005 and 2009 , women were selected by convenience at the postmenopausal clinic of the instituto nacional de perinatologa isidro espinosa de los reyes , where they were receiving medical treatment . during the first visit , a clinical dietitian using a random number list and sequentially numbered files randomly assigned women ( simple randomization ) to study groups ( parallel design ) . the recruitment protocol included an initial visit where suitability for randomization was evaluated and women were invited to participate . women were allocated into two groups : group 1 : women received a structured hypocaloric diet with serving sizes based on cardioprotective dietary recommendations ( 25%-35% fat intake ; < 7% saturated fat intake ; increased intake of monounsaturated and polyunsaturated fat ; fiber 25 - 30 g / d ; high intake of fruits / vegetables [ 5 servings daily ] , whole grains , low - fat animal products , healthy fats [ avocado , canola oil , and seeds ] ; and low intake of high - energy refined grains , red and processed meats , added sugars , and soda and sweetened beverages ) . they received lifestyle recommendations and established a physical activity goal of 30 minutes three times a week . group 2 : women did not receive a structured diet but received a bt intervention . they were evaluated using a traffic - light questionnaire ( details of dietary risk assessment were published recently ) , where dietary and lifestyle behaviors associated with cardiovascular disease were included.16 the main objective was to gradually promote a change in these behaviors from the high - risk category ( red light ) to the low - risk category ( green light ) . based on the risk factors detected , short - term goals were established for the next visit . to improve success in goal achievement , we used other behavioral strategies such as problem solving and stimulus control . in both groups , initial visits lasted 60 to 70 minutes , and follow - up visits were 45 minutes long . all measurements ( metabolic assessment : blood pressure , fasting glucose , total cholesterol , hdl cholesterol , ldl cholesterol , and tg ; nutrition assessment : weight , height , waist circumference , and 24-h food recalls ) were performed at baseline and after 2 , 4 , and 6 months of intervention . both groups received routine medical care and the same individual nutrition education at each visit . dietary assessment was performed using a five - step multiple - pass 24-hour recall.18 estimation of food servings was performed using measuring cups and spoons , and food replicas ( life / form ; nasco , fort atkinson , wi ) . the food processor sql software ( version 10.4 , 2008 ; esha research ) , which included mexican foods , daily intake of high - fat dairy , high - energy refined grains , soda and sweetened beverages , added sugars , low - fat dairy , and fruits / vegetables was recorded as the number of servings using the mexican food exchange system . in addition , cardioprotective dietary goals20,21 were established on a daily basis as follows : ( a ) fruits / vegetables ( 5 servings ) ; ( b ) high - fat dairy ( 0 servings ) ; ( c ) low - fat dairy ( 1 servings ) ; ( d ) added sugars and sweetened beverages ( < 100 kcal ) ; and ( e ) high - energy refined grains ( 0 servings ) . adequate metabolic status was considered when women achieved optimal blood pressure ( systolic blood pressure < 130 mm hg , diastolic blood pressure < 85 mm hg ) , optimal blood lipid concentrations ( total cholesterol < 200 mg / dl , hdl cholesterol > 50 mg / dl , ldl cholesterol < 130 mg / dl , tg < 150 mg / dl ) , normal fasting glucose ( < 100 mg / dl ) , and/or adequate waist circumference ( < 88 cm ; adult treatment panel iii ) . methods for analyzing each metabolic marker have been described elsewhere.16 analysis included data from all women in the study . last - observation - carried - forward imputation data were assumed when women were lost to follow - up . dietary data were analyzed at baseline ( t0 ) , 2 months ( t1 ) , 4 months ( t2 ) , and 6 months ( t3 ) . except for baseline , mean differences were analyzed using student s t test or mann - whitney u test to assess correct randomization and to compare changes in food intake ( t3 t0 ) . test and fisher s exact test were used to evaluate the association between the number of women who achieved dietary goals and the number of women who had adequate metabolic status ( both independent observations ) . mcnemar s test was used to evaluate change in the number of women who met dietary goals from baseline to the end of the study . repeated - measures analysis of variance was performed to assess food intake changes throughout the intervention . multiple logistic regression models were used to evaluate the association between change in food intake and each metabolic goal , adjusting for confounding factors ( energy intake , use of medication , study group , and/or baseline value ) . statistics were performed with the statistical package for the social sciences software ( version 17.0 , 2008 ; spss inc ) . between 2005 and 2009 , women were selected by convenience at the postmenopausal clinic of the instituto nacional de perinatologa isidro espinosa de los reyes , where they were receiving medical treatment . during the first visit , a clinical dietitian using a random number list and sequentially numbered files randomly assigned women ( simple randomization ) to study groups ( parallel design ) . the recruitment protocol included an initial visit where suitability for randomization was evaluated and women were invited to participate . women were allocated into two groups : group 1 : women received a structured hypocaloric diet with serving sizes based on cardioprotective dietary recommendations ( 25%-35% fat intake ; < 7% saturated fat intake ; increased intake of monounsaturated and polyunsaturated fat ; fiber 25 - 30 g / d ; high intake of fruits / vegetables [ 5 servings daily ] , whole grains , low - fat animal products , healthy fats [ avocado , canola oil , and seeds ] ; and low intake of high - energy refined grains , red and processed meats , added sugars , and soda and sweetened beverages ) . they received lifestyle recommendations and established a physical activity goal of 30 minutes three times a week . group 2 : women did not receive a structured diet but received a bt intervention . they were evaluated using a traffic - light questionnaire ( details of dietary risk assessment were published recently ) , where dietary and lifestyle behaviors associated with cardiovascular disease were included.16 the main objective was to gradually promote a change in these behaviors from the high - risk category ( red light ) to the low - risk category ( green light ) . based on the risk factors detected , short - term goals were established for the next visit . to improve success in goal achievement , we used other behavioral strategies such as problem solving and stimulus control . in both groups , initial visits lasted 60 to 70 minutes , and follow - up visits were 45 minutes long . all measurements ( metabolic assessment : blood pressure , fasting glucose , total cholesterol , hdl cholesterol , ldl cholesterol , and tg ; nutrition assessment : weight , height , waist circumference , and 24-h food recalls ) were performed at baseline and after 2 , 4 , and 6 months of intervention . both groups received routine medical care and the same individual nutrition education at each visit . dietary assessment was performed using a five - step multiple - pass 24-hour recall.18 estimation of food servings was performed using measuring cups and spoons , and food replicas ( life / form ; nasco , fort atkinson , wi ) . the food processor sql software ( version 10.4 , 2008 ; esha research ) , which included mexican foods , was used for nutrient analysis . daily intake of high - fat dairy , high - energy refined grains , soda and sweetened beverages , added sugars , low - fat dairy , and fruits / vegetables was recorded as the number of servings using the mexican food exchange system . in addition , cardioprotective dietary goals20,21 were established on a daily basis as follows : ( a ) fruits / vegetables ( 5 servings ) ; ( b ) high - fat dairy ( 0 servings ) ; ( c ) low - fat dairy ( 1 servings ) ; ( d ) added sugars and sweetened beverages ( < 100 kcal ) ; and ( e ) high - energy refined grains ( 0 servings ) . adequate metabolic status was considered when women achieved optimal blood pressure ( systolic blood pressure < 130 mm hg , diastolic blood pressure < 85 mm hg ) , optimal blood lipid concentrations ( total cholesterol < 200 mg / dl , hdl cholesterol > 50 mg / dl , ldl cholesterol < 130 mg / dl , tg < 150 mg / dl ) , normal fasting glucose ( < 100 mg / dl ) , and/or adequate waist circumference ( < 88 cm ; adult treatment panel iii ) . last - observation - carried - forward imputation data were assumed when women were lost to follow - up . dietary data were analyzed at baseline ( t0 ) , 2 months ( t1 ) , 4 months ( t2 ) , and 6 months ( t3 ) . except for baseline , mean differences were analyzed using student s t test or mann - whitney u test to assess correct randomization and to compare changes in food intake ( t3 t0 ) . test and fisher s exact test were used to evaluate the association between the number of women who achieved dietary goals and the number of women who had adequate metabolic status ( both independent observations ) . mcnemar s test was used to evaluate change in the number of women who met dietary goals from baseline to the end of the study . repeated - measures analysis of variance was performed to assess food intake changes throughout the intervention . multiple logistic regression models were used to evaluate the association between change in food intake and each metabolic goal , adjusting for confounding factors ( energy intake , use of medication , study group , and/or baseline value ) . statistics were performed with the statistical package for the social sciences software ( version 17.0 , 2008 ; spss inc ) . we approached 528 women ; 357 women did not meet the inclusion criteria or were excluded , and 53 women refused to participate . a total of 118 women met our eligibility criteria and agreed to participate ; 63 women were randomly assigned to group 1 , and 55 women were randomly assigned to group 2 ( fig . ) . as previously reported , the mean ( sd ) age of our population was 53.81 ( 6.43 ) years ( range , 40 - 75 y ) . at baseline , the second most frequent alteration was increased hdl cholesterol ( 86.4% ) , followed by increased tg ( 82.1% ) and total cholesterol ( 78.6% ) . there were no baseline differences in metabolic , clinical , or dietary data between groups.16 at the beginning of the study , 75% of all women consumed 3 or fewer servings of fruits , 2.5 or fewer servings of vegetables , and 1 or fewer servings of low - fat dairy daily . both groups started with similar food serving intake , except for vegetables ( p = 0.044 ; table 1 ) . the proportions of women who met established cardioprotective dietary goals at baseline were as follows : fruits / vegetables , 33.1% ; high - fat dairy , 35.5% ; low - fat dairy , 41.5% ; added sugars and sweetened beverages , 39.8% ; high - energy refined grains , 41.5% . no differences were observed between groups , except for fruits / vegetables and high - energy refined grains ( table 2 ) . food servings eaten at the beginning of , during , and at the end of the study women meeting cardioprotective recommendations before and after intervention throughout the study , women in both groups significantly decreased energy intake , total fat intake , saturated fat intake , and added sugars intake ( data not shown).16 women in group 1 increased their intake of fruits ( p = 0.005 ) and low - fat dairy ( p = 0.001 ) , and decreased their intake of high - fat dairy ( p = 0.002 ) , high - energy refined grains ( p < 0.001 ) , added sugars ( p = 0.011 ) , and soda and sweetened beverages ( p = 0.005 ) . women in group 2 increased their intake of vegetables ( p = 0.001 ) and decreased their intake of added sugars ( p = 0.003 ) and soda and sweetened beverages ( p < 0.001 ) . there were no between - group differences in change in food serving intake after 6 months of intervention , except for a greater decrease in high - energy refined grains observed in group 1 ( p = 0.013 ; table 1 ) . at the end of the intervention , there was a significant increase in the number of women who met the following dietary goals : five or more servings of fruits / vegetables , one or more servings of low - fat dairy , and less than 100 kcal from added sugars and soda and sweetened beverages in both groups . in group 1 , more women met the recommended intake of high - fat dairy and low - fat dairy ; in group 2 , more women achieved the five - a - day recommendation for fruits / vegetables ( table 2 ) . in group 1 , significant positive correlations were observed between intake of high - energy refined grains and waist circumference ( r = 0.324 ; p = 0.010 ) , high - fat dairy intake and hdl cholesterol ( r = 0.255 ; p = 0.044 ) , and low - fat dairy intake and fasting glucose ( r = 0.279 ; p = 0.031 ) . women who ate one or more servings of low - fat dairy had lower mean ( sd ) diastolic blood pressure ( 1 serving vs < 1 serving : 4.6735 [ 10.18 ] vs 6.8571 [ 9.58 ] mm hg ; p = 0.012 ) and higher mean ( sd ) hdl cholesterol ( 1 serving vs < 1 serving : + 1.6178 [ 8.41 ] vs 7.9071 [ 5.99 ] mg / dl ; p = 0.001 ) . in this same group , women who did not eat any high - energy refined grains had greater probability of having normal fasting glucose ( relative risk , 1.514 ; 95% ci , 0.989 - 2.316 ; p = 0.035 ) . in group 2 , significant correlations were observed between intake of high - energy refined grains and body weight ( r = 0.266 ; p = 0.049 ) , intake of low - fat dairy and systolic blood pressure ( r = 0.325 ; p = 0.017 ) , and intake of high - fat dairy and total cholesterol ( r = 0.349 ; p = 0.009 ) , ldl cholesterol ( r = 0.274 ; p = 0.043 ) , and tg ( r = 0.307 ; p = 0.023 ) . women who did not eat any high - fat dairy had a greater probability of having a fasting glucose level lower than100 mg / dl ( relative risk , 1.915 ; 95% ci , 1.123 - 3.266 ; p = 0.026 ) ; those who ate five or more servings of fruits / vegetables had greater probability of having altered ldl cholesterol , although this last odds was not statistically significant ( relative risk , 1.583 ; 95% ci , 0.951 - 2.637 ; p = 0.040 ) . in all women , exclusion of high - fat dairy from the diet decreases by 60% the probability of having impaired fasting glucose ( > 100 mg / dl ; relative risk , 0.40 ; 95% ci , 0.181 - 0.906 ; p = 0.028 ) . in all women , exclusion of high - fat dairy from the diet decreases by 60% the probability of having impaired fasting glucose ( > 100 mg / dl ; relative risk , 0.40 ; 95% ci , 0.181 - 0.906 ; p = 0.028 ) . this study presents new evidence showing that two different nutrition interventions promote positive dietary changes that modify mets components in postmenopausal women . overall , these two nutrition interventions were effective in achieving the recommended intakes of fruits / vegetables , low - fat dairy , added sugars , soda and sweetened beverages , and/or high - energy refined grains . the observed improvements in food intake throughout the intervention seem to have positive effects on metabolic components such as glucose , blood pressure , and hdl cholesterol . our findings on fruits women in group 2 , who ate at least five servings of fruits / vegetables daily , were more likely to have altered ldl cholesterol , although this probability did not reach statistical significance . in mexico , intake of high - glycemic - index fruits some evidence has shown an inverse association between dietary glycemic index / glycemic load and total cholesterol and ldl cholesterol22,23 ; this effect has been reported in hispanic postmenopausal women.24 another factor that may affect this association is the fact that we quantified total fruits / vegetables intake without specifying type or preparation . in mexico , it is common to eat fruits with yogurt , honey , or syrups ; thus , the addition of other foods , such as high - fat dairy and added sugars , may be exerting a negative effect on ldl cholesterol . on the other hand , some studies have reported a lack of effect of increased fruits / vegetables intake on lipid profile in the studied population.25 we also found a higher probability of achieving normal fasting glucose at 6 months with elimination of high - energy refined grains from the diet ( not statistically significant ) ; the intake of these types of grains also correlated positively with waist circumference . refined grains tend to be high in energy and low in nutrient density ( fiber , magnesium , vitamin d , folic acid , and potassium ) and may be associated with insulin resistance and excess weight.26 fiber may act in a protective manner by slowing absorption and digestion of carbohydrates , leading to a reduced demand for insulin and lowering glucose responses.27 in previous studies , high intake of refined grains has been associated with insulin resistance , higher waist circumference , and various components of mets , leading to higher risk of t2 dm and cardiovascular disease.26,28 we observed that avoiding consumption of high - fat dairy decreases by 60% the risk of having impaired fasting glucose , after adjusting for energy intake and study group . this finding is in line with the fact that promoting the exchange of high - fat dairy to low - fat dairy may be a promising strategy for preventing and/or controlling t2 dm . overall , there is evidence for the beneficial effect of dairy intake on glucose homeostasis . observational studies have found that eating higher amounts of dairy products decrease the risk of t2 dm by 8% to 14%.29,30 in other clinical trials , a high - dairy diet ( 3 servings ) resulted in improved insulin sensitivity , lower plasma insulin , decrease in total body fat , decrease in waist circumference , decrease in trunk fat , and increase in lean mass.30 dairy products are good sources of vitamins ( a , d , k2 , b12 , and riboflavin ) and minerals ( calcium , magnesium , and potassium ) , which may play a role in insulin sensitivity and/or resistance . the protective effect of dairy products could also be explained by their whey content , which may stimulate insulin secretion.29,31,32 another link could be that , in addition to medium - chain saturated fatty acids , dairy fat contains bioactive lipids , including conjugated linoleic acid , that have the potential to improve insulin resistance.33 - 35 another finding was the positive correlation between high - fat dairy and blood lipids . whole - fat dairy products are a major source of saturated fat , which has been associated with higher risk of mets and cardiovascular disease , mainly by increasing total cholesterol and ldl cholesterol in blood . although some studies have found higher concentrations of total cholesterol and ldl cholesterol with a high - fat dairy diet versus a low - fat dairy diet,36 the latest evidence has failed to demonstrate this link.30,37,38 further research is needed to clarify the role of different bioactive substances in dairy products and their interactions with other macronutrients in the diet.36 the bt intervention ( group 2 ) was designed to promote gradual behavioral change and was conducted according to individual women s baseline food behaviors and their motivation to commit to specific diet and lifestyle goals . bt is an excellent approach that may promote healthy dietary behaviors , similar to a stricter , less individualized diet intervention . increasing evidence highlights the importance of including behavioral and motivational strategies to improve adherence because the latter remains an issue of concern when treatment involves diet and/or exercise.39 women in the bt group were able to meet similar dietary goals ( fruits / vegetables , low - fat dairy , and sugars ) as women following the structured hypocaloric diet . considering these results , the bt intervention seems to be an excellent approach to promoting cardioprotective dietary behaviors . the primary outcome of the clinical trial was the effectiveness of two different dietary strategies on the prevalence of mets , and not the achievement of specific dietary changes that influence mets components . therefore , the sample size in each group and the low event rates may partially explain some inconsistent findings from this study and the difficulty of establishing strong associations . the observed associations from bivariate analysis may be confounded , but the study did not have enough power to conduct multivariate analysis . in addition , we could not perform cluster or factor analysis to evaluate dietary patterns . this methodology would have been ideal because we do not eat isolated nutrients but a variety of foods that are consumed in many complex combinations and may synergically interact to reduce mets risk . we analyzed foods as separate entities and how they can affect some biochemical makers , and this relationship may be confounded by other factors . on the other hand , although a well - trained dietitian uses food replicas , kitchen utensils , and other methods to help reduce bias in serving estimation , this method relies mainly on the participant s memory . the multiple - pass methodology has been shown to improve the accuracy of recalls.40 finally , underreporting may be an issue . when nutrition education is given within a period , it is probable that the individual may be reporting what she learned rather than it would have been interesting to evaluate whether the dietary goals achieved could be maintained for a longer period and whether the bt intervention would promote better sustainment of the observed behaviors . the different associations between dietary and metabolic changes observed in both study groups should be taken into consideration when designing nutrition interventions . nutrition care at clinic level should be flexible , with individualized dietary recommendations based on metabolic risk factors . a structured hypocaloric diet and a bt intervention promote achievement of cardioprotective dietary goals for fruits / vegetables , sugars , soda and sweetened beverages , low - fat dairy , and high - energy refined grains , which result in improvement of some mets components . elimination of high - fat dairy from the diet decreases the risk of impaired fasting glucose , independent of total energy intake and the nutrition intervention . empowerment to achieve self - confidence and to develop motivation is essential for promoting healthy food choices and behavioral change .

abstractobjectivethis study aims to examine the association between dietary changes and improvement of metabolic syndrome components in mexican postmenopausal women receiving two different nutrition interventions.methodswomen ( n = 118 ) with metabolic syndrome were randomly assigned to group 1 ( n = 63 ; structured hypocaloric diet ) or group 2 ( n = 55 ; behavioral therapy ) . metabolic and nutrition assessment was performed at baseline and after 2 , 4 , and 6 months of intervention . dietary changes throughout the study and achievement of cardioprotective dietary goals were assessed at the end of the intervention.resultsthere was a significant increase in the number of women who met recommended servings for fruits / vegetables , low - fat dairy , and sugars in both groups . in group 1 , elimination of high - energy refined grains increased the probability of having normal fasting glucose ( relative risk , 1.514 ; 95% ci , 0.989 - 2.316 ; p = 0.035 ) . in this group , women who met the low - fat dairy goal at the end of the study had lower diastolic blood pressure ( p = 0.012 ) and higher high - density lipoprotein cholesterol ( p = 0.001 ) . in group 2 , women who met the high - fat dairy goal had greater probability of having normal fasting glucose ( relative risk , 1.915 ; 95% ci , 1.123 - 3.266 ; p = 0.026 ) . in all women , exclusion of high - fat dairy decreased by 60% the probability of having impaired fasting glucose ( relative risk , 0.40 ; 95% ci , 0.181 - 0.906 ; p = 0.028).conclusionsboth strategies promote achievement of cardioprotective dietary goals for fruits / vegetables , sugars , soda and sweetened beverages , low - fat dairy , and high - energy refined grains , and improve some metabolic syndrome components . elimination of high - fat dairy decreases the risk of impaired fasting glucose . dietary strategies should be flexible and individualized based on metabolic profile .

METHODS Ethics and study participants Participants Design Dietary assessment MetS components Statistical analysis RESULTS Multivariate analysis DISCUSSION CONCLUSIONS

the institutional review board at the instituto nacional de perinatologa isidro espinosa de los reyes postmenopausal women were included if they were overweight or obese ( body mass index 25 kg / m ) and had mets ( three or more adult treatment panel iii risk criteria).17 women were excluded if they had type 2 diabetes mellitus ( t2 dm ) or a fasting glucose concentration higher than 126 mg / dl ( 7.0 mmol / l ) , had thyroid problems , or were taking medications that may affect nutrient metabolism or absorption . between 2005 and 2009 , women were selected by convenience at the postmenopausal clinic of the instituto nacional de perinatologa isidro espinosa de los reyes , where they were receiving medical treatment . women were allocated into two groups : group 1 : women received a structured hypocaloric diet with serving sizes based on cardioprotective dietary recommendations ( 25%-35% fat intake ; < 7% saturated fat intake ; increased intake of monounsaturated and polyunsaturated fat ; fiber 25 - 30 g / d ; high intake of fruits / vegetables [ 5 servings daily ] , whole grains , low - fat animal products , healthy fats [ avocado , canola oil , and seeds ] ; and low intake of high - energy refined grains , red and processed meats , added sugars , and soda and sweetened beverages ) . they were evaluated using a traffic - light questionnaire ( details of dietary risk assessment were published recently ) , where dietary and lifestyle behaviors associated with cardiovascular disease were included.16 the main objective was to gradually promote a change in these behaviors from the high - risk category ( red light ) to the low - risk category ( green light ) . based on the risk factors detected , short - term goals were established for the next visit . in both groups , initial visits lasted 60 to 70 minutes , and follow - up visits were 45 minutes long . all measurements ( metabolic assessment : blood pressure , fasting glucose , total cholesterol , hdl cholesterol , ldl cholesterol , and tg ; nutrition assessment : weight , height , waist circumference , and 24-h food recalls ) were performed at baseline and after 2 , 4 , and 6 months of intervention . dietary assessment was performed using a five - step multiple - pass 24-hour recall.18 estimation of food servings was performed using measuring cups and spoons , and food replicas ( life / form ; nasco , fort atkinson , wi ) . the food processor sql software ( version 10.4 , 2008 ; esha research ) , which included mexican foods , daily intake of high - fat dairy , high - energy refined grains , soda and sweetened beverages , added sugars , low - fat dairy , and fruits / vegetables was recorded as the number of servings using the mexican food exchange system . in addition , cardioprotective dietary goals20,21 were established on a daily basis as follows : ( a ) fruits / vegetables ( 5 servings ) ; ( b ) high - fat dairy ( 0 servings ) ; ( c ) low - fat dairy ( 1 servings ) ; ( d ) added sugars and sweetened beverages ( < 100 kcal ) ; and ( e ) high - energy refined grains ( 0 servings ) . adequate metabolic status was considered when women achieved optimal blood pressure ( systolic blood pressure < 130 mm hg , diastolic blood pressure < 85 mm hg ) , optimal blood lipid concentrations ( total cholesterol < 200 mg / dl , hdl cholesterol > 50 mg / dl , ldl cholesterol < 130 mg / dl , tg < 150 mg / dl ) , normal fasting glucose ( < 100 mg / dl ) , and/or adequate waist circumference ( < 88 cm ; adult treatment panel iii ) . methods for analyzing each metabolic marker have been described elsewhere.16 analysis included data from all women in the study . dietary data were analyzed at baseline ( t0 ) , 2 months ( t1 ) , 4 months ( t2 ) , and 6 months ( t3 ) . test and fisher s exact test were used to evaluate the association between the number of women who achieved dietary goals and the number of women who had adequate metabolic status ( both independent observations ) . mcnemar s test was used to evaluate change in the number of women who met dietary goals from baseline to the end of the study . repeated - measures analysis of variance was performed to assess food intake changes throughout the intervention . multiple logistic regression models were used to evaluate the association between change in food intake and each metabolic goal , adjusting for confounding factors ( energy intake , use of medication , study group , and/or baseline value ) . between 2005 and 2009 , women were selected by convenience at the postmenopausal clinic of the instituto nacional de perinatologa isidro espinosa de los reyes , where they were receiving medical treatment . women were allocated into two groups : group 1 : women received a structured hypocaloric diet with serving sizes based on cardioprotective dietary recommendations ( 25%-35% fat intake ; < 7% saturated fat intake ; increased intake of monounsaturated and polyunsaturated fat ; fiber 25 - 30 g / d ; high intake of fruits / vegetables [ 5 servings daily ] , whole grains , low - fat animal products , healthy fats [ avocado , canola oil , and seeds ] ; and low intake of high - energy refined grains , red and processed meats , added sugars , and soda and sweetened beverages ) . they were evaluated using a traffic - light questionnaire ( details of dietary risk assessment were published recently ) , where dietary and lifestyle behaviors associated with cardiovascular disease were included.16 the main objective was to gradually promote a change in these behaviors from the high - risk category ( red light ) to the low - risk category ( green light ) . based on the risk factors detected , short - term goals were established for the next visit . in both groups , initial visits lasted 60 to 70 minutes , and follow - up visits were 45 minutes long . all measurements ( metabolic assessment : blood pressure , fasting glucose , total cholesterol , hdl cholesterol , ldl cholesterol , and tg ; nutrition assessment : weight , height , waist circumference , and 24-h food recalls ) were performed at baseline and after 2 , 4 , and 6 months of intervention . dietary assessment was performed using a five - step multiple - pass 24-hour recall.18 estimation of food servings was performed using measuring cups and spoons , and food replicas ( life / form ; nasco , fort atkinson , wi ) . daily intake of high - fat dairy , high - energy refined grains , soda and sweetened beverages , added sugars , low - fat dairy , and fruits / vegetables was recorded as the number of servings using the mexican food exchange system . in addition , cardioprotective dietary goals20,21 were established on a daily basis as follows : ( a ) fruits / vegetables ( 5 servings ) ; ( b ) high - fat dairy ( 0 servings ) ; ( c ) low - fat dairy ( 1 servings ) ; ( d ) added sugars and sweetened beverages ( < 100 kcal ) ; and ( e ) high - energy refined grains ( 0 servings ) . adequate metabolic status was considered when women achieved optimal blood pressure ( systolic blood pressure < 130 mm hg , diastolic blood pressure < 85 mm hg ) , optimal blood lipid concentrations ( total cholesterol < 200 mg / dl , hdl cholesterol > 50 mg / dl , ldl cholesterol < 130 mg / dl , tg < 150 mg / dl ) , normal fasting glucose ( < 100 mg / dl ) , and/or adequate waist circumference ( < 88 cm ; adult treatment panel iii ) . dietary data were analyzed at baseline ( t0 ) , 2 months ( t1 ) , 4 months ( t2 ) , and 6 months ( t3 ) . test and fisher s exact test were used to evaluate the association between the number of women who achieved dietary goals and the number of women who had adequate metabolic status ( both independent observations ) . mcnemar s test was used to evaluate change in the number of women who met dietary goals from baseline to the end of the study . repeated - measures analysis of variance was performed to assess food intake changes throughout the intervention . multiple logistic regression models were used to evaluate the association between change in food intake and each metabolic goal , adjusting for confounding factors ( energy intake , use of medication , study group , and/or baseline value ) . a total of 118 women met our eligibility criteria and agreed to participate ; 63 women were randomly assigned to group 1 , and 55 women were randomly assigned to group 2 ( fig . ) at baseline , the second most frequent alteration was increased hdl cholesterol ( 86.4% ) , followed by increased tg ( 82.1% ) and total cholesterol ( 78.6% ) . there were no baseline differences in metabolic , clinical , or dietary data between groups.16 at the beginning of the study , 75% of all women consumed 3 or fewer servings of fruits , 2.5 or fewer servings of vegetables , and 1 or fewer servings of low - fat dairy daily . both groups started with similar food serving intake , except for vegetables ( p = 0.044 ; table 1 ) . the proportions of women who met established cardioprotective dietary goals at baseline were as follows : fruits / vegetables , 33.1% ; high - fat dairy , 35.5% ; low - fat dairy , 41.5% ; added sugars and sweetened beverages , 39.8% ; high - energy refined grains , 41.5% . no differences were observed between groups , except for fruits / vegetables and high - energy refined grains ( table 2 ) . food servings eaten at the beginning of , during , and at the end of the study women meeting cardioprotective recommendations before and after intervention throughout the study , women in both groups significantly decreased energy intake , total fat intake , saturated fat intake , and added sugars intake ( data not shown).16 women in group 1 increased their intake of fruits ( p = 0.005 ) and low - fat dairy ( p = 0.001 ) , and decreased their intake of high - fat dairy ( p = 0.002 ) , high - energy refined grains ( p < 0.001 ) , added sugars ( p = 0.011 ) , and soda and sweetened beverages ( p = 0.005 ) . women in group 2 increased their intake of vegetables ( p = 0.001 ) and decreased their intake of added sugars ( p = 0.003 ) and soda and sweetened beverages ( p < 0.001 ) . there were no between - group differences in change in food serving intake after 6 months of intervention , except for a greater decrease in high - energy refined grains observed in group 1 ( p = 0.013 ; table 1 ) . at the end of the intervention , there was a significant increase in the number of women who met the following dietary goals : five or more servings of fruits / vegetables , one or more servings of low - fat dairy , and less than 100 kcal from added sugars and soda and sweetened beverages in both groups . in group 1 , more women met the recommended intake of high - fat dairy and low - fat dairy ; in group 2 , more women achieved the five - a - day recommendation for fruits / vegetables ( table 2 ) . in group 1 , significant positive correlations were observed between intake of high - energy refined grains and waist circumference ( r = 0.324 ; p = 0.010 ) , high - fat dairy intake and hdl cholesterol ( r = 0.255 ; p = 0.044 ) , and low - fat dairy intake and fasting glucose ( r = 0.279 ; p = 0.031 ) . women who ate one or more servings of low - fat dairy had lower mean ( sd ) diastolic blood pressure ( 1 serving vs < 1 serving : 4.6735 [ 10.18 ] vs 6.8571 [ 9.58 ] mm hg ; p = 0.012 ) and higher mean ( sd ) hdl cholesterol ( 1 serving vs < 1 serving : + 1.6178 [ 8.41 ] vs 7.9071 [ 5.99 ] mg / dl ; p = 0.001 ) . in this same group , women who did not eat any high - energy refined grains had greater probability of having normal fasting glucose ( relative risk , 1.514 ; 95% ci , 0.989 - 2.316 ; p = 0.035 ) . in group 2 , significant correlations were observed between intake of high - energy refined grains and body weight ( r = 0.266 ; p = 0.049 ) , intake of low - fat dairy and systolic blood pressure ( r = 0.325 ; p = 0.017 ) , and intake of high - fat dairy and total cholesterol ( r = 0.349 ; p = 0.009 ) , ldl cholesterol ( r = 0.274 ; p = 0.043 ) , and tg ( r = 0.307 ; p = 0.023 ) . women who did not eat any high - fat dairy had a greater probability of having a fasting glucose level lower than100 mg / dl ( relative risk , 1.915 ; 95% ci , 1.123 - 3.266 ; p = 0.026 ) ; those who ate five or more servings of fruits / vegetables had greater probability of having altered ldl cholesterol , although this last odds was not statistically significant ( relative risk , 1.583 ; 95% ci , 0.951 - 2.637 ; p = 0.040 ) . in all women , exclusion of high - fat dairy from the diet decreases by 60% the probability of having impaired fasting glucose ( > 100 mg / dl ; relative risk , 0.40 ; 95% ci , 0.181 - 0.906 ; p = 0.028 ) . in all women , exclusion of high - fat dairy from the diet decreases by 60% the probability of having impaired fasting glucose ( > 100 mg / dl ; relative risk , 0.40 ; 95% ci , 0.181 - 0.906 ; p = 0.028 ) . this study presents new evidence showing that two different nutrition interventions promote positive dietary changes that modify mets components in postmenopausal women . overall , these two nutrition interventions were effective in achieving the recommended intakes of fruits / vegetables , low - fat dairy , added sugars , soda and sweetened beverages , and/or high - energy refined grains . the observed improvements in food intake throughout the intervention seem to have positive effects on metabolic components such as glucose , blood pressure , and hdl cholesterol . our findings on fruits women in group 2 , who ate at least five servings of fruits / vegetables daily , were more likely to have altered ldl cholesterol , although this probability did not reach statistical significance . in mexico , intake of high - glycemic - index fruits some evidence has shown an inverse association between dietary glycemic index / glycemic load and total cholesterol and ldl cholesterol22,23 ; this effect has been reported in hispanic postmenopausal women.24 another factor that may affect this association is the fact that we quantified total fruits / vegetables intake without specifying type or preparation . in mexico , it is common to eat fruits with yogurt , honey , or syrups ; thus , the addition of other foods , such as high - fat dairy and added sugars , may be exerting a negative effect on ldl cholesterol . on the other hand , some studies have reported a lack of effect of increased fruits / vegetables intake on lipid profile in the studied population.25 we also found a higher probability of achieving normal fasting glucose at 6 months with elimination of high - energy refined grains from the diet ( not statistically significant ) ; the intake of these types of grains also correlated positively with waist circumference . refined grains tend to be high in energy and low in nutrient density ( fiber , magnesium , vitamin d , folic acid , and potassium ) and may be associated with insulin resistance and excess weight.26 fiber may act in a protective manner by slowing absorption and digestion of carbohydrates , leading to a reduced demand for insulin and lowering glucose responses.27 in previous studies , high intake of refined grains has been associated with insulin resistance , higher waist circumference , and various components of mets , leading to higher risk of t2 dm and cardiovascular disease.26,28 we observed that avoiding consumption of high - fat dairy decreases by 60% the risk of having impaired fasting glucose , after adjusting for energy intake and study group . this finding is in line with the fact that promoting the exchange of high - fat dairy to low - fat dairy may be a promising strategy for preventing and/or controlling t2 dm . observational studies have found that eating higher amounts of dairy products decrease the risk of t2 dm by 8% to 14%.29,30 in other clinical trials , a high - dairy diet ( 3 servings ) resulted in improved insulin sensitivity , lower plasma insulin , decrease in total body fat , decrease in waist circumference , decrease in trunk fat , and increase in lean mass.30 dairy products are good sources of vitamins ( a , d , k2 , b12 , and riboflavin ) and minerals ( calcium , magnesium , and potassium ) , which may play a role in insulin sensitivity and/or resistance . the protective effect of dairy products could also be explained by their whey content , which may stimulate insulin secretion.29,31,32 another link could be that , in addition to medium - chain saturated fatty acids , dairy fat contains bioactive lipids , including conjugated linoleic acid , that have the potential to improve insulin resistance.33 - 35 another finding was the positive correlation between high - fat dairy and blood lipids . whole - fat dairy products are a major source of saturated fat , which has been associated with higher risk of mets and cardiovascular disease , mainly by increasing total cholesterol and ldl cholesterol in blood . although some studies have found higher concentrations of total cholesterol and ldl cholesterol with a high - fat dairy diet versus a low - fat dairy diet,36 the latest evidence has failed to demonstrate this link.30,37,38 further research is needed to clarify the role of different bioactive substances in dairy products and their interactions with other macronutrients in the diet.36 the bt intervention ( group 2 ) was designed to promote gradual behavioral change and was conducted according to individual women s baseline food behaviors and their motivation to commit to specific diet and lifestyle goals . increasing evidence highlights the importance of including behavioral and motivational strategies to improve adherence because the latter remains an issue of concern when treatment involves diet and/or exercise.39 women in the bt group were able to meet similar dietary goals ( fruits / vegetables , low - fat dairy , and sugars ) as women following the structured hypocaloric diet . the primary outcome of the clinical trial was the effectiveness of two different dietary strategies on the prevalence of mets , and not the achievement of specific dietary changes that influence mets components . when nutrition education is given within a period , it is probable that the individual may be reporting what she learned rather than it would have been interesting to evaluate whether the dietary goals achieved could be maintained for a longer period and whether the bt intervention would promote better sustainment of the observed behaviors . the different associations between dietary and metabolic changes observed in both study groups should be taken into consideration when designing nutrition interventions . nutrition care at clinic level should be flexible , with individualized dietary recommendations based on metabolic risk factors . a structured hypocaloric diet and a bt intervention promote achievement of cardioprotective dietary goals for fruits / vegetables , sugars , soda and sweetened beverages , low - fat dairy , and high - energy refined grains , which result in improvement of some mets components . elimination of high - fat dairy from the diet decreases the risk of impaired fasting glucose , independent of total energy intake and the nutrition intervention .

the correct identification of dementia subtypes represents a major challenge for clinicians , since an early differential diagnosis may be difficult on clinical grounds only . the use of biomarkers in the different neurodegenerative processes ( e.g. , alzheimer 's disease ( ad ) , frontotemporal lobar degeneration ( fltd ) or dementia with lewy bodies ( dlb ) ) can help obtain more accurate diagnosis , also in the prodromal stages of the diseases ( anchisi et al . distinctive topographic and pathophysiological markers have been thus included in the new proposed research and clinical criteria for dementias ( sperling et al . , 2011 ; albert et al . , 2011 ; jack et al . , 2011 ; , 2010 ; dubois et al . , 2014 ; gorno - tempini et al . , 2011 ; rascovsky et al . , 2011 ; mckeith et al . , 2005 ) , comprising changes in a and tau protein values in the cerebrospinal fluid , structural brain changes visible on brain magnetic resonance imaging ( mri ) and metabolic changes seen with 18-fluorodeoxyglucose positron emission tomography ( fdg - pet ) . in ad , the pathophysiological process starts decades before the clinical onset of cognitive impairments in at risk individuals ( bateman et al . therefore , the concept of the ad pathophysiological process has been teased apart from that of ad dementia ( dubois et al . , 2013 ) . the current research criteria for ad encourage translating the results from biomarker studies into guidelines for diagnosis ( sperling et al . , 2011 ; albert et al . , 2011 ; jack et al . , 2011 ; as far as imaging markers of ad are concerned , there is a widespread evidence for an increase in diagnostic accuracy for metrics - based assessment ( frisoni et al . , 2013 ) . among the topographic imaging markers , the best mri marker in ad , even at the prodromal stage , i.e. hippocampal volume , might be influenced by several potential confounders , such as aging ( fjell et al . , 2013 ) , concomitant pathologies affecting the medial temporal lobe ( e.g. , diabetes or sleep apnoea ) ( fotuhi et al . , 2012 ) , and the presence of hippocampal sclerosis or other dementia types that might also show hippocampal atrophy ( de souza et al . , 2013 ) . fdg - pet instead shows high sensitivity in detecting typical and reliable patterns of brain metabolic dysfunction , also in the early ad phase ( anchisi et al . , 2005 ; it has been considered to be more sensitive than mri in the typical ad pathological cascade ( jack et al . , 2010 ) , since the pathological phenomena leading to neuronal synaptic dysfunction affect glucose consumption prior to causing cell death and detectable atrophy ( bateman et al . , 2012 ; the typical ad pattern represented by hypometabolism in temporo - parietal regions , precuneus and posterior cingulate cortex is closely related to cognitive impairment ( perani , 2008 ) and allows the early identification of the downstream neuronal degeneration , in prodromal phase before the full onset of dementia ( mosconi et al . , 2008 ; herholz , 2010 ) , and many years before in at risk individuals ( mckeith et al . , 2005 ; bateman et al . , moreover , on the basis of the specific patterns of topographic distribution of the metabolic changes , fdg - pet can also help to recognize and differentiate other dementia types ( teune et al . therefore , fdg - pet imaging has been included also in the supportive criteria for non - ad dementias , such as ftld and dlb ( gorno - tempini et al . , 2011 ; the pet imaging recommendations for dementia diagnosis are largely referring to fdg - pet literature based on subjective methods for evaluating metabolic fdg - pet changes ( i.e. , visual inspection ) . these approaches greatly depend on the observer 's experience and may reduce sensitivity and specificity especially in those centres where advanced expertise in image reading is unavailable , due to the lack of an objective cut - off between normal and pathological findings . a lower diagnostic confidence may arise , especially for the earliest disease stages when only subtle metabolic abnormalities may be present . to overcome these limitations , fdg - pet images have been assessed using statistical image analysis , such as ad t - sum ( herholz et al . , 2002 ) , stereotactic surface projection ( ssp ) statistics ( neurostat ) ( foster et al . , 2007 ; minoshima et al . , 2001 ) and statistical parametrical mapping ( spm ) ( signorini et al . other methods have been reported and compared in accuracy ( caroli et al . , 2012 ) . ad t - sum score provides a measure of scan abnormality associated with a preset threshold for discrimination between ad patients and controls . this method , however , based on the sum of t - values in predefined regions typically affected by ad , does not allow differential diagnosis in dementia conditions and the identification of whole - brain patterns of hypometabolism ( herholz et al . 2013 ) at difference with spm procedures ( signorini et al . , 1999 ; patterson et al . , 2010 ) . neurostat ( minoshima et al . , 2001 ) is an atlas - based method allowing a whole - brain parametric analysis of fdg - uptake , yielding z - scores as a result of the voxel by voxel comparison between an individual subject and a predefined control group . package uses surface projection technology to generate and display an entire statistical map on a projection of brain surfaces in different views . neurostat ( minoshima et al . , 2001 ) uses four different intensity normalization methods , including global intensity normalization , as well as normalization to the thalamus , cerebellum and pons . a limit is that the individual fdg - pet scan is normalized to the talairach and tournoux atlas space and 3d rendering of statistical map of glucose hypometabolism are visualized through a cortical surface projection technology . in addition , despite the possibility of statistically inferring upon the pattern of fdg metabolism through a colour bar , p - values for single voxels or clusters of voxels are not available and this procedure does not allow the detection of metabolic changes in deep brain structures ( i.e. , basal ganglia or midbrain ) . spm analysis of fdg - pet images has been also used in the evaluation of brain metabolic changes in neurodegenerative conditions showing specific topographic patterns associated with cognitive decline and dementia ( anchisi et al . each individual fdg - pet scan is first warped in the standard mni space using a template image for spatial normalization and subsequently smoothed with a 3d gaussian kernel . parametric analysis of fdg - uptake in spm is obtained using voxel - level statistical parametric mapping at the whole - brain level , in the framework of the general linear model by means of a two - sample t - test , comparing each subject against images pertaining to a reference control group . the comparison between each individual and the reference group yields a contrast t - map testing for areas with relative decreases in metabolism ( i.e. , hypometabolism ) compared to the controls . one of the main advantages of the spm approach resides in the use of metrics evaluated at a predefined significance statistical threshold to define the topography of hypometabolism , thus drastically reducing the chance for false positives and increasing specificity ( silverman et al . , the presence of false positives is particularly detrimental at the single - subject level , when it is crucial to exclude neurodegenerative conditions and to effectively discriminate between the different dementia conditions . the aim of this study was to assess and validate at a single - subject level the sensitivity and specificity of a new spm procedure compared to subjective visual inspection of fdg - uptake distribution maps and to the sole clinical information . voxel - based spm hypometabolism maps were produced by means of spatial normalization to a new standardized fdg - pet template that showed to increase reliability and accuracy of estimated brain metabolic patterns ( della rosa et al . , 2014 ) . statistical analysis was based on a large normal dataset ( 112 control scans ) for single - subject comparisons . a group of neuroimaging experts was individually asked to provide a forced diagnosis and indicate their level of confidence on the basis of a ) summary of clinical data , b ) visual inspection of standard fdg images , and c ) assessment of spm t - maps , in a large series of patients with clinical diagnosis of neurodegenerative disorders ( i.e. , ad , ftld and dlb ) , and in subjects with mild cognitive impairment mci . a series of 88 subjects was retrospectively pooled from the population database of the neurology centres for cognitive disorders in the san raffaele hospital ( milan , italy ) . medical history and neurological examination the clinical information ( medical history , neurological examination and neuropsychological assessment ) was evaluated by three neurologist experts in dementia diagnosis ( am , sfc , gm ) that examined the whole medical records related to the entire clinical course of each subject in order to correctly assign the clinical diagnosis at the follow - up , which was used as gold - standard . only subjects with the clinical follow - up and complete diagnostic agreement by the three expert neurologists ( am , sfc , gm ) were included . according to the diagnosis at the clinical follow - up ( 27.6 4.1 months ; range 2235 months ) ( gold - standard ) , we identified patients fulfilling consensus criteria for ad ( jack et al . , 2011 ; mckhann et al . , 2011 ; dubois et al . , 2010 ; dubois et al . , 2014 ) ( n = 27 ; men 16 , female 11 ; mean age = 67.40 6.68 ; mmse < 22 ; cdr global score range 12 ) , dlb ( mckeith et al . , 2005 ) ( n = 9 ; men 3 , female 6 ; mean age = 74.11 6.90 ; mmse < 23 ; cdr global score range 12 ) and for the ftld spectrum ( n = 24 ; men 11 , female 13 ; mean age = 68 6 ) . ftld patients included 10 behavioural variant of frontotemporal dementia ( bvftd ) ( rascovsky et al . , 2011 ) ( mmse < 28 ; cdr global score range 0.51 ) , 5 primary progressive aphasias ( ppa ) ( gorno - tempini et al . , 2011 ) ( mmse range 2329 ; cdr global score range 0.51 ) , and 9 corticobasal degeneration syndrome ( cbs ) ( armstrong et al . , 2013 ) ( mmse < 21 ; cdr global score range 12 ) . in addition , 28 subjects were classified at the baseline as mci ( petersen et al . , 2009 ) ( amnestic single - domain , non - amnestic single domain and multidomain ; men 16 , female 12 ; mean age = 71.32 5.67 ; mmse range 2528 ; cdr global score 0.5 ) . among these , 7 subjects progressed to dementia at follow - up ( 27.6 4.1 months ) ; while 6 reverted to cognitive normal condition and 15 remained stable . we selected 93 cognitively normal subjects by the european alzheimer disease consortium ( eadc)-pet dataset ( http://www.eadc.info/ ) and 19 cognitively normal subjects that were previously acquired in the nuclear medicine dept of the san raffaele hospital . cognitive health was established in each pet centre by means of a structured clinical and a neuropsychological battery and subjects were followed up for more than a year , as specified in a previous paper ( morbelli et al . , 2012 ) . the institutional ethical committee at the university - hospital san raffaele of milan approved this study . fdg - pet acquisitions of the whole patient group and of 112 cognitively normal subjects were performed according to the guidelines of the european association of nuclear medicine ( eanm ) ( morbelli et al . , 2012 ; varrone et al . , , san raffaele hospital ( milan , italy ) , with a discovery ste ( ge medical systems , milwaukee , wi ) multi - ring pet tomography ( pet - ct ) system ( time interval between injection and scan start = 45 min ; scan duration = 15 min ) . in order to obtain voxel - based statistical parametric hypometabolic maps , each fdg - pet brain image scan was pre - processed using spm5 ( http://www.fil.ion.ucl.ac.uk/spm/software/spm5/ ) , running in matlab 7.6 ( mathworks inc . , functional normalization procedure allowed to place the images in the standard mni space using a new fdg - pet dementia - specific template for spatial normalization , based on images derived from both neurological patients ( sample of scans representative of the various forms of dementia in the population ) and age - matched controls , developed by della rosa et al . ( 2014 ) , available to download in the templates section ( on the spm official website http://www.fil.ion.ucl.ac.uk/spm/ ) . we created this new [ f]-fdg pet population - specific template for spatial normalization , since the standard spatial normalization requires a non - linear registration of pet images to a [ o]-h2o template provided with the spm software . fdg uptake ( i.e. , the pattern of signal intensities in the [ f]-fdg pet image ) may not be represented properly by the pattern of signal intensities in the standard [ o]-h2o pet template which is based on blood flow . this dementia - specific fdg - pet template ( della rosa et al . , 2014 ) was built by averaging and smoothing ( with an 8-mm fwhm gaussian filter ) a number of 100 intensity and spatially normalized fdg - pet images ( 50 controls and 50 patients ) . this template was used to spatially transform fdg - pet patient images to the mni ( montreal neurological institute ) reference space , as it has been shown to provide a higher degree of accuracy for spatial normalization of fdg - pet scans and a higher statistical sensitivity at the single - subject level in spm5 , useful for clinical purposes ( della rosa et al . , 2014 ) . prior to normalization , we first performed approximate manual image re - orientation and positioning to mni space of each subject fdg - pet image . to spatially normalize the fdg - pet patient images to the dementia - specific fdg - pet template , we used the normalization algorithm provided by spm5 with the following parameter settings including 12-parameters ' affine transformation , 7 8 7 discrete cosine transform basis functions , no template and source weighting ; discrete cosine transform cut - off : 25 mm ; 16 non - linear iterations and the non - linear regularization term set to 1 . no modulation ( preserve concentrations ) and trilinear interpolation were used during final reslicing . normalized images were written in the default spm5 bounding box with an isotropic voxel size of 2 mm . visual inspections of normalized images allowed to ensure registration quality and convergence of the normalization procedure . spatially normalized images of all subjects were subsequently smoothed with an isotropic 3d gaussian kernel of 8 mm fwhm before entering statistical analysis . a single - subject analysis for comparison to a cognitively normal control group ( n = 112 ; age range 5080 years ) was performed using spm5 for all the included subjects . in the application of spm approach , usually the number of images included as control reference is very small ( 20 ) ( signorini et al . , 1999 ) , and limited by ethical constraints dealing with the pet acquisition of normal controls . it has been shown however , that increasing the sample size of control database for comparison , improves the diagnostic performances ( chen et al . the control database used for comparison in our new approach amounts to 112 images , which allows to use higher and more conservative significance thresholds at both the voxel ( p = 0.05 fwe - corrected ) and cluster level ( extent > 100 voxels ) and , even more importantly , to correct for multiple comparisons ( i.e. , fwe : family - wise error correction ) . this reduces the false positive rate and allows for more robust inference on fdg patterns of hypometabolism when assessing neurodegenerative diseases . furthermore , each fdg - pet scan of the healthy control group ( n = 112 ) underwent an intensity rescaling and global count intensity normalization ( della rosa et al . , 2014 ) to have the same mean intensities ( friston et al . , 1994 ; buchert et al . , 2005 ) in order to standardize the magnitude of all voxel values in every image , thus accounting for potential sources of variability both between scanners ( i.e. , centre - specific image scaling ) or between individuals ( i.e. , patient weight , cardiac output , the amount of injected radioactivity ) . then , each fdg - pet scan of the control group was normalized to the dementia - specific fdg - pet template and tested for normality in a jack - knife approach , where every normalized fdg pet scan was evaluated with respect to the remaining sample via the two sample t - test in spm5 ( della rosa et al . , 2014 ) . hypometabolism by comparison with the reference group of 112 controls on a voxel - by - voxel basis using the general linear model , by means of the two sample t - test design of spm5 . age was included as a covariate . due to the lack of any significant difference in metabolic activity of male and female demented patients ( minoshima et al . , 1997 ) , global normalization of voxel values used proportional scaling to a mean voxel value of 6.5 mg/100 ml / min ( see signorini et al . proportional scaling basically scales each image according to a reference count , which is the global brain activity to a physiologically realistic reference value of 6.5 mg/100 ml / min . the threshold was left at the default 0.8 value ( i.e. , the mean brain intensity was computed from only those voxels with intensity above 0.8 of the mean over the entire scan ) . voxel - wise comparisons were made using an explicit fdg - pet mask ( ridgway et al . , 2009 ) . this mask was created using the spm masking toolbox to produce an average binary mask , where the voxels from which to determine the fdg - metabolism parameter estimates were restricted to an explicit mask . the latter resulted from optimal thresholding of voxels in each image based on their correlation with an average image ( i.e. , the average of fdg - pet images from the 112 healthy control scans ) with voxels not meeting the optimality criterion set to zero ( masked ) . this mask was applied ( i.e. , explicit masking option in spm5-glm models ) to restrict subsequent single - subject statistical analyses only to within - brain voxels in order to eliminate variance due to inter - subject variation and noise from outside the brain ( spence et al . , 2006 ) . the spm comparison between each single fdg - pet scan and the healthy control group of scans essentially provides regional differences in relative glucose metabolism by means of a t - statistic for each voxel ( spm - t maps ) . clusters of decreased metabolism were considered significant when they met a significance level of p = 0.05 , corrected for multiple comparisons with the family - wise error ( fwe ) option at the voxel level , and contained more than 100 voxels . nine neuroimaging experts with an extensive experience in the dementia diagnosis ( dp , ff , egv , ap , fn , sp , cc , vg , lg ) were presented with clinical and neuropsychological information ( clinical scenarios ) ( fig . 1a ) , standard clinical display of fdg - pet images ( standard fdg images ) ( fig . 1b ) , and voxel - based statistical parametric hypometabolic maps ( spm maps ) ( see fig . they had a long lasting experience in reporting of fdg - pet scans ( i.e. , visual assessment of standard fdg images and spm maps ) together with the clinical information . they were informed that the study included patients who had at follow - up a clinically confirmed diagnosis of ad , ftld spectrum ( including different subtypes ) , dlb , as well as mci subjects . an anonymized score sheet grid was built for each patient at baseline ( near and before the fdg - pet scan ) . it summarized general clinical information ( onset , disease duration , age , education , comorbidity ) , neurological signs , cognitive symptoms ( impairments of memory , executive functions , language , visuo - spatial , orientation , praxia ) , behavioural changes ( positive and negative symptoms , as well as sleep disorders ) , global cognitive efficiency ( i.e. , mini mental state examination ( mmse ) score ) , and functionality ( i.e. , activities of daily living ( adl ) and instrumental activities of daily living ( iadl ) scores ) ( fig . results of a detailed neuropsychological battery were also provided for each subject on a separate sheet . in particular , the administered tests were rey auditory verbal learning test , rey 's figure recall test , verbal and visual digit span tasks , attentive matrices , phonological and semantic fluency , token test , aachener aphasie test ( aat ) or batteria per l'analisi dei deficit afasici ( bada ) subtests , rey 's figure copy and raven 's progressive matrices scores . each image was reoriented to the ac pc line and then displayed in a 5 5 matrix of transaxial images ( i.e. , 25 axial slices ) from rostral to ventral brain sections in a radiological convention ( i.e. , left = right ; right = left ) . images were shown as relative metabolic rates with the highest and lowest pixel values in the scan placed at the highest value ( 100% red pink ) or lowest value ( 0% blue purple ) according to a rainbow colour scale . we provided raters with spm - t coloured anonymous displays of fdg - pet hypometabolic overlaid on a canonical mri t1-weighted structural brain scan in the mni space . each spm map included the same matrix ( i.e. , 5 5 matrix of transaxial images ) used for the visual inspection of the standard fdg images . spm maps were displayed in a neurological convention ( i.e. , left = left ; right = right ) . each axial slice covered 4 mm on the z - axis ranging from 40 to + 56 . images showed statistically significant reductions of metabolism with the significant t - values in yellow / red scale . each rater independently evaluated one type of information for each subject : a ) clinical scenarios ; or b ) standard fdg images ; or c ) spm maps . thus , the raters were presented with the standard fdg images or spm maps independently for any given subject and in both situations were blinded to any clinical data . raters were asked to make a forced diagnosis of ad , ftld spectrum , or dlb , and also of negative pet scan , and to indicate the degree of diagnostic confidence on a 3-point scale ( 1poor confidence , 2medium confidence or 3high confidence ) . the diagnostic labels assigned by each rater were compared to the clinical diagnosis at follow - up ( the gold standard ) . all raters were then informed that all patients included in this study had a follow - up clinically confirmed diagnosis of ad or dlb , ftld subtypes , stable mci or mci conversion or reversion , but they did not know the proportion of subjects with each diagnosis . based solely upon the standard fdg images or the spm maps , raters were first asked to grade the level of overall scan abnormality as normal , uncertain or abnormal . second , raters were instructed to report more extensive details on brain metabolism , encompassing the involved brain lobes , ( i.e. , frontal , temporal , parietal , occipital ) and they had to decide whether they were hypometabolic or not and /or if the pattern of hypometabolism was bilateral or distributed in asymmetrical way ( i.e. , > left hemisphere or > right hemisphere ) . third , raters were asked to focus on more specific brain areas critical to the differential diagnosis of specific neurodegenerative diseases . hypometabolism in specific brain regions was rated , namely in the anterior cingulate cortex ( acc ) , medial frontal cortex , orbito - frontal cortex , frontal operculum ; in the inferior and superior parietal lobules , parietal operculum , precuneus , posterior cingulate cortex ; in the temporal lateral cortex , temporo - medial cortex and temporal pole and ; in the medial and lateral occipital cortices . subcortical structures , namely the thalamus and basal ganglia , as well as the midbrain were also included in the list of specific areas to be rated . as in the former case , raters had to decide whether these areas were hypometabolic or not and/or if the pattern of hypometabolism was bilateral or asymmetrical . the procedure for evaluation of the clinical scenarios , the standard fdg images and the spm maps was identical as those outlined above for step 1 . the only difference was that each rater saw for each subject the clinical scenarios first and initially entered their diagnosis and degree of diagnostic certainty based exclusively upon this information . second , the rater could be presented with the standard fdg images or spm maps along with the clinical scenarios and was asked to evaluate the combination of both types of information , either clinical scenarios plus standard images or clinical scenarios plus spm maps , asking them for assessments of overall degree of scan normality , the presence of hypometabolism in more extensive or specific regions and of a potential asymmetrical pattern . finally , they were asked to make a forced diagnosis among the predefined disease categories added with their degree of confidence independently from the one indicated solely on the basis of the clinical information , thus maybe resulting in a potentially different diagnostic choice . the aim of step 2 was to assess the differential value of adding qualitative ( standard fdg images ) or quantitative ( spm maps ) information to clinical scenarios as a support for both diagnostic accuracy and confidence . clinical scenarios , standard fdg images and spm maps for steps 1 and 2 were sent independently to raters on separate dates ( i.e. , a 6 month time gap ) and had different alphanumeric codes . in this way , raters could not associate or compare information across subjects and between the two steps . the three types of information ( i.e. , clinical scenarios , standard fdg images , and spm maps ) were associated to each subject in a factorial manner and arranged according to six possible permutations ( i.e. , clinical scenariosstandard fdg imagesspm maps , clinical scenariosspm mapsstandard fdg images , standard fdg imagesclinical scenariosspm maps , standard fdg imagesspm mapsclinical scenarios , spm mapsclinical scenariosstandard fdg images , and spm mapsstandard fdg imagesclinical scenarios ) . for each diagnostic group , of permutations ) , in order for each permutation to be equally represented among considered dementia conditions . the sequence according to which each permutation was assigned to each successive subject in each diagnostic group was randomized . this design was implemented in order to have the same amount of subjects rated for each of the three types of information , and to test agreement between different pairs of raters on the same information and between the same pair of raters on different types of information . overall rater consensus was also evaluated independently for each type of information by computing a contingency coefficient measuring the association between the follow - up diagnostic category and the rated disease category as indicated in the study . a set of twelve subjects was used as a consistency sample in order to assess reliability of the rating procedure . the diagnostic labels assigned by each rater were evaluated according to the clinical diagnosis at follow - up ( gold standard ) . in order to assess the diagnostic power of each type of information , sensitivity and specificity were computed independently for clinical scenarios , standard fdg images and spm maps information and expressed as percentages , averaged across the nine raters and weighted by the number of subjects evaluated by each rater for each type of information . moreover , for each type of information , we calculated both positive and negative likelihood ratios ( lr+ and lr ) , which provide the ratio between the probability of positive or negative test outcome in patients and the probability of positive or negative test outcome in true negatives . lr+ 5 and lr 0.2 are agreed upon as being diagnostically useful ( frisoni et al . , 2013 ) . the inter - rater agreement was calculated by rater for all possible rater pairs evaluating the same subjects for each type of information . an average phi - value between rater pairs and a chi - transform was computed independently for clinical scenarios , standard fdg images and spm maps and tested for significance . inter - rater reliability by subjects was assessed using k - statistics calculated on the basis of the agreement between the two raters judging each subject , independently from the designated pair among all the possible rater pairs . the level of agreement based on the k - statistics was classified as fair ( k = 0.200.39 ) , moderate ( k = 0.400.59 ) , substantial ( k = 0.600.79 ) , and almost perfect ( k = 0.801.00 ) ( foster et al . , 2007 ) . diagnostic accuracy was also assessed for clinical scenarios , standard fdg images and spm maps taking in consideration the percentage of rated false negatives ( fn% ) for the two major dementia categories here represented ( i.e. , ad and ftld ) , and by means of a concordance index using an unanimity rule for the rater pair ( number of correctly classified subjects when the rater pair is concordant ) . three independent logistical models were fit to a categorical variable representing an ad or ftld diagnosis at follow - up , using as predictor variable only the correctly and concordantly classified ad or ftld subjects by the rater pair through clinical scenarios , standard fdg images or spm maps in order to estimate a concordance index for each type of information on the basis of diagnostic accuracy . predicted group classification probabilities were then used for roc curves corresponding to clinical scenarios , standard fdg images and spm maps in order to assess the average performance of the three types of information . the area under the curve ( auc ) was computed in order to test the accuracy of each information type . clinical information combined with the standard fdg images ( clinical scenarios plus standard fdg image ) or spm maps ( clinical scenarios plus spm maps ) was evaluated for each subject . the randomized procedure for evaluation of the three types of information was identical for both steps 1 and 2 . in order to measure the level of agreement between the raters for clinical scenarios plus standard fdg images or clinical scenarios plus spm maps on consistency subjects , we computed a generalized kappa coefficient and used the z - statistic to test the null hypothesis , given the equal number of ratings . we then compared in an anova model the level of confidence ( loc ) in making a diagnostic choice only for correctly classified subjects in each disease category . in addition , considering only ad and ftld groups , i.e. the two major dementia categories here represented , we assessed the strength of standard fdg images or spm maps in terms of correctly classifying those ad or ftld subjects , which were misclassified based solely upon clinical scenarios . for both combinations ( i.e. , clinical scenarios plus standard fdg images or clinical scenarios plus spm maps ) , raters were also asked to specify the hypometabolic brain regions as well as the whole - brain distribution of the pattern ( i.e. , bilateral or asymmetrical ) . in order to quantify the contribution of the identification of selected hypometabolic patterns through standard fdg images or spm maps to the correct classification of ad and ftld spectrum , we computed an index , expressed as the differential % of incidence ( di ) of localized hypometabolism in disease - specific areas between standard fdg images and spm maps for either ad or ftld . only the ad and ftld subjects the % difference between the number of times an area was rated as hypometabolic through standard fdg images and spm maps was calculated for each specific area weighted by the total number of correct classifications for either ad or ftld , independently for standard fdg images and spm maps . only areas with an index above 10% were considered . finally , loc for correctly evaluating mci was also compared between standard fdg images ( clinical scenarios plus standard fdg images ) or spm maps ( clinical scenarios plus spm maps ) . raters ' performance for diagnostic accuracy and confidence levels is illustrated in figs . 2 and 3 analysis of raters ' performances for diagnostic accuracy indicated higher sensitivity and specificity values for spm maps ( 96% and 84% ) , compared to clinical scenarios ( 91% and 40% ) and standard fdg images ( 78% and 50% ) , considering the total sample of 88 subjects . diagnostic lr+ was better for spm maps ( 6.08 ) than for standard fdg images ( 1.55 ) or clinical scenarios ( 1.52 ) , exceeding the lr+ accuracy cut - off ( 5 ) only for spm maps . lr - values were best for spm maps ( 0.05 ) and very poor for standard fdg images ( 0.45 ) and clinical scenarios ( 0.22 ) , with only spm maps fell below lr - cut off ( 0.2 ) ( see fig . 4a and 4b ) . the chi - transforms of average phi - values measuring inter - rater agreement between each rater pair were higher for spm maps ( = 173.53 , p < 0.001 ) than for clinical scenarios ( = 126.04 , p = 0.01 ) and lowest for standard fdg images ( = 79.29 , p = 0.9 ) . the k - statistic measuring inter - rater reliability by subjects independently of the rater pair was substantial for spm maps ( k = 0.6 ) , moderate for clinical scenarios ( k = 0.4 ) and fair for standard fdg images ( k = 0.3 ) . the contingency coefficient ( c ) measuring general association between diagnosis at follow - up and rated category for all disease categories and overall rater consensus was higher for spm maps ( c = 0.722 , p < 0.0001 ) than for clinical scenarios ( c = 0.673 , p < 0.0001 ) or standard fdg images ( c = 0.678 , p < 0.0001 ) . when considering only ad and ftld , the average false - negative rate for spm maps was 0% , for clinical scenarios 2% and for standard fdg images 16% . logistic regressions tested whether spm maps is a more robust metric than clinical scenarios or standard fdg images in terms of both concordance and accuracy for the classification of ftld with respect to ad . it revealed that the spm maps model ( beta = 1.414 ; p = 0.019 ) correctly classified a significantly higher number of concordant subjects in the ftld group , compared to the clinical scenarios ( beta = 0.671 ; p = 0.291 ) or standard fdg images ( beta = 0.041 ; p = 0.945 ) , considering ad classification as the reference . roc curves measuring of the goodness - of - fit of the spm maps , clinical scenarios and standard fdg images models indicated that the auc was 0.67 for spm maps , 0.57 for clinical scenarios and 0.50 for standard fdg images . the asymptotic significance was inferior to 0.05 only for the spm maps model ( p = 0.039 ; clinical scenarios : p = 0.416 ; standard fdg images : p = 0.955 ) . this means that the logistic regression using the spm maps information classified correctly and concordantly the ftld group significantly better than chance ( see fig . the overall inter - rater diagnostic agreement based on a generalized kappa coefficient for consistently rated subjects was substantial for clinical scenarios plus spm maps combination ( k = 0.6 ; z = 10.57 , p < 0.0001 ) and only moderate for clinical scenarios plus standard fdg images ( k = 0.4 ; z = 5.99 , p < 0.0001 ) . viewing the clinical scenarios plus spm maps combination ( mean loc = 2.4 ) significantly increased the diagnostic confidence as compared to clinical scenarios plus standard fdg images ( mean loc = 2.07 ) ( p = 0.003 ) . in the case of the ftld spectrum only a trend was present in the same direction ( clinical scenarios plus spm maps mean loc = 2.26 ; clinical scenarios plus standard fdg images mean loc = 1.97 , p = 0.078 ) . in the subjects with a correct diagnosis of ad , raters found hypometabolism in the temporo - parietal ( di = + 26% ) and posterior cingulate regions ( di = + 38% ) more frequently with spm maps than standard fdg images . in the subjects with correct diagnosis of ftld , the anterior cingulate cortex ( di = + 16% ) , the medial frontal cortex ( di = + 12% ) and the superior anterior temporal cortex ( di = + 18% ) in the left hemisphere were identified as hypometabolic much more frequently with spm maps than with standard fdg images . within the mci group , the 7 subjects who progressed to dementia ( i.e. , 5 ad and 2 ftld ) were all correctly classified by spm maps at baseline . only 2 mci subjects who progressed to ad were correctly classified by the standard fdg images . all mci subjects classified as ad at follow - up showed the typical hypometabolic pattern suggestive for ad ( anchisi et al . , 2005 ; de souza et al . , 2013 ; herholz et al . , 2002 ; 2010 ) and characterized by bilateral temporo - parietal hypometabolism , and also involving precuneus / posterior cingulate cortex . one subject classified at baseline as executive non - amnestic mci presented frontal and anterior temporal hypometabolism with prevalent involvement of ventro - medial prefrontal cortex , a pattern suggestive of bvftd ( rascovsky et al . , 2011 ; teune et al . one additional subject , clinically classified as amnestic single - domain mci at baseline showed predominant anterior temporal polar hypometabolism , typically described in association with the semantic variant of primary progressive aphasia ( svppa ) ( gorno - tempini et al . , 2011 ) . the latter two subjects both fulfilled criteria for respectively bvftd and svppa at follow - up ( gorno - tempini et al . spm analysis identified also specific hypometabolic patterns suggestive for different neurodegenerative conditions ( i.e. , ad , dlb , ftld ) in 15 mci subjects who did not convert within the time frame of the present clinical study . a longer follow - up would be necessary in these subjects to obtain values in the prediction of disease progression . the spm maps of six mci individuals ( 3 amnestic single domain , 2 amnestic multi - domain and 1 visuo - spatial non - amnestic ) showed normal brain metabolism at baseline . on the basis of this information they were all classified as negative by each rater . at follow - up , all these subjects reverted to normal cognition . compared to clinical scenarios plus standard fdg images , the effect of adding spm maps to clinical scenarios on ratings was significantly more beneficial for correctly classifying both positive mci who progressed and the negative mci category ( clinical scenarios plus spm maps mean loc = 2.46 ; clinical scenarios plus standard fdg images mean loc = 1.81 , p < 0.001 ) . the main result of this single - subject study is that the spm - based tool for the analysis of fdg - pet imaging improved diagnostic accuracy in dementia and pre - dementia conditions compared to visual inspection of fdg - uptake distribution , providing also additional value to clinical information . our data add solid evidence to the importance of metrics in the clinical setting ( frisoni et al . , 2013 ; foster et al . , 2007 ; perani et al . , 2014 ) analysis of performances showed very high sensitivity and specificity for spm maps ( 96% and 84% ) as compared to clinical scenarios ( 91% and 40% ) and standard fdg images ( 78% and 50% ) ( fig . independent measures of diagnostic accuracy ( lr+ and lr ) were better for spm maps than standard fdg images with only spm maps exceeding the proposed cut - off value for dementia diagnosis ( fig . 4b ) . noteworthy , spm maps showed higher inter - rater agreement compared to the other types of information . all this results in a substantial reliability of spm analysis for the diagnostic classification , confirming that the assessment of spm disease - specific hypometabolic patterns is less influenced by the specific reader expertise than visual inspection . in the case of the differential diagnosis between ad and ftld , there is evidence of a high percentage of false - negative with visual inspection ( 16% ) . on the contrary , roc curves showed that spm information is a very robust metric to correctly and concordantly differentiate among diagnostic groups ( see fig . in particular , the roc area under the curve ( auc ) proves that spm - t maps classified ftld subtypes better than visual inspection , underlining the importance of voxel - based analysis for differential diagnosis , especially in the case of dementia subtypes with more heterogeneous genetic and pathological signatures , such as those within the ftld spectrum . moreover , spm single subject information provides more diagnostic strength to clinical information than standard fdg images . noteworthy , viewing the spm maps in combination with clinical scenarios significantly increased diagnostic confidence as compared to the combination of clinical scenarios and standard fdg images . finally , spm analysis allowed a better delineation of the anatomical signatures specific for ad and ftld spectrum , as shown by the disease differential indexes . crucially , hypometabolism in the temporo - parietal and posterior cingulate for ad subjects , and the anterior cingulate , medial frontal and superior anterior temporal cortices for bvftd subjects was more frequently identified by spm maps than standard fdg images . the assessment of measured level of confidence revealed that diagnostic confidence of clinical information was lower in ftld variants , in dlb and in some ad patients with atypical presentation . this could be partially ascribed to the fixed format of the clinical scenario that might have neglected some crucial clinical details . nevertheless , the use of spm maps added a further value to clinical evaluation providing the clinician a higher degree of accuracy compared to standard fdg images . the results of raters ' performances on the mci group proved that spm maps allow identifying with significantly stronger confidence distinct patterns of hypometabolism underlying mci condition at the baseline , which predicted the further progression of cognitive decline to different dementia conditions at the clinical follow - up . according to previous findings , heterogeneous hypometabolic profiles may be recognized using fdg - pet in mci subjects developing into different dementia diseases ( mosconi et al . , 2008 ; in addition , the mci individuals who reverted to normal cognition at the follow - up were all negative on spm analysis at the baseline , thus supporting the role of our new spm tool as an exclusionary test . it has been highlighted that a cognitively impaired subject with a negative pet scan has a low chance in the progression of cognitive disorders towards dementia ( silverman et al . , 2008 ) . it is now known that different operating procedures of imaging biomarkers ( visual inspection vs. semi - quantitative / quantitative method ) can be responsible for the heterogeneous levels of their estimated diagnostic and prognostic accuracy ( see frisoni et al . , 2013 and perani et al . , 2014 for meta - analyses ) . within the different operating procedures for fdg - pet imaging , voxel - based analyses at a single - subject level are the most accurate , thus mandatorily calling for an objective statistical analysis of fdg - pet brain images that allows to reach the highest sensitivity and specificity ( perani et al . , 2014 ) . given that accuracy in dementia diagnosis highly depends not only on which marker ( topographic or pathological ) is used , but also on how it is measured ( qualitative or semi - quantitative / quantitative ) ( frisoni et al . , 2013 ) , in this context , we showed that spm maps specifically improve fdg - pet imaging in dementias . the main limitation of this study that resides in the lack of pathological confirmation needs to be acknowledged . further studies are necessary and some are in due course , in order to replicate the results in multi - site trials .

diagnostic accuracy in fdg - pet imaging highly depends on the operating procedures . in this clinical study on dementia , we compared the diagnostic accuracy at a single - subject level of a ) clinical scenarios , b ) standard fdg images and c ) statistical parametrical ( spm ) maps generated via a new optimized spm procedure . we evaluated the added value of fdg - pet , either standard fdg images or spm maps , to clinical scenarios . in 88 patients with neurodegenerative diseases ( alzheimer 's disease ad , frontotemporal lobar degeneration ftld , dementia with lewy bodies dlb and mild cognitive impairment mci ) , 9 neuroimaging experts made a forced diagnostic decision on the basis of the evaluation of the three types of information . there was also the possibility of a decision of normality on the fdg - pet images . the clinical diagnosis confirmed at a long - term follow - up was used as the gold standard . spm maps showed higher sensitivity and specificity ( 96% and 84% ) , and better diagnostic positive ( 6.8 ) and negative ( 0.05 ) likelihood ratios compared to clinical scenarios and standard fdg images . spm maps increased diagnostic accuracy for differential diagnosis ( ad vs. ftd ; beta 1.414 , p = 0.019 ) . the auc of the roc curve was 0.67 for spm maps , 0.57 for clinical scenarios and 0.50 for standard fdg images . in the mci group , spm maps showed the highest predictive prognostic value ( mean loc = 2.46 ) , by identifying either normal brain metabolism ( exclusionary role ) or hypometabolic patterns typical of different neurodegenerative conditions .

Introduction Material and methods Results Discussion

, alzheimer 's disease ( ad ) , frontotemporal lobar degeneration ( fltd ) or dementia with lewy bodies ( dlb ) ) can help obtain more accurate diagnosis , also in the prodromal stages of the diseases ( anchisi et al . , 2005 ) , comprising changes in a and tau protein values in the cerebrospinal fluid , structural brain changes visible on brain magnetic resonance imaging ( mri ) and metabolic changes seen with 18-fluorodeoxyglucose positron emission tomography ( fdg - pet ) . , moreover , on the basis of the specific patterns of topographic distribution of the metabolic changes , fdg - pet can also help to recognize and differentiate other dementia types ( teune et al . , 2001 ) and statistical parametrical mapping ( spm ) ( signorini et al . spm analysis of fdg - pet images has been also used in the evaluation of brain metabolic changes in neurodegenerative conditions showing specific topographic patterns associated with cognitive decline and dementia ( anchisi et al . parametric analysis of fdg - uptake in spm is obtained using voxel - level statistical parametric mapping at the whole - brain level , in the framework of the general linear model by means of a two - sample t - test , comparing each subject against images pertaining to a reference control group . one of the main advantages of the spm approach resides in the use of metrics evaluated at a predefined significance statistical threshold to define the topography of hypometabolism , thus drastically reducing the chance for false positives and increasing specificity ( silverman et al . the aim of this study was to assess and validate at a single - subject level the sensitivity and specificity of a new spm procedure compared to subjective visual inspection of fdg - uptake distribution maps and to the sole clinical information . a group of neuroimaging experts was individually asked to provide a forced diagnosis and indicate their level of confidence on the basis of a ) summary of clinical data , b ) visual inspection of standard fdg images , and c ) assessment of spm t - maps , in a large series of patients with clinical diagnosis of neurodegenerative disorders ( i.e. , ad , ftld and dlb ) , and in subjects with mild cognitive impairment mci . medical history and neurological examination the clinical information ( medical history , neurological examination and neuropsychological assessment ) was evaluated by three neurologist experts in dementia diagnosis ( am , sfc , gm ) that examined the whole medical records related to the entire clinical course of each subject in order to correctly assign the clinical diagnosis at the follow - up , which was used as gold - standard . according to the diagnosis at the clinical follow - up ( 27.6 4.1 months ; range 2235 months ) ( gold - standard ) , we identified patients fulfilling consensus criteria for ad ( jack et al . , functional normalization procedure allowed to place the images in the standard mni space using a new fdg - pet dementia - specific template for spatial normalization , based on images derived from both neurological patients ( sample of scans representative of the various forms of dementia in the population ) and age - matched controls , developed by della rosa et al . this template was used to spatially transform fdg - pet patient images to the mni ( montreal neurological institute ) reference space , as it has been shown to provide a higher degree of accuracy for spatial normalization of fdg - pet scans and a higher statistical sensitivity at the single - subject level in spm5 , useful for clinical purposes ( della rosa et al . the control database used for comparison in our new approach amounts to 112 images , which allows to use higher and more conservative significance thresholds at both the voxel ( p = 0.05 fwe - corrected ) and cluster level ( extent > 100 voxels ) and , even more importantly , to correct for multiple comparisons ( i.e. , the average of fdg - pet images from the 112 healthy control scans ) with voxels not meeting the optimality criterion set to zero ( masked ) . clusters of decreased metabolism were considered significant when they met a significance level of p = 0.05 , corrected for multiple comparisons with the family - wise error ( fwe ) option at the voxel level , and contained more than 100 voxels . nine neuroimaging experts with an extensive experience in the dementia diagnosis ( dp , ff , egv , ap , fn , sp , cc , vg , lg ) were presented with clinical and neuropsychological information ( clinical scenarios ) ( fig . 1a ) , standard clinical display of fdg - pet images ( standard fdg images ) ( fig . , visual assessment of standard fdg images and spm maps ) together with the clinical information . they were informed that the study included patients who had at follow - up a clinically confirmed diagnosis of ad , ftld spectrum ( including different subtypes ) , dlb , as well as mci subjects . each rater independently evaluated one type of information for each subject : a ) clinical scenarios ; or b ) standard fdg images ; or c ) spm maps . thus , the raters were presented with the standard fdg images or spm maps independently for any given subject and in both situations were blinded to any clinical data . the diagnostic labels assigned by each rater were compared to the clinical diagnosis at follow - up ( the gold standard ) . based solely upon the standard fdg images or the spm maps , raters were first asked to grade the level of overall scan abnormality as normal , uncertain or abnormal . the procedure for evaluation of the clinical scenarios , the standard fdg images and the spm maps was identical as those outlined above for step 1 . second , the rater could be presented with the standard fdg images or spm maps along with the clinical scenarios and was asked to evaluate the combination of both types of information , either clinical scenarios plus standard images or clinical scenarios plus spm maps , asking them for assessments of overall degree of scan normality , the presence of hypometabolism in more extensive or specific regions and of a potential asymmetrical pattern . finally , they were asked to make a forced diagnosis among the predefined disease categories added with their degree of confidence independently from the one indicated solely on the basis of the clinical information , thus maybe resulting in a potentially different diagnostic choice . the aim of step 2 was to assess the differential value of adding qualitative ( standard fdg images ) or quantitative ( spm maps ) information to clinical scenarios as a support for both diagnostic accuracy and confidence . clinical scenarios , standard fdg images and spm maps for steps 1 and 2 were sent independently to raters on separate dates ( i.e. the three types of information ( i.e. , clinical scenarios , standard fdg images , and spm maps ) were associated to each subject in a factorial manner and arranged according to six possible permutations ( i.e. , clinical scenariosstandard fdg imagesspm maps , clinical scenariosspm mapsstandard fdg images , standard fdg imagesclinical scenariosspm maps , standard fdg imagesspm mapsclinical scenarios , spm mapsclinical scenariosstandard fdg images , and spm mapsstandard fdg imagesclinical scenarios ) . this design was implemented in order to have the same amount of subjects rated for each of the three types of information , and to test agreement between different pairs of raters on the same information and between the same pair of raters on different types of information . overall rater consensus was also evaluated independently for each type of information by computing a contingency coefficient measuring the association between the follow - up diagnostic category and the rated disease category as indicated in the study . the diagnostic labels assigned by each rater were evaluated according to the clinical diagnosis at follow - up ( gold standard ) . in order to assess the diagnostic power of each type of information , sensitivity and specificity were computed independently for clinical scenarios , standard fdg images and spm maps information and expressed as percentages , averaged across the nine raters and weighted by the number of subjects evaluated by each rater for each type of information . moreover , for each type of information , we calculated both positive and negative likelihood ratios ( lr+ and lr ) , which provide the ratio between the probability of positive or negative test outcome in patients and the probability of positive or negative test outcome in true negatives . an average phi - value between rater pairs and a chi - transform was computed independently for clinical scenarios , standard fdg images and spm maps and tested for significance . the level of agreement based on the k - statistics was classified as fair ( k = 0.200.39 ) , moderate ( k = 0.400.59 ) , substantial ( k = 0.600.79 ) , and almost perfect ( k = 0.801.00 ) ( foster et al . diagnostic accuracy was also assessed for clinical scenarios , standard fdg images and spm maps taking in consideration the percentage of rated false negatives ( fn% ) for the two major dementia categories here represented ( i.e. three independent logistical models were fit to a categorical variable representing an ad or ftld diagnosis at follow - up , using as predictor variable only the correctly and concordantly classified ad or ftld subjects by the rater pair through clinical scenarios , standard fdg images or spm maps in order to estimate a concordance index for each type of information on the basis of diagnostic accuracy . predicted group classification probabilities were then used for roc curves corresponding to clinical scenarios , standard fdg images and spm maps in order to assess the average performance of the three types of information . clinical information combined with the standard fdg images ( clinical scenarios plus standard fdg image ) or spm maps ( clinical scenarios plus spm maps ) was evaluated for each subject . the randomized procedure for evaluation of the three types of information was identical for both steps 1 and 2 . in order to measure the level of agreement between the raters for clinical scenarios plus standard fdg images or clinical scenarios plus spm maps on consistency subjects , we computed a generalized kappa coefficient and used the z - statistic to test the null hypothesis , given the equal number of ratings . the two major dementia categories here represented , we assessed the strength of standard fdg images or spm maps in terms of correctly classifying those ad or ftld subjects , which were misclassified based solely upon clinical scenarios . , clinical scenarios plus standard fdg images or clinical scenarios plus spm maps ) , raters were also asked to specify the hypometabolic brain regions as well as the whole - brain distribution of the pattern ( i.e. in order to quantify the contribution of the identification of selected hypometabolic patterns through standard fdg images or spm maps to the correct classification of ad and ftld spectrum , we computed an index , expressed as the differential % of incidence ( di ) of localized hypometabolism in disease - specific areas between standard fdg images and spm maps for either ad or ftld . only the ad and ftld subjects the % difference between the number of times an area was rated as hypometabolic through standard fdg images and spm maps was calculated for each specific area weighted by the total number of correct classifications for either ad or ftld , independently for standard fdg images and spm maps . finally , loc for correctly evaluating mci was also compared between standard fdg images ( clinical scenarios plus standard fdg images ) or spm maps ( clinical scenarios plus spm maps ) . 2 and 3 analysis of raters ' performances for diagnostic accuracy indicated higher sensitivity and specificity values for spm maps ( 96% and 84% ) , compared to clinical scenarios ( 91% and 40% ) and standard fdg images ( 78% and 50% ) , considering the total sample of 88 subjects . diagnostic lr+ was better for spm maps ( 6.08 ) than for standard fdg images ( 1.55 ) or clinical scenarios ( 1.52 ) , exceeding the lr+ accuracy cut - off ( 5 ) only for spm maps . lr - values were best for spm maps ( 0.05 ) and very poor for standard fdg images ( 0.45 ) and clinical scenarios ( 0.22 ) , with only spm maps fell below lr - cut off ( 0.2 ) ( see fig . the chi - transforms of average phi - values measuring inter - rater agreement between each rater pair were higher for spm maps ( = 173.53 , p < 0.001 ) than for clinical scenarios ( = 126.04 , p = 0.01 ) and lowest for standard fdg images ( = 79.29 , p = 0.9 ) . the k - statistic measuring inter - rater reliability by subjects independently of the rater pair was substantial for spm maps ( k = 0.6 ) , moderate for clinical scenarios ( k = 0.4 ) and fair for standard fdg images ( k = 0.3 ) . the contingency coefficient ( c ) measuring general association between diagnosis at follow - up and rated category for all disease categories and overall rater consensus was higher for spm maps ( c = 0.722 , p < 0.0001 ) than for clinical scenarios ( c = 0.673 , p < 0.0001 ) or standard fdg images ( c = 0.678 , p < 0.0001 ) . when considering only ad and ftld , the average false - negative rate for spm maps was 0% , for clinical scenarios 2% and for standard fdg images 16% . logistic regressions tested whether spm maps is a more robust metric than clinical scenarios or standard fdg images in terms of both concordance and accuracy for the classification of ftld with respect to ad . it revealed that the spm maps model ( beta = 1.414 ; p = 0.019 ) correctly classified a significantly higher number of concordant subjects in the ftld group , compared to the clinical scenarios ( beta = 0.671 ; p = 0.291 ) or standard fdg images ( beta = 0.041 ; p = 0.945 ) , considering ad classification as the reference . roc curves measuring of the goodness - of - fit of the spm maps , clinical scenarios and standard fdg images models indicated that the auc was 0.67 for spm maps , 0.57 for clinical scenarios and 0.50 for standard fdg images . the asymptotic significance was inferior to 0.05 only for the spm maps model ( p = 0.039 ; clinical scenarios : p = 0.416 ; standard fdg images : p = 0.955 ) . the overall inter - rater diagnostic agreement based on a generalized kappa coefficient for consistently rated subjects was substantial for clinical scenarios plus spm maps combination ( k = 0.6 ; z = 10.57 , p < 0.0001 ) and only moderate for clinical scenarios plus standard fdg images ( k = 0.4 ; z = 5.99 , p < 0.0001 ) . viewing the clinical scenarios plus spm maps combination ( mean loc = 2.4 ) significantly increased the diagnostic confidence as compared to clinical scenarios plus standard fdg images ( mean loc = 2.07 ) ( p = 0.003 ) . in the case of the ftld spectrum only a trend was present in the same direction ( clinical scenarios plus spm maps mean loc = 2.26 ; clinical scenarios plus standard fdg images mean loc = 1.97 , p = 0.078 ) . in the subjects with a correct diagnosis of ad , raters found hypometabolism in the temporo - parietal ( di = + 26% ) and posterior cingulate regions ( di = + 38% ) more frequently with spm maps than standard fdg images . in the subjects with correct diagnosis of ftld , the anterior cingulate cortex ( di = + 16% ) , the medial frontal cortex ( di = + 12% ) and the superior anterior temporal cortex ( di = + 18% ) in the left hemisphere were identified as hypometabolic much more frequently with spm maps than with standard fdg images . compared to clinical scenarios plus standard fdg images , the effect of adding spm maps to clinical scenarios on ratings was significantly more beneficial for correctly classifying both positive mci who progressed and the negative mci category ( clinical scenarios plus spm maps mean loc = 2.46 ; clinical scenarios plus standard fdg images mean loc = 1.81 , p < 0.001 ) . the main result of this single - subject study is that the spm - based tool for the analysis of fdg - pet imaging improved diagnostic accuracy in dementia and pre - dementia conditions compared to visual inspection of fdg - uptake distribution , providing also additional value to clinical information . , 2014 ) analysis of performances showed very high sensitivity and specificity for spm maps ( 96% and 84% ) as compared to clinical scenarios ( 91% and 40% ) and standard fdg images ( 78% and 50% ) ( fig . independent measures of diagnostic accuracy ( lr+ and lr ) were better for spm maps than standard fdg images with only spm maps exceeding the proposed cut - off value for dementia diagnosis ( fig . noteworthy , spm maps showed higher inter - rater agreement compared to the other types of information . in the case of the differential diagnosis between ad and ftld , there is evidence of a high percentage of false - negative with visual inspection ( 16% ) . in particular , the roc area under the curve ( auc ) proves that spm - t maps classified ftld subtypes better than visual inspection , underlining the importance of voxel - based analysis for differential diagnosis , especially in the case of dementia subtypes with more heterogeneous genetic and pathological signatures , such as those within the ftld spectrum . noteworthy , viewing the spm maps in combination with clinical scenarios significantly increased diagnostic confidence as compared to the combination of clinical scenarios and standard fdg images . crucially , hypometabolism in the temporo - parietal and posterior cingulate for ad subjects , and the anterior cingulate , medial frontal and superior anterior temporal cortices for bvftd subjects was more frequently identified by spm maps than standard fdg images . nevertheless , the use of spm maps added a further value to clinical evaluation providing the clinician a higher degree of accuracy compared to standard fdg images . the results of raters ' performances on the mci group proved that spm maps allow identifying with significantly stronger confidence distinct patterns of hypometabolism underlying mci condition at the baseline , which predicted the further progression of cognitive decline to different dementia conditions at the clinical follow - up . within the different operating procedures for fdg - pet imaging , voxel - based analyses at a single - subject level are the most accurate , thus mandatorily calling for an objective statistical analysis of fdg - pet brain images that allows to reach the highest sensitivity and specificity ( perani et al . , 2013 ) , in this context , we showed that spm maps specifically improve fdg - pet imaging in dementias .

changes in the person - environment relationship as well as the negative outcomes of shrinkage in life space ( i.e. , the extent of mobility of older adults as measured by the range of places in which a person engages in activities within a designated time frame ) associated with aging , particularly among seniors with mobility limitations , have been long conceptualized and widely documented [ 13 ] . in fact , restricted life space has been recently linked to increased risk of alzheimer 's disease . older adults have been reported to spend 80 percent of their time in their primary residence and have demonstrated an environmental centralization of behaviors ( i.e. , the tendency of using a few preferred places at home where necessary or desired items are located ) to maintain control and competence over the living environment [ 2 , 6 ] . with almost 9 out of 10 ( 86% ) older americans reporting that they want to spend the rest of their lives in the homes and communities in which the majority of their daily activities take place , a robust life space is essential for older adults to continue to engage and participate in as many home and community activities as independently and safely as possible . while prior work has consistently linked supportive home and community settings to continued performance of home activities and participation in community roles , respectively , evidence suggests that community participation , which is dependent on maintaining a wide range of life spaces outside the home , may also be affected by one 's level of dependence and difficulty in performance of daily activities in the home . further , activity dependence and difficulty may be differentially affected by the same environmental features . to develop a more comprehensive understanding of the factors that affect performance or activities in the home and participation in the community among older adults with limitations in mobility , this paper will describe the relationships among ( 1 ) home environmental features and performance of routine activities in the home as measured by task dependence and difficulty , ( 2 ) home and community environmental features and opportunities for community participation as measured by the frequency of travel to community life space destinations , such as restaurants , grocery stores , doctor 's offices , and recreational areas , and ( 3 ) dependence and difficulty in home activities and opportunities for community participation ( see figure 1 ) . the theoretical bases of this study are derived from models of person - environment interaction , including two ecological models , the international classification of functioning , disability , and health ( icf ) and the environmental press model and the life space model [ 12 , 13 ] . the icf provides a model that defines performance and participation as the interaction between the context , including the physical environment , and an individual 's functional ability . based on these underlying principles , this study examined the association between environmental factors and performance at home and participation in the community as well as interactions between home performance and community participation . the second ecological model , the environmental press model , has long played a major role in defining environmental contributions to activity and participation . derived from the environmental press model , the environmental docility hypothesis suggests that the impact of demands is a function of an individual 's ability . in other words , individuals with less ability will be impacted more by the same environmental demands than individuals with greater levels of ability . based on the environmental docility hypothesis , this study examined the effects of environmental features on activity performance and community participation outcomes of older adults with and without mobility limitations . the life space questionnaire , as developed by stalvey et al . , illustrates the movement trajectory of older adults in nine environmental zones from the bedroom , immediately outside the home ( e.g. , porch / patio ) , outside the home ( e.g. , yard or parking ) , immediate neighborhood , outside immediate neighborhood , outside the town , outside the county , outside the state , to outside the country . due to the increased time spent inside home and decreased participation in the community among older adults , this paper used a simplified version of the life space concept that focuses on older adults ' ability to move from inside their homes to life spaces in the community ( including both immediate neighborhood and outside immediate neighborhood ) . as a result , use of community life space reflects the potential for community participation . this dichotomization from home to community spaces is critical as increasingly accessing community spaces provides and enhances opportunities for continued participation in societal roles . most previous studies of environmental supports for aging in place have separately examined associations between either the home environment and activity performance or the community environments and participation . as a result , a comprehensive understanding of the interrelationships among home and community environments , performance , and participation is still lacking . in home settings , unsupportive home features have been linked to greater difficulty and dependence in daily household activities [ 10 , 16 , 17 ] . in contrast , the provision of supportive environmental features , such as grab bars and home modifications , has been shown to enhance independence in activities , reduce caregiver burden , and decrease home care costs [ 1820 ] . most of these studies tend to associate the number of barriers / facilitators , a global score of environmental misfit [ 16 , 17 ] , or broad domains of home barriers / facilitators ( e.g. , overall bathroom barriers ) [ 18 , 19 ] to performance outcomes rather than linking specific home features ( e.g. , bathtub space or bathtub height ) to specific performance outcomes ( e.g. , getting in and out of tub ) . as a result , these studies generally do not depict the differential impacts of specific home environmental features on explicit performance outcomes . one of the few exceptions was a study that described correlations between home facilitators ( i.e. , home modifications ) with both activity independence , and ease of performance in 15 home tasks by individuals with mobility impairments . however , none of the factors , home environmental features , activity independence or ease of performance were linked to community participation . in public settings , physical environmental factors , such as mixed land uses , highly connected street networks , availability ( e.g. , number and types ) of stores and services , pedestrian - friendly streets and sidewalks , neighborhood attractiveness , and transportation , were linked not only to engagement in activity ( e.g. , exercise or walking to community destinations ) of older adults , but also to their propensity to participate in society [ 21 , 22 ] . conversely , a study that examined the impact of specific environmental factors on activity and participation of seniors who used wheeled mobility aids reported that among the 50 factors examined , including 17 sidewalks , 17 crossings , 10 curb ramps , and 6 ramp characteristics , all 50 significantly prevented the 95 percentile of older wheelchair population from going out into the community , thus restricting opportunities for participation when those barriers were present . yet , other studies have found that the overall impact of the environment on participation was smaller than expected [ 2427 ] . while mobility and balance ( as opposed to other personal factors ) explained 24% of participation in one study and activity limitations explained much of community participation in another , in a third study , community environments , such as governmental and public services and physical environment and accessibility , only accounted for 6% of the variance in participation . these data suggest that perhaps other factors , such as environmental factors in the home , may play a key role in community participation . in contrast to studies that focused on community features alone , haak et al . reported that a continuum of home to community features was significantly correlated with participation . however , while the study examined the impact of mostly social environmental supports in the community , such as good medical care in the vicinity , living close to friends and relatives , cultural opportunities in the vicinity , and having good local transport , specific physical environmental factors were not included . moreover , although physical barriers in the home environments were included , these were aggregated to a number of environmental barriers and magnitude of accessibility problems . as a result , the impact of specific features on participation could not be assessed . to date , only one study has examined the interaction between home environmental factors and community participation . in a pre - post study prior to and after receiving home modifications for getting in and out of the house , moving around the house , and using the bathroom , hammel et al . examined older adults ' ability to use a range of life spaces within and outside the home when they wanted and with whom they wanted . after receiving home modifications , participants not only reported an increased use of community life spaces , but they also used more distant life spaces . most importantly , among all of the types of home modifications made , toilet and bathtub modifications , even more so than ramps and lifts for getting in and out of the house , demonstrated the largest effect on going out into the community . recognizing the complex interactions among environments , home activity performance , and community participation and the potential impacts on the ability of older georgians to successfully age in place , the georgia council on aging , which serves in an advisory capacity on aging issues to the governor and general assembly of georgia , supported a survey to identify and prioritize the environmental and performance correlates of unmet home activity and community participation needs of georgia 's seniors . the goal of the survey was to develop a comprehensive understanding of home and community environmental barriers and facilitators that impact the activity and participation of georgia 's seniors to inform policy and prioritize service delivery needs for the state of georgia . in addition , the data are useful in developing a more comprehensive understanding of community participation potential and life space restriction as a function of the interrelationships among home and community environmental features and home activity performance . the data reported here described relationships between the home setting ( i.e. , environmental features and activity performance ) and community participation potential of georgia 's seniors with mobility limitations compared to those without mobility limitations . mobility limitation was selected as a subset of interest because this group is more likely to experience more environmental barriers and life space restriction than older adults with other limitations ( i.e. , hearing , vision , speech , and dexterity ) , but without mobility limitations . specifically , the paper will address three key research questions by describing the associations among ( 1 ) home environmental features ( i.e. , barriers and facilitators ) and activity performance in the home as measured by dependence and difficulty in home activities of seniors with mobility limitations compared to those with other limitations , ( 2 ) home and community environmental features and community participation potential as measured by usage of community life space ( i.e. , the frequency of going into community destinations , such as restaurants , grocery stores , doctor 's offices , and recreational areas among older adults with mobility limitations compared those with other limitations ) of seniors with mobility limitations compared to those with other limitations , and ( 3 ) home activity ( i.e. , dependence and difficulty ) and community participation potential of seniors with mobility limitations compared to those with other limitations ( see table 1 ) . the study employed a cross - sectional survey design to explore the relationships among environmental features , dependence and difficulty in activity performance in the home , and life space usage in the community participation to understand the met and unmet activity and participation needs of older georgians . a web - based survey hosted by survey gizmo survey gizmo was chosen because it is compliant with section 508 of the rehabilitation act and is generally the most accessible and usable online survey platform . in addition , it utilizes an encrypted connection to ensure confidentiality of data . to ensure inclusion of older adults who did not have access to an online platform , alternative a total of 239 individuals with and without functional limitations who were 60 + , living in the state of georgia and had resided in their current residence for at least one year , were recruited for the survey . seven out of 179 online surveys and 6 out of 54 written surveys were eliminated due to large amounts of missing data , resulting in a total of 226 participants in the study . participants were asked to indicate whether they experienced one or more of five functional limitations , including vision , hearing , speaking , moving around , and hand manipulation . among the total of 226 participants , 122 had at least one of the five functional limitations . the 63 respondents who answered yes to the question : the 59 participants who responded with a yes to any of the other 4 limitations were included in the other limitation ( ol ) group . it should be noted that because participants could respond to more than one limitation , the ol group included 52 individuals who had vision , 60 who had hearing , 26 who had hand manipulation , and 4 who had speaking limitations . a convenience sampling technique was employed in order to reach the required sample of 200 participants to achieve a statistical power of 80 . participants were recruited through a variety of methods , including subject registries maintained by the center for assistive technology and environmental access at georgia tech , as well as through email invitations and posts at aaas , aarp , norcs , senior centers , and other senior - related organizations throughout georgia . both email invitations and posts were provided with a brief study description and researchers ' contact information so that potential subjects who were interested in the study could actively contact the researchers by phone or email . all subjects that expressed an interest in the study were given a more detailed study description and a written informed consent form . those who consented to participate were given the choice of taking an on - line , written , or telephone survey . those who chose an on - line method ( n = 179 ) were emailed with the link to the on - line survey . hardcopy , text versions of the survey ( n = 54 ) were mailed out with a self - addressed stamped envelope . the telephone survey ( n = 6 ) was scheduled with the participant at a time that was mutually agreeable . the survey took approximately 20 minutes online and 3040 minutes in a written or telephone format . the survey gathered self - perceive information on a variety of factors , including ( 1 ) activity performance , ( 2 ) community participation potential , ( 3 ) environmental barriers and facilitators , and ( 4 ) participant demographics . all survey questions were answered by participants without assistance from the researchers . based on a comprehensive review of literature , the survey borrowed from and adapted questions from a number of existing instruments , including comprehensive assessment and solution process for aging residents ( caspar ) , the healthy aging research network ( han ) environmental audit tool and protocol , the facilitators and barriers survey of environmental influences on participation among people with lower limb mobility impairments and limitations ( fabs / m ) , and the participation survey : mobility ( parts / m ) . activity performance and environmental barriers / facilitators in the home were adapted from the caspar . caspar was chosen because it associates demand - producing environmental attributes ( which could be barriers or facilitators ) with actual activity performance [ 28 , 32 ] . in contrast , other existing home assessment instruments that compare environmental attributes to performance , such as the housing enabler , focus on environmental barriers and not assess actual performance . rather , activity performance ( e.g. , can not go up and down the ramp to get in and out of the house ) is predicted from a comparison of environmental attributes that are expected to be barriers ( e.g. , ramp slope ) to an individual with functional limitations that interact with those attributes ( e.g. , lower body motor limitation ) . this approach is useful in informing decisions about home modification needs when actual performance can not be observed , such as prior to an individual 's discharge from a rehabilitation facility , but because it only predicts performance that may or may not actually occur , it may result in false positives as well as underestimating problems . as a result , these types of predictive assessments did not provide sufficient information to make decisions about environmental modification needs . caspar , in contrast , which associates environmental attributes against actual activity performance , results in information that could be used by the georgia council on aging to determine actual environmental modification needs . the caspar includes self - reported information on functional abilities , types of performance problems with person - environmental transactions , such as getting on and off toilet , and detailed measures of activity - relevant environmental attributes of the home , such as height and location of toilet . the sections of performance and environmental attributes were utilized and adapted for the current study . for example , in the environmental section , direct measurements of home attributes such as bathtub dimensions were omitted as provision of modification intervention was not the intent of this study . instead , perceived impacts of environmental attributes on matching task performance were surveyed . that is , participants were asked to rate on a 5-point likert scale from limits a lot to helps a lot to be consistent with the response system in the measure of community environment . in addition , to shorten the time required for survey administration , the number of home environmental features in caspar was reduced from features in eight activity areas ( i.e. , getting in and out of the house , using interior stairs , moving around the house , using the bathroom , using the bedroom , using the kitchen , using the laundry , and controlling ambient conditions ) to those in four activity areas that were considered to be the most crucial for daily home activities ( i.e. , getting into and around the home , using the bathroom , using the kitchen , and using the bedroom ) . interrater reliability and criterion validity of caspar were moderate to high on the majority of items . measures of community environmental barriers / facilitators were adapted from the healthy aging research network ( han ) environmental audit tool and protocol and the facilitators and barriers survey ( fabs / m ) of environmental influences on participation among people with lower limb mobility impairments and limitations . the han environmental audit tool and protocol was designed for research purposes and developed through both qualitative interviews and quantitative reliability testing at multiple sites . this tool was chosen because it covers both physical and social attributes in the community . the total number of attributes in the original tool was 55 , and they were grouped by the study researchers into a final list of 7 community features ( i.e. , stores , streets , sidewalks , visual appeal , public transit , and destination physical and social support ) with descriptions of the original attributes under each of the seven categories in order to reduce survey burden placed on the participants . in addition , because the han environmental audit tool used both ordinal and categorical data , it did not lend itself to the ordinal scoring system needed to measure the magnitude of environmental features as barriers or facilitators to performance . as a result , the ordinal response options in the fabs / m were adopted to measure person - environmental transactions ( i.e. , impact of community features and attributes on corresponding community activities or behaviors ) . the response options utilized a 5-point likert scale from limits a lot to helps a lot . the fabs / m is a widely used measurement on community environments with sound psychometric properties ; however , as the fabs / m was not originally developed for the older population , its survey questions could not fully capture barriers and facilitators encountered by older adults . finally , the participation survey : mobility ( parts / m ) was used to develop measures of community participation potential . the parts / m was developed based on the international classification of functioning , disability , and health ( icf ) and had good internal consistency and stability . the parts / m measures the frequency of traveling to various community settings ( e.g. , restaurants , bank , doctor , and grocery ) as an indicator of the potential for participation . in other words , travel to community destinations is a perquisite to participation in societal roles . the more frequently individuals traveled to community destinations , the greater the likelihood that they would participate in societal roles . conversely , the less often they traveled to community destinations , the fewer opportunities they would have to participate in societal roles . independent variables included both environmental features rated as either barriers or facilitators and functional limitations . environmental features included 17 features ( e.g. , steps , toilets , kitchen appliances , and bedroom closets ) in four areas of the home ( i.e. , circulation , bathroom , kitchen , and bedroom ) and 7 features in the community ( i.e. , stores , streets , sidewalks , visual appeal , public transit , and destination physical and social support ) . the degree to which any environmental feature was perceived to be a barrier or facilitator was defined by the perceived level of support , on a 5-point likert scale from 1 = helps a lot to 5 = limits a lot , that was afforded by any particular feature . functional limitations were divided into two groups : mobility limitation group ( ml ) as defined by difficulty moving around and other limitations group ( ol ) , as defined by having difficulty with vision , hearing , speaking , and/or hand manipulation , were used as the other independent variable . each of the limitations was measured dichotomously ( i.e. , with or without a specific functional limitation ) . activity performance was measured by activity independence / dependence as well as ease / difficulty . activity independence / dependence was defined as needing personal assistance while performing an activity , regardless of the use of assistive technology . activity dependence was reported by subjects on a 3-point likert scale from 1 = independent , 2 = dependent , to 3 = unable to perform the activity . activity ease / difficulty was defined as self - reported ease or difficulty in performing each activity in the usual way ( i.e. , with or without assistance of another person ) . four levels of perceived difficulty from 1 = no difficulty , 2 = somewhat difficult , 3 = very difficult , to 4 = unable to perform the activity were assessed . both activity independence and difficulty address routine performance , that is , actual performance rather capacity to perform . the activities queried in the survey were adapted from the caspar , including three circulation tasks ( getting in and out of the house , going up and down interior stairs , and moving around inside the house ) , two tasks for using the bathroom ( getting on and off a toilet , getting in and out of a bathtub or shower ) , three for using the kitchen ( using kitchen appliances , getting items in and out of upper cabinets , and getting items in and out of lower cabinets ) , and the two for using the bedroom ( getting on and off a bed and using the closet ) . participation potential , adopted from the parts / m , was defined as self - reported frequency of actual community participation . it was assessed by one question , how often do you actually go into destinations ( such as restaurants , banks , churches , and recreational areas ) in your community ? on six levels of frequency from daily , several times a week , several times a month , less than once a month , and do not participate in the community . demographic data were used to describe the study sample , including age ( i.e. , year born ) , gender , ethnicity ( i.e. , white / caucasian , african american , hispanic or latino , asian , native american / alaskan native , native hawaiian / other pacific islander , and other ) , education levels ( i.e. , no high school , some high school , high school diploma / ged , associate degree , bachelor 's degree , master 's degree , and doctorate degree ) , community types ( i.e. , urban , suburban , and rural areas ) , mobility aids ( i.e. , cane , crutch , walker , manual wheelchair , power wheelchair , and scooter ) and sensory devices ( i.e. , hearing aids and glasses ) . data from the online survey ( n = 172 ) were automatically entered into an online database . data from the written ( n = 48 ) and telephone survey ( n = 6 ) were hand entered . spearman rho correlations were conducted for all three research questions , that is , to associate ( 1 ) home environmental features to independence and to difficulty of home activities , ( 2 ) home and community environmental features to participation potential , and ( 3 ) independence and difficulty of home activities to participation potential . since matched sets of activity and activity - related environmental features ( e.g. , getting on / off toilet and toilet space and toilet ) were used in research question 1 , stepwise regressions were only conducted for research questions 2 and 3 . four stepwise regressions were further conducted to individually identify which ( 1 ) home and ( 2 ) community environmental feature that explain community participation for research question 2 , and ( 3 ) independence and ( 4 ) difficulty in which home activities explain community participation for research question 3 . cohen effect size conventions of small = 0.10 , medium = 0.30 , and large = 0.50 were used for both correlation and regression analyses . both moderate and large descriptive comparisons of all independent and outcome variables between the mobility and other limitation groups were all conducted by chi - square analyses . due to an exploratory and descriptive nature of this paper , results were considered to be significant at p < .05 . because the analysis of this data employed multiple independent analyses , uncorrected significance tests are not appropriate for inferential interpretation . however , significance is reported here with uncorrected p values to be interpreted as an arbitrary criterion of effect size strength in deference to its widespread use in social science for exploratory analyses . a total of 122 participants met the criteria of having functional limitations and were included in this analysis . among these , the sample was approximately equally divided between the ml ( n = 63 , 51.6% ) and ol groups ( n = 59 , 48.4% ) . more than 4 out of 10 ( 41% ) did not use mobility aids , while an almost equivalent percentage ( 38% ) used a cane . only 18% used walkers , 8% used power wheelchairs , 3% used manual wheelchairs , and 2% each used crutches and scooters . almost two - thirds ( 64% ) of the ol group had a hearing limitation ( see table 2 ) . = 8.50 ) with the ml group being 71.2 years of age and the ol group being slightly older at 73.9 years of age , although the difference was not significant . the majority of the respondents was female ( 64% ) , caucasian ( 74% ) , and living in suburban ( 51% ) areas ( see table 2 ) . in addition , almost one - third ( 32% ) had an associate or bachelor degree . there were no significant differences in race or residence between the ml and ol groups ( 73% and 74% caucasian ; 50% and 51% living in suburban areas , resp . ) ( p < .01 ) in gender ( 73% female in the ml group versus 54% in the ol group ) . overall , only approximately one in five respondents perceived barriers in either the home ( n = 18 , 14.8% ) or in the community ( n = 26 , 21.3% ) . the most common home barriers reported by the whole sample included kitchen cabinets ( 24.8% ) , bathtubs or showers ( 23.5% ) , bedroom closets ( 23.5% ) , and steps ( 19.2% ) . the most common community barriers were streets ( 28.0% ) , sidewalks ( 28.0% ) , and number and of stores ( 23.5% ) . conversely , the bathroom sink was the feature perceived by the lowest percentage ( 8.4% ) of the whole sample in the home , whereas social environments in community destinations were perceived by the lowest percentage of respondents ( 14.4% ) as a barrier in the community . similar to the whole sample , both ml and ol groups perceived more barriers in the community than in the home . although the ml group perceived more barriers in each of the settings with 29% perceiving community barriers to 18% in the ol group and 17% perceiving home barriers to 12% in the ol group , neither was statistically significant . the home and community barriers cited most often by the largest percentage of the ml and ol groups were also similar to the whole sample . however , all home and community features were perceived as barriers by higher percentages in the ml group than the ol group with the exception of public transportation , which had equal percentages in both groups . however , among these features , only steps ( p < .05 ) and kitchen cabinets ( p < .05 ) in the home and the physical environment in community destinations ( p < .01 ) were significantly higher in the ml than the ol group ( see table 3 ) . dependence in each of the ten home activities ranged from 4.3% to 33.6% for the overall sample , with the largest percentage of respondents ( 33.6% ) being dependent in getting items in and out of upper cabinets in the kitchen and the smallest percentage of respondents being dependent in getting on and off a toilet ( 4.3% ) . a significantly higher percentage of the ml group reported being more dependent than the ol group in eight of the ten home activities ( p = .000.038 ) . moreover , the trend continued with a higher frequency of respondents in the ml group reporting greater dependence in the other two activities , moving around inside house and getting items in and out of a closet , although the differences between groups were not significant . compared to activity dependence , higher percentages of the overall study sample reported having difficulty with the 10 activities ranging from 13% to 53.0% . the largest percentage of respondents had difficulty going up and down stairs ( 53% ) , whereas the smallest percentage had difficulty getting on and off a bed ( 13% ) . similar to activity dependence , higher percentages of the ml group reported having difficulty in all ten home activities , although in this case , all activities were significantly ( p = .000.022 ) more difficult in the ml than the ol group ( see table 4 ) . in general , older adults in the study sample were generally active . almost three - quarters ( n = 88 , 72.5% ) of the overall study sample went into community at least several times a week ( n = 48 , 39.7% ) or everyday ( n = 40 , 32.8% ) . despite the large number of participants who were active , more than one - quarter demonstrated restricted life space by traveling to community destinations less than weekly ( 18.1% , n = 22 ) or once a month or less ( 9.5% , n = 12 ) . when the ml and ol groups were compared , as expected , a lower percentage of the ml group participated in the community everyday ( 28.6% ) compared to the ol group ( 37.7% ) , although the differences were not statistically significant . the differences in community participation between the ml and ol groups were most evident among the least active community participants , with a trend ( p = .054 ) toward more participants in the ml group ( 14.3% ) demonstrating life space restriction ( i.e. , going into community every month or less ) than the ol group ( 3.8% ) . rq1 : what is the relationship between home environmental features and activity performance in the home?while almost none of the home features were significantly correlated with activity performance in the ol group , over half of the home barriers were significantly correlated with either activity dependence or difficulty in the ml group . more than three - quarters ( 76.6% ) of the 17 home barriers were significantly correlated with activity difficulty , while 58.8% ( n = 10 ) were significantly correlated with dependence . while almost none of the home features were significantly correlated with activity performance in the ol group , over half of the home barriers more than three - quarters ( 76.6% ) of the 17 home barriers were significantly correlated with activity difficulty , while 58.8% ( n = 10 ) were significantly correlated with dependence . among the features in the four home spaces included in the study , all features in the kitchen , including kitchen space , appliances , and cabinets , positively ( r = 268.627 ) and significantly ( p < .001<.05 ) correlated with both difficulty and dependence in performing the corresponding activities ( i.e. , using kitchen appliances , and getting items in / out of upper cabinets , getting items in / out of lower drawers ) . two of the circulation features , steps and going up and down stairs , were significantly correlated with both the dependence and difficulty in going up and down stairs and moving around the house , respectively . steps were positively and significantly correlated to dependence ( r = .520 ; p < .001 ) , and difficulty ( r = .303 ; p < .05 ) in going up and down stairs , whereas home space barriers were positively and significantly correlated with dependence ( r = .377 , p < .01 ) and difficulty ( r = .364 , p < the other four circulation features were correlated with either dependence ( i.e. , walkway and doorway ) or difficulty ( i.e. , pathway and door ) with r values ranging from r = .276.434 and significance ranging from p < .001 to p < .05 . in the bathroom , toilet features barriers were significantly correlated with both dependence ( r = .327 ; p < .01 ) and difficulty ( r = .268 ; p < .05 ) in getting on and off toilet ; however , tub / shower features were only significantly correlated ( r = .257 ; p < .05 ) with difficulty in getting in and out of bathtub / shower . interestingly , neither space at the toilet nor at the tub / shower was significantly correlated with dependence or difficulty in getting on or off the toilet or in and out of the shower . finally , in the bedroom , closet features were significantly ( r = .503 ; p < .001 ) correlated with both dependence and difficulty in getting items in and out of a bedroom closet . the other two bedroom features , bedroom space ( r = .393 , p < .01 ) and bed ( r = .468 , p < .001 ) , were significantly correlated with activity difficulty ( see table 5 ) . in the ol group , only three home features , kitchen cabinets , bed , and steps , were significantly correlated with activity performance at home . two features , kitchen cabinets and bed , were positively correlated with difficulty in getting items in and out of lower drawers ( r = .292 ; p < .05 ) and difficulty getting out of bed ( r = .316 ; p < .05 ) , respectively . in contrast , two home features , kitchen cabinets and steps , were negatively correlated with activity . kitchen cabinets were significantly correlated ( r = .342 ; p < .05 ) with getting items in and out of upper cabinets , whereas steps were significantly correlated ( r = .355 ; p < .05 ) with independence in going up and down stairs . rq2:what is the relationship between home and community environmental features and potential for community participation?whereas community environmental features were significantly correlated to frequency of travel to community destinations in the ml group , they were not significantly correlated in the ol group . although no environmental features in the home were significantly correlated with frequency of travel to community destinations in either the ml or ol groups , among the 7 community features , three , including streets ( r = .294 ; p < .05 ) , sidewalks ( r = .283 ; p < .05 ) , and social environments of community destinations ( r = .346 ; p < .01 ) , were significantly correlated with frequency of going into community in the ml group . whereas community environmental features were significantly correlated to frequency of travel to community destinations in the ml group , they were not significantly correlated in the ol group . although no environmental features in the home were significantly correlated with frequency of travel to community destinations in either the ml or ol groups , among the 7 community features , three , including streets ( r = .294 ; p < .05 ) , sidewalks ( r = .283 ; p < .05 ) , and social environments of community destinations ( r = .346 ; p < .01 ) , were significantly correlated with frequency of going into community in the ml group . stepwise regressions were undertaken to further identify home and community features that explained travel frequency among the ml group . results indicate that bathtub / shower was the only home feature that explains any significant amount , although slightly less than 6% ( adjusted r = .055 ; p < .05 ) of travel frequency . in the community , social environments at a destination was the only feature that accounts for a significant amount of variance ( adjusted r = .130 ; p < .01 ) . however , when frequency of travel to community destinations is dichotomized into frequent travelers ( i.e. , more than once a month ) and infrequent travelers ( once a month and less ) , frequency was significantly correlated with the majority of both home ( 64.7% , n = 11 ) and community ( 71.4% , n = 5 ) features in the ml group . among home features , kitchen and bathroom features had the highest percentage of features that were significantly correlated with infrequency of travel to community destinations in the ml group , including four out of five ( 80.0% ) bathroom features ( r = .289.401 ; p < .01 to p < .05 ) and all three ( 100.0% ) kitchen features ( r = .252.301 , p < .05 ) . among community features , stores , sidewalks , visual appeal , physical environments , and social environments were significantly correlated ( r = .268.431 , p < .001 to p < .05 ) with infrequent travel in the ml group ( see table 6 ) . in contrast , no home or community features were significantly correlated with dichotomized frequency of travel in the ol group . in addition to correlations between environmental features and dichotomized participation , odds ratios were calculated for dichotomized environmental features ( i.e. , barriers / facilitators ) and dichotomized participation ( i.e. , frequent / infrequent travel ) . in the home setting , all bathroom features , with the exception of bathroom sinks , had significant odds ratio results ( i.e. , the upper and lower ci95% did not overlap 1.00 ) in the ml group , while there were no significant results in the ol group . when toilet space , toilet , tub / shower space , and tub / shower were perceived as barriers , the odds of infrequent travel were 46.7 , 25.0 , 29.0 , and 8.0 times higher , respectively , compared to when they were perceived as facilitators . among community features , the odds of infrequent community travel were 17.8 times higher in the ml group when sidewalks were perceived as barriers and 21.3 times higher when social environments at the destination were perceived as barriers . similarly , there were no significant odds ratios in the ol group in community settings . rq3 : what is the relationship between activity performance in the home and community participation potential?as in other analyses , significant correlations were only found in the ml group . again , in the ml group , there were a greater number of significant correlations between frequency of travel to community destinations and both kitchen and bathroom activities than circulation activities . there were no significant correlations between bedroom activities and frequency of travel to community destinations ( see table 7 ) . again , in the ml group , there were a greater number of significant correlations between frequency of travel to community destinations and both kitchen and bathroom activities than circulation activities . there were no significant correlations between bedroom activities and frequency of travel to community destinations ( see table 7 ) . comparing kitchen and bathroom activities , dependence of all kitchen activities and difficulty in all bathroom activities dependence in using kitchen appliances , getting items in and out of upper cabinets and of lower drawers were significantly correlated with less community participation ( r = .272.417 ; p < .01 to p < .05 ) . among these three activities , getting items in and out of upper cabinets showed the strongest correlation ( r = .417 ) . in the bathroom , difficulty in getting on and off a toilet and getting in and out of a bathtub / shower were significantly correlated with less frequent travel to community destinations ( r = .259 and .438 , p < .05 and p < .001 , resp . ) . in circulation - related activities , both dependence in getting in and out of the house and going up and down stairs , as well as difficulty in getting in and out of the house , is significantly correlated ( r = .406.463 ; p < .001 to p < .01 ) with less frequent travel to community destinations . to identify the home activities that account for the largest variance in frequency of travel to community destinations getting items in and out of upper cabinets and getting in and out of house explained approximately one - fourth ( adjusted r = .246 ; p < .001 ) of the variance in travel frequency . for activity difficulty , getting in and out of a bathtub / shower is the only significant activity in the model , accounting for almost one - third of frequency of going into community ( adjusted r = . 306 ; this study described relationships among home and community environmental features , dependence and difficulty in activity performance at home , and frequency of travel to community life space as an indicator of participation potential . specifically , the study demonstrated that home environmental features were not only significantly associated with activity difficulty and dependence in the home , but also with less restriction in life space . in fact , the latter was positively related to home features and home performance as well as community environmental features . these results clearly demonstrated our primary hypothesis that remotely located home environmental features and activity performance can impact community participation . as expected , correlations were only significant among the ml group . however , this group also had lower performance and participation outcomes , which supports findings by anaby and colleagues that mobility and balance , more so than any other limitations , account for the largest variance in performance and participation . the lower performance and participation outcomes as well as the stronger link between environment , performance , and participation not only indicate the higher vulnerability in people with mobility limitations to age in place , but also postulate that both performance and environmental intervention are a potentially important strategies to facilitate aging in place . rq1 : home features and activity performance at homeprevious studies have identified top barriers to activity performance at home as well as environmental features to reduce dependence and difficulty [ 10 , 1620 ] . this study not only provides further evidence that home features impact difficulty and dependence among mobility impaired seniors , but also suggests that features can be either barriers or facilitators . this is not surprising as research and practice suggest that kitchen features are low - priority modifications as they are expensive , and kitchen activities are perceived to be easily substituted or skipped without impacting daily functions greatly , particularly in comparison to more critical bathroom and entry / exit modifications . as a result , the data suggest that many of the participants had modification needs in the kitchen . surprisingly , bathroom features ( i.e. , tubs , showers , and toilets ) , rather than space , were significantly correlated to activity performance , which perhaps reflects the vast majority ( almost 90% ) of the sample that was ambulatory . since maneuvering space is a factor that primarily affects wheelchair users , the results may be different if the sample had a larger number of wheelchair users . previous studies have identified top barriers to activity performance at home as well as environmental features to reduce dependence and difficulty [ 10 , 1620 ] . this study not only provides further evidence that home features impact difficulty and dependence among mobility impaired seniors , but also suggests that features can be either barriers or facilitators . this is not surprising as research and practice suggest that kitchen features are low - priority modifications as they are expensive , and kitchen activities are perceived to be easily substituted or skipped without impacting daily functions greatly , particularly in comparison to more critical bathroom and entry / exit modifications . as a result , the data suggest that many of the participants had modification needs in the kitchen . surprisingly , bathroom features ( i.e. , tubs , showers , and toilets ) , rather than space , were significantly correlated to activity performance , which perhaps reflects the vast majority ( almost 90% ) of the sample that was ambulatory . since maneuvering space is a factor that primarily affects wheelchair users , the results may be different if the sample had a larger number of wheelchair users . rq2 : environmental features and community participation potentialnot surprisingly , community features were more strongly related with overall community travel than home features . nonetheless , like their impact on home activity , the majority of bathroom and kitchen features were also significantly related to infrequent community travel . this was particularly true of bathtub or shower design ( e.g. , size of bathtub or shower , height of bathtub edge , or shower threshold ) which accounted for a significant amount of the variance in travel frequency . moreover , when the four toilet and bathing features , toilet space , toilet design , tub / shower space , and tub / shower design , were perceived as barriers , respondents were 8 to 46.7 times more likely to travel into the community only once a month or less . not surprisingly , community features were more strongly related with overall community travel than home features . nonetheless , like their impact on home activity , the majority of bathroom and kitchen features were also significantly related to infrequent community travel . this was particularly true of bathtub or shower design ( e.g. , size of bathtub or shower , height of bathtub edge , or shower threshold ) which accounted for a significant amount of the variance in travel frequency . moreover , when the four toilet and bathing features , toilet space , toilet design , tub / shower space , and tub / shower design , were perceived as barriers , respondents were 8 to 46.7 times more likely to travel into the community only once a month or less . these findings are consistent with a previous study , which reported large effect sizes of toilet and bathing interventions on community travel . one possible explanation is the toilet and bathtub create such significant barriers such that the amount of time and energy required to toilet and bathe limits the amount of time that can be spent in the community . alternatively , people may feel that the barriers limit their personal hygiene activities and are therefore less willing to travel into the community . in addition to home environmental features , the social environment at community destinations , such as staff 's willingness to offer assistance in a restaurant , not only showed the strongest correlation among all community features , but also it was the only community feature that attained significance in explaining the variance in community travel frequency . social environment at community destinations also shows stronger odds ratio to community participation between the only two significant community features . however , together bathtub / shower and destination social environments only explained little ( 5.5% to13% ) of community travel . such findings are consistent with previous literature , in which community accessibility only accounted for 6% of the variances in participation . our finding of positive correlations between home barriers and less community participation was also consistent with results from haak and colleagues that significant correlations between the number of home barriers and community participation were reported . despite this , our findings identified specific home and community features that were significantly correlated to participation , which suggests a potential direction for further research , if not environmental intervention . rq3 : activity performance at home and community participation potentialsimilar to home barriers , performance challenges at home were positively correlated to less community participation , especially in kitchen , bathroom , and circulation - related activities . dependence in getting items in and out of upper kitchen cabinets and getting in and out of the house explained 24.6% of community travel patterns . the former was significantly related to barriers of upper cabinets in the kitchen , including height of cabinets and handle styles , and it entails the likelihood of a positive association between decreased mobility / balance function and increased difficulty in community participation . the latter was significantly related to barriers of walkways such as physical condition or material of the street , driveway , and lighting , and it points out the importance of achieving independence in getting in and out of the house because assistance can not be always handy . similar to home barriers , performance challenges at home were positively correlated to less community participation , especially in kitchen , bathroom , and circulation - related activities . dependence in getting items in and out of upper kitchen cabinets and getting in and out of the house explained 24.6% of community travel patterns . the former was significantly related to barriers of upper cabinets in the kitchen , including height of cabinets and handle styles , and it entails the likelihood of a positive association between decreased mobility / balance function and increased difficulty in community participation . the latter was significantly related to barriers of walkways such as physical condition or material of the street , driveway , and lighting , and it points out the importance of achieving independence in getting in and out of the house because assistance can not be always handy . in addition to dependence at home , difficulty in getting in and out of bathtub or shower also explained 30.5% of community travel . one possible explanation is the increased time and energy spent in the bathroom that reduces the time and energy available for going out into the community . difficulty in getting in and out of the tub or shower was significantly correlated to barriers in the tub or shower , which also corresponds to predictors of home barriers to community participation . while previous research has shown the strong contribution of activity limitation to participation , our findings further described the type of home activity as well as the kind of performance indicator related to community participation . this study provides the first step to comprehensively understand the relationships between home and community environments , home performance , and community participation as they impact aging in place . however , the study was limited by a number of factors including a small sample of convenience , which resulted in small effect sizes on many correlation results , and , ultimately limited generalizability of the data . the sample itself was an artifact of the time frame and funding allocated to the project , which limited the sample size and the data collection options . although many of the correlations in the results had small to medium effect sizes , the prediction of difficulty in using the tub / shower to community participation achieved a moderate to large effect size . further studies should also include and control for covariates , such as functional level and living situation , in the examination of the environment and performance predictors for aging in place . however , despite the limitations , to the authors ' knowledge , this is one of the first studies that provides a more robust and comprehensive understanding of the impact of home and community environmental factors on home activity performance as well as community participation of older adults . such an understanding of the impact of home and community features as well as itemized home activities provides a more viable recipe for intervention to facilitate aging in place . first , individuals with mobility limitations were found to be more vulnerable to the environment than those with other types of limitations , which suggests that environmental interventions for aging in place should first target at older adults who have difficulty moving around as they are the most environmentally vulnerable . most importantly , environmental modifications should be conceived as a continuum of interventions from home to community in order to support both the activities and community participation that are necessary for successfully aging in place . in doing so , understanding the effects of interventions across settings is an important tool in identifying and prioritizing environmental modification needs for making decisions in policy and practice . in addition , findings from this study suggest that contrary to current policy and practice that focus on independence as the primary intervention goal , both dependence and difficulty in activity performance predicted much of community participation . in fact , more home barriers were correlated with difficulty than dependence at home in our sample . many older adults may not ask for assistance at the onset of functional declinations but may have already started experiencing difficulty in basic home activities . this may not only reduce the time they have available for community participation and other meaningful activities but may also pose potential safety hazards . therefore , reducing activity difficulty should be a directed intervention goal in order to detect early unmet needs for aging in place . finally , our results provide more detailed information about environmental features that can be prioritized as interventions for aging in place . targeted home features to enhance both home performance and community included accessible bathtubs or shower , walkways , and kitchen features . in the community , it is important to pay more attention to the social environment in the destinations to promote participation . ultimately , a good physical environment will never overcome a bad social environment , but a good social environment can overcome a bad physical environment . possible interventions could include disability awareness training for all community members and community social support system and network , to enable older adults to participate in the community and successfully age in place .

this paper describes relationships among home and community environmental features , activity performance in the home , and community participation potential to support aging in place . a subset of data on older adults with functional limitations ( n = 122 ) , sixty three ( 63 ) with mobility and 59 with other limitations , were utilized in this study from a larger project 's subject pool . results showed significant and positive correlations between environmental barriers , activity dependence and difficulty at home , and less community participation in the mobility limitation group . while kitchen and bathroom features were most limiting to home performance , bathtub or shower was the only home feature , and destination social environment was the only community feature , that explained community participation . compared to environmental features , home performance explained much more community participation . study results provide detailed information about environmental features as well as types of home activities that can be prioritized as interventions for aging in place .

1. Introduction 2. Purpose 3. Methods 4. Results 5. Discussion 6. Implications

changes in the person - environment relationship as well as the negative outcomes of shrinkage in life space ( i.e. with almost 9 out of 10 ( 86% ) older americans reporting that they want to spend the rest of their lives in the homes and communities in which the majority of their daily activities take place , a robust life space is essential for older adults to continue to engage and participate in as many home and community activities as independently and safely as possible . while prior work has consistently linked supportive home and community settings to continued performance of home activities and participation in community roles , respectively , evidence suggests that community participation , which is dependent on maintaining a wide range of life spaces outside the home , may also be affected by one 's level of dependence and difficulty in performance of daily activities in the home . further , activity dependence and difficulty may be differentially affected by the same environmental features . to develop a more comprehensive understanding of the factors that affect performance or activities in the home and participation in the community among older adults with limitations in mobility , this paper will describe the relationships among ( 1 ) home environmental features and performance of routine activities in the home as measured by task dependence and difficulty , ( 2 ) home and community environmental features and opportunities for community participation as measured by the frequency of travel to community life space destinations , such as restaurants , grocery stores , doctor 's offices , and recreational areas , and ( 3 ) dependence and difficulty in home activities and opportunities for community participation ( see figure 1 ) . based on these underlying principles , this study examined the association between environmental factors and performance at home and participation in the community as well as interactions between home performance and community participation . based on the environmental docility hypothesis , this study examined the effects of environmental features on activity performance and community participation outcomes of older adults with and without mobility limitations . due to the increased time spent inside home and decreased participation in the community among older adults , this paper used a simplified version of the life space concept that focuses on older adults ' ability to move from inside their homes to life spaces in the community ( including both immediate neighborhood and outside immediate neighborhood ) . most previous studies of environmental supports for aging in place have separately examined associations between either the home environment and activity performance or the community environments and participation . as a result , a comprehensive understanding of the interrelationships among home and community environments , performance , and participation is still lacking . , home modifications ) with both activity independence , and ease of performance in 15 home tasks by individuals with mobility impairments . however , none of the factors , home environmental features , activity independence or ease of performance were linked to community participation . while mobility and balance ( as opposed to other personal factors ) explained 24% of participation in one study and activity limitations explained much of community participation in another , in a third study , community environments , such as governmental and public services and physical environment and accessibility , only accounted for 6% of the variance in participation . these data suggest that perhaps other factors , such as environmental factors in the home , may play a key role in community participation . recognizing the complex interactions among environments , home activity performance , and community participation and the potential impacts on the ability of older georgians to successfully age in place , the georgia council on aging , which serves in an advisory capacity on aging issues to the governor and general assembly of georgia , supported a survey to identify and prioritize the environmental and performance correlates of unmet home activity and community participation needs of georgia 's seniors . the goal of the survey was to develop a comprehensive understanding of home and community environmental barriers and facilitators that impact the activity and participation of georgia 's seniors to inform policy and prioritize service delivery needs for the state of georgia . in addition , the data are useful in developing a more comprehensive understanding of community participation potential and life space restriction as a function of the interrelationships among home and community environmental features and home activity performance . , environmental features and activity performance ) and community participation potential of georgia 's seniors with mobility limitations compared to those without mobility limitations . mobility limitation was selected as a subset of interest because this group is more likely to experience more environmental barriers and life space restriction than older adults with other limitations ( i.e. , barriers and facilitators ) and activity performance in the home as measured by dependence and difficulty in home activities of seniors with mobility limitations compared to those with other limitations , ( 2 ) home and community environmental features and community participation potential as measured by usage of community life space ( i.e. , the frequency of going into community destinations , such as restaurants , grocery stores , doctor 's offices , and recreational areas among older adults with mobility limitations compared those with other limitations ) of seniors with mobility limitations compared to those with other limitations , and ( 3 ) home activity ( i.e. , dependence and difficulty ) and community participation potential of seniors with mobility limitations compared to those with other limitations ( see table 1 ) . the study employed a cross - sectional survey design to explore the relationships among environmental features , dependence and difficulty in activity performance in the home , and life space usage in the community participation to understand the met and unmet activity and participation needs of older georgians . to ensure inclusion of older adults who did not have access to an online platform , alternative a total of 239 individuals with and without functional limitations who were 60 + , living in the state of georgia and had resided in their current residence for at least one year , were recruited for the survey . participants were recruited through a variety of methods , including subject registries maintained by the center for assistive technology and environmental access at georgia tech , as well as through email invitations and posts at aaas , aarp , norcs , senior centers , and other senior - related organizations throughout georgia . the survey gathered self - perceive information on a variety of factors , including ( 1 ) activity performance , ( 2 ) community participation potential , ( 3 ) environmental barriers and facilitators , and ( 4 ) participant demographics . based on a comprehensive review of literature , the survey borrowed from and adapted questions from a number of existing instruments , including comprehensive assessment and solution process for aging residents ( caspar ) , the healthy aging research network ( han ) environmental audit tool and protocol , the facilitators and barriers survey of environmental influences on participation among people with lower limb mobility impairments and limitations ( fabs / m ) , and the participation survey : mobility ( parts / m ) . activity performance and environmental barriers / facilitators in the home were adapted from the caspar . the caspar includes self - reported information on functional abilities , types of performance problems with person - environmental transactions , such as getting on and off toilet , and detailed measures of activity - relevant environmental attributes of the home , such as height and location of toilet . functional limitations were divided into two groups : mobility limitation group ( ml ) as defined by difficulty moving around and other limitations group ( ol ) , as defined by having difficulty with vision , hearing , speaking , and/or hand manipulation , were used as the other independent variable . the activities queried in the survey were adapted from the caspar , including three circulation tasks ( getting in and out of the house , going up and down interior stairs , and moving around inside the house ) , two tasks for using the bathroom ( getting on and off a toilet , getting in and out of a bathtub or shower ) , three for using the kitchen ( using kitchen appliances , getting items in and out of upper cabinets , and getting items in and out of lower cabinets ) , and the two for using the bedroom ( getting on and off a bed and using the closet ) . spearman rho correlations were conducted for all three research questions , that is , to associate ( 1 ) home environmental features to independence and to difficulty of home activities , ( 2 ) home and community environmental features to participation potential , and ( 3 ) independence and difficulty of home activities to participation potential . four stepwise regressions were further conducted to individually identify which ( 1 ) home and ( 2 ) community environmental feature that explain community participation for research question 2 , and ( 3 ) independence and ( 4 ) difficulty in which home activities explain community participation for research question 3 . overall , only approximately one in five respondents perceived barriers in either the home ( n = 18 , 14.8% ) or in the community ( n = 26 , 21.3% ) . conversely , the bathroom sink was the feature perceived by the lowest percentage ( 8.4% ) of the whole sample in the home , whereas social environments in community destinations were perceived by the lowest percentage of respondents ( 14.4% ) as a barrier in the community . however , all home and community features were perceived as barriers by higher percentages in the ml group than the ol group with the exception of public transportation , which had equal percentages in both groups . however , among these features , only steps ( p < .05 ) and kitchen cabinets ( p < .05 ) in the home and the physical environment in community destinations ( p < .01 ) were significantly higher in the ml than the ol group ( see table 3 ) . similar to activity dependence , higher percentages of the ml group reported having difficulty in all ten home activities , although in this case , all activities were significantly ( p = .000.022 ) more difficult in the ml than the ol group ( see table 4 ) . rq1 : what is the relationship between home environmental features and activity performance in the home?while almost none of the home features were significantly correlated with activity performance in the ol group , over half of the home barriers were significantly correlated with either activity dependence or difficulty in the ml group . while almost none of the home features were significantly correlated with activity performance in the ol group , over half of the home barriers more than three - quarters ( 76.6% ) of the 17 home barriers were significantly correlated with activity difficulty , while 58.8% ( n = 10 ) were significantly correlated with dependence . two of the circulation features , steps and going up and down stairs , were significantly correlated with both the dependence and difficulty in going up and down stairs and moving around the house , respectively . steps were positively and significantly correlated to dependence ( r = .520 ; p < .001 ) , and difficulty ( r = .303 ; p < .05 ) in going up and down stairs , whereas home space barriers were positively and significantly correlated with dependence ( r = .377 , p < .01 ) and difficulty ( r = .364 , p < the other four circulation features were correlated with either dependence ( i.e. in the bathroom , toilet features barriers were significantly correlated with both dependence ( r = .327 ; p < .01 ) and difficulty ( r = .268 ; p < .05 ) in getting on and off toilet ; however , tub / shower features were only significantly correlated ( r = .257 ; p < .05 ) with difficulty in getting in and out of bathtub / shower . finally , in the bedroom , closet features were significantly ( r = .503 ; p < .001 ) correlated with both dependence and difficulty in getting items in and out of a bedroom closet . in the ol group , only three home features , kitchen cabinets , bed , and steps , were significantly correlated with activity performance at home . two features , kitchen cabinets and bed , were positively correlated with difficulty in getting items in and out of lower drawers ( r = .292 ; p < .05 ) and difficulty getting out of bed ( r = .316 ; p < .05 ) , respectively . rq2:what is the relationship between home and community environmental features and potential for community participation?whereas community environmental features were significantly correlated to frequency of travel to community destinations in the ml group , they were not significantly correlated in the ol group . although no environmental features in the home were significantly correlated with frequency of travel to community destinations in either the ml or ol groups , among the 7 community features , three , including streets ( r = .294 ; p < .05 ) , sidewalks ( r = .283 ; p < .05 ) , and social environments of community destinations ( r = .346 ; p < .01 ) , were significantly correlated with frequency of going into community in the ml group . whereas community environmental features were significantly correlated to frequency of travel to community destinations in the ml group , they were not significantly correlated in the ol group . although no environmental features in the home were significantly correlated with frequency of travel to community destinations in either the ml or ol groups , among the 7 community features , three , including streets ( r = .294 ; p < .05 ) , sidewalks ( r = .283 ; p < .05 ) , and social environments of community destinations ( r = .346 ; p < .01 ) , were significantly correlated with frequency of going into community in the ml group . results indicate that bathtub / shower was the only home feature that explains any significant amount , although slightly less than 6% ( adjusted r = .055 ; p < .05 ) of travel frequency . , more than once a month ) and infrequent travelers ( once a month and less ) , frequency was significantly correlated with the majority of both home ( 64.7% , n = 11 ) and community ( 71.4% , n = 5 ) features in the ml group . among home features , kitchen and bathroom features had the highest percentage of features that were significantly correlated with infrequency of travel to community destinations in the ml group , including four out of five ( 80.0% ) bathroom features ( r = .289.401 ; p < .01 to p < .05 ) and all three ( 100.0% ) kitchen features ( r = .252.301 , p < .05 ) . among community features , stores , sidewalks , visual appeal , physical environments , and social environments were significantly correlated ( r = .268.431 , p < .001 to p < .05 ) with infrequent travel in the ml group ( see table 6 ) . in the home setting , all bathroom features , with the exception of bathroom sinks , had significant odds ratio results ( i.e. rq3 : what is the relationship between activity performance in the home and community participation potential?as in other analyses , significant correlations were only found in the ml group . again , in the ml group , there were a greater number of significant correlations between frequency of travel to community destinations and both kitchen and bathroom activities than circulation activities . again , in the ml group , there were a greater number of significant correlations between frequency of travel to community destinations and both kitchen and bathroom activities than circulation activities . comparing kitchen and bathroom activities , dependence of all kitchen activities and difficulty in all bathroom activities dependence in using kitchen appliances , getting items in and out of upper cabinets and of lower drawers were significantly correlated with less community participation ( r = .272.417 ; p < .01 to p < .05 ) . 306 ; this study described relationships among home and community environmental features , dependence and difficulty in activity performance at home , and frequency of travel to community life space as an indicator of participation potential . specifically , the study demonstrated that home environmental features were not only significantly associated with activity difficulty and dependence in the home , but also with less restriction in life space . in fact , the latter was positively related to home features and home performance as well as community environmental features . the lower performance and participation outcomes as well as the stronger link between environment , performance , and participation not only indicate the higher vulnerability in people with mobility limitations to age in place , but also postulate that both performance and environmental intervention are a potentially important strategies to facilitate aging in place . rq1 : home features and activity performance at homeprevious studies have identified top barriers to activity performance at home as well as environmental features to reduce dependence and difficulty [ 10 , 1620 ] . , tubs , showers , and toilets ) , rather than space , were significantly correlated to activity performance , which perhaps reflects the vast majority ( almost 90% ) of the sample that was ambulatory . previous studies have identified top barriers to activity performance at home as well as environmental features to reduce dependence and difficulty [ 10 , 1620 ] . , tubs , showers , and toilets ) , rather than space , were significantly correlated to activity performance , which perhaps reflects the vast majority ( almost 90% ) of the sample that was ambulatory . rq2 : environmental features and community participation potentialnot surprisingly , community features were more strongly related with overall community travel than home features . moreover , when the four toilet and bathing features , toilet space , toilet design , tub / shower space , and tub / shower design , were perceived as barriers , respondents were 8 to 46.7 times more likely to travel into the community only once a month or less . moreover , when the four toilet and bathing features , toilet space , toilet design , tub / shower space , and tub / shower design , were perceived as barriers , respondents were 8 to 46.7 times more likely to travel into the community only once a month or less . in addition to home environmental features , the social environment at community destinations , such as staff 's willingness to offer assistance in a restaurant , not only showed the strongest correlation among all community features , but also it was the only community feature that attained significance in explaining the variance in community travel frequency . our finding of positive correlations between home barriers and less community participation was also consistent with results from haak and colleagues that significant correlations between the number of home barriers and community participation were reported . rq3 : activity performance at home and community participation potentialsimilar to home barriers , performance challenges at home were positively correlated to less community participation , especially in kitchen , bathroom , and circulation - related activities . similar to home barriers , performance challenges at home were positively correlated to less community participation , especially in kitchen , bathroom , and circulation - related activities . in addition to dependence at home , difficulty in getting in and out of bathtub or shower also explained 30.5% of community travel . difficulty in getting in and out of the tub or shower was significantly correlated to barriers in the tub or shower , which also corresponds to predictors of home barriers to community participation . while previous research has shown the strong contribution of activity limitation to participation , our findings further described the type of home activity as well as the kind of performance indicator related to community participation . this study provides the first step to comprehensively understand the relationships between home and community environments , home performance , and community participation as they impact aging in place . further studies should also include and control for covariates , such as functional level and living situation , in the examination of the environment and performance predictors for aging in place . however , despite the limitations , to the authors ' knowledge , this is one of the first studies that provides a more robust and comprehensive understanding of the impact of home and community environmental factors on home activity performance as well as community participation of older adults . such an understanding of the impact of home and community features as well as itemized home activities provides a more viable recipe for intervention to facilitate aging in place . first , individuals with mobility limitations were found to be more vulnerable to the environment than those with other types of limitations , which suggests that environmental interventions for aging in place should first target at older adults who have difficulty moving around as they are the most environmentally vulnerable . most importantly , environmental modifications should be conceived as a continuum of interventions from home to community in order to support both the activities and community participation that are necessary for successfully aging in place . in addition , findings from this study suggest that contrary to current policy and practice that focus on independence as the primary intervention goal , both dependence and difficulty in activity performance predicted much of community participation . finally , our results provide more detailed information about environmental features that can be prioritized as interventions for aging in place . targeted home features to enhance both home performance and community included accessible bathtubs or shower , walkways , and kitchen features . possible interventions could include disability awareness training for all community members and community social support system and network , to enable older adults to participate in the community and successfully age in place .

integrated by 3 serine proteases , factor xii ( fxii ) , factor xi ( fxi ) , and plasma prekallikrein ( pk ) and by one nonenzymatic cofactor , high molecular weight kininogen ( hk ) , the kallikrein - kinin system ( kks ) , also referred to as the plasma contact pathway of coagulation , is assembled and activated when the blood comes in contact with negatively charged polymers of endogenous origin or microbial surfaces [ 1 , 2 ] . upon binding to these negatively charged structures , the zymogen fxii undergoes a conformational change that endows the unstable proenzyme with limited enzymatic activity . activated fxii ( fxiia ) then cleaves prekallikrein ( complexed to the cofactor hk ) , generating pka . reciprocal cleavage reactions between fxiia and pka amplify the proteolytic cascade , leading to downstream ( i ) generation of fibrin via the fxiia / fxia - dependent procoagulative pathway , ( ii ) release of the internal bradykinin ( bk ) moiety of hk by pka . once liberated , the short - lived bk induces vasodilation and increases microvascular permeability through the activation of bradykinin b2 receptors ( b2r ) expressed in the endothelium lining . in addition , the multifunctional pka generates plasmin , an effector of fibrinolysis , and cleaves native c3 of the complement system c3 [ 3 , 4 ] . although hk is classically regarded as the parental precursor of proinflammatory kinins , the cleaved form of hk ( hka ) , a disulfide linked two - chain structure , has additional biological functions . for example , it has been reported that hka reduces neutrophil adhesive functions upon binding to 2-integrin mac-1 ( cr3 , cd11b / cd18 , m2 ) [ 1 , 5 ] . more recently , yang et al . appointed hk / hka as the plasma - borne opsonins that drive efferocytosis of apoptotic cells via plasminogen activator receptor ( upar)/rac1-pathway . after binding to phosphatidyl serine ( ps ) exposed by apoptotic neutrophils , the surface - bound hk binds to upar before switching off proinflammatory responses of macrophages . in contrast to this novel immunoregulatory function , hk ( and low molecular weight kininogen , lk ) are traditionally viewed as precursors of proinflammatory kinins . once leaked into extravascular tissues , the plasma - borne hk / lk undergo proteolytic cleavage by tissue kallikrein , releasing the b2r agonist lysyl - bk ( lbk ) in inflammatory exudates . it is noteworthy that oxidized forms of kininogens may release bioactive kinins as result of cooperation between neutrophil elastase ( ne ) and mast cell tryptase [ 7 , 8 ] . acting as paracrine hormones , the short - lived kinins ( bk or lbk ) swiftly activate g - protein coupled bradykinin b2 receptors ( b2r ) , a subtype of receptor constitutively expressed by endothelial cells , nociceptive neurons , macrophages , and dcs [ 1 , 911 ] . the long - range signaling activity of intact kinins is controlled by kinin - degrading metallopeptidases , such as the angiotensin converting enzyme ( ace / kininase ii ) . in addition , the liberated kinin peptides are metabolized by kininase i ( carboxypeptidase n or m ) ; removal of the c - terminal arg residue generates des - arg - kinins , the high - affinity ligands of b1r , a gpcr subtype whose expression is strongly upregulated in injured / inflamed tissues . during the last decade , research conducted in our laboratory showed that kinins proteolytically released in peripheral sites of t. cruzi or leishmania chagasi infection reversibly couple inflammation to antiparasite immunity [ 1317 ] . another interesting twist came from studies showing that activation of the contact system / kks promotes bacterial entrapment within fibrin meshes , thus providing a physical barrier against the systemic spread of microbial pathogens . to this date , however , it is unclear whether the contact pathway modulates immunity at early stages of cutaneous leishmaniasis . intravital microscopy studies conducted in the mouse ear model of l. major infection [ 1921 ] have shown that infiltrating neutrophils engulf the promastigotes before expressing the apoptotic markers required for efferocytosis by dermal dcs . after internalizing the parasitized / apoptotic neutrophils , the dermal dcs are no longer capable of steering protective th1-responses in the draining lymph node [ 1921 ] . although efferocytosis has strong impact on dc function and th development in l. major infection , independent studies showed that macrophage clearance of apoptotic neutrophils may either induce pro- or anti - inflammatory responses in ne - dependent manner , the intracellular fate of the parasite being influenced by the host genetic background [ 22 , 23 ] . in natural infection by blood - feeding arthropods , insect proboscis inevitably causes bleeding , which then causes the mixing of plasma and sandfly saliva substances with parasites deposited in the injured dermis . interestingly , phlebotomy duboscq , a vector of leishmania species , contains high levels of a salivary protein ( pdsp15 ) that inhibits the contact pathway by binding to negatively charged polymers of endogenous origin such as platelet - derived polyphosphates [ 2 , 18 , 26 ] . considering that activation of the procoagulative contact system induces microvascular leakage through pka - mediated release of bk , it is conceivable that sandfly - transmitted leishmania promastigotes have evolved the means to subvert the innate effector function of the kinin pathway at early stages of infection . the current study was motivated by the recent discovery that leishmania has three genes encoding ecotin - like inhibitors of serine peptidases ( isps ) . previous studies with the archetype of the family escherichia coli ecotin showed that this inhibitor targets neutrophil elastase ( ne ) a member of the trypsin - fold serine peptidases of clan pa / family s1a . after noting that the leishmania genome lacks these endogenous serine peptidase targets , eschenlauer et al . predicted that l. major isps might target s1a - family serine peptidases expressed by cells of the innate immune system , such as ne , tryptase , and cathepsin g . in a series of elegant studies , [ 31 , 32 ] addressed this issue using l. major lines lacking isp2 and isp3 ( isp2/3 ) . after studying the outcome of interactions between l. major promastigotes and elicited macrophages , these authors found that these phagocytes internalized the isp2/3 promastigotes far more efficiently than isp - expressing wild - type ( wt ) parasites [ 27 , 31 ] and linked the upregulated cr3-dependent phagocytosis of the isp2/3 l. major mutants to ne - dependent activation of innate immunity via the tlr4/pkr / tnf-/ifn- , a prooxidative pathway that limits intracellular parasite survival [ 31 , 32 ] . notably , the phenotype of isp2/3 promastigotes was reversed by supplementing the macrophage cultures with purified ( recombinant ) isp-2 or with the synthetic ne inhibitor ( meosuc - aapv - cmk ) , at the onset of infection . based on these collective findings , these authors suggested that isp-2 expressing l. major promastigotes might downmodulate phagocytosis and limit microbicidal responses of macrophages by preventing ne - dependent activation of tlr4 [ 31 , 32 ] . more recently , we have documented that macrophages internalize and limit intracellular t. cruzi growth in resident macrophages through activation pathways forged by the cross - talk between bradykinin b2 receptors and c5a receptors . intrigued by the similarities that exist between the phenotype of the l. major isp2/3 mutant and l. chagasi promastigotes and t. cruzi trypomastigotes ( dm28 strain ) [ 33 , 34 ] , in the current work we interrogated whether isp - expressing l. major and the isp-2 isp2/3 mutants differ in their ability to activate the kks in vivo and in vitro . using intravital microscopy , we first showed that l. major promastigotes topically applied to the hamster cheek pouch potently activate the kks extravascularly , irrespective of presence / absence of isp . in the second part of this study , we present evidence indicating that isp - expressing l. major may subvert innate immunity by targeting kinin - releasing serine proteases ( s1a family ) exposed at the cell - surface of macrophages . l. major promastigotes of friedlin ( mhom / jl/80/friedlin ) were grown in modified eagle 's medium ( homem , sigma ) supplemented with 10% heat - inactivated fetal bovine serum ( fbs , gibco ) at 25c , as previously described [ 31 , 32 ] . suspensions of promastigotes were washed twice with pbs before being used either in vitro or in vivo . leishmania major deficient in isp2 and isp3 ( isp2/3 ) were generated as previously described by eschenlauer et al . . the following antibiotics were used at the indicated concentration for the selection of transfectants : 50 mg / ml hygromycin b ( roche ) , 25 mg / ml g418 ( invitrogen ) , 10 mg / ml phleomycin ( invivogen ) , and 50 mg / ml puromycin dihydrochloride ( calbiochem ) . syrian hamsters , 3-month - old males , were maintained and anesthetized according to regulations given by the local ethical committee ( ibccf , protocol-014 , 23/02/2008 ) . altogether 65 hamsters ( 114 18 g ) ( anilab , so paulo , brazil ) were used . anesthesia was induced by intraperitoneal injection of sodium pentobarbital 3% that was supplemented with i.v . -chloralose ( 2.5% w / v , solution in saline ) through a femoral vein catheter . a tracheal cannula ( pe 190 ) was inserted to facilitate spontaneous breathing and the body temperature was maintained at 37c by a heating pad monitored with a rectal thermistor . the hamster cheek pouch ( hcp ) was prepared and used for intravital microscopy as previously reported [ 34 , 35 ] . the microcirculation of the hcp was observed using an axioskop 40 microscope , objective 4x , and oculars 10x equipped with a led light source colibri ( carl zeiss , germany ) and appropriate filters ( 490/520 nm and 540/580 nm , rhodamine ) for observations of fluorescence in epiluminescence . a digital camera , axiocam hrc , and a computer with the axiovision 4.4 software program ( carl zeiss , germany ) were used for image analysis of arteriolar diameter and total fluorescence in a representative rectangular area ( 5 mm ) of the prepared hcp . fluorescence was recorded for 30 min prior to experimental interventions to secure normal blood flow and unaltered vascular permeability and the fluorescence measured at 30 min after fitc - dextran ( fitc - dextran 150 kda , 100 mg / kg bodyweight , tdb consultancy , uppsala , sweden ) injection was adjusted to 2000 fluorescent units ( rfu = relative fluorescent units ) for statistical reasons . leukocytes were labeled in vivo by injecting rhodamine 100 g / kg b.w i.v . ( 10 min prior to experimental interventions ) , reinforced by injection of the same tracer at 10 g / kg b.w . the recorded fluorescence at 10 min after rhodamine injection in each experiment was adjusted to 3000 fluorescent units ( rfu ) for statistical reasons . two images of exactly the same area were recorded at every 5 min interval during the entire experiment . one was used to measure plasma leakage and arteriolar diameter ( 490/520 nm ) and the other to measure total fluorescence of rhodamine - labeled leukocytes in circulation , rolling , adherence , and migration ( 540/580 nm ) in the observed area ( 5 mm ) here defined as leukocyte accumulation . exposure time was limited to 15 s for each captured image in order to avoid phototoxicity . following 30 min control period after fitc - dextran injection hcps were topically exposed to wt l. major promastigotes or isp2/3 ( 7.5 10/500 l ) during interruption of the superfusion for 10 min . ( 40 mg / kg b.w . ) at time of pentobarbital anesthesia and dextran sulfate 500 kda ( tdb consultancy , uppsala , sweden ) was injected i.v . hoe-140 tested at 0.5 m and the histamine receptor h1 mepyramine ( 10 m ) were applied locally via a syringe pump into the superfusion during 10 min prior to application of promastigotes . c57bl/6 mice received an intraperitoneal injection of 2 ml of 3% thioglycolate and macrophages were harvested from peritoneal lavage 3 days later . macrophages were plated on 13 mm coverslips in 24-well plate and after 20 h of incubation at 37c in complete medium ( rpmi + 10% fbs , 100 u / ml penicillin , and 100 g / ml streptomycin ) the nonadherent cells were removed by washing the monolayer of cells with pbs . invasion assays were performed by adding stationary phase promastigotes to the monolayers at a ratio of 5 : 1 ( parasite / macrophage ) in medium containing 1 mg / ml albumin from bovine serum ( bsa , sigma ) . the interaction was performed during 3 h in a humidified chamber containing 5% co2 at 37c . when indicated , the culture medium was supplemented with 100 nm of b2r antagonist ( hoe-140 , sigma ) or 1 m of b1r antagonist des - arg-[leu]-bk ( dal - bk ; sigma ) , 5 minutes before addition of parasites . after interaction , extracellular promastigotes were removed by washing the monolayers twice with pbs , which were then fixed with bouin overnight and stained with giemsa ( merck ) . the number of intracellular amastigotes was determined by counting at least 100 cells per replicate under the light microscope . all assays were done in triplicates and results were expressed as mean values sd . promastigotes were preincubated with pbs-1% bsa for 1 hour to avoid unspecific binding and then incubated for 1 h with monoclonal antibody mbk3 ( igg11 : 50 ) or hkh4 ( igg2a1 : 50 ) , kindly provided by dr . w. mller - esterl from frankfurt university . mbk3 recognizes the bk epitope in domain d4 of human and bovine h-/l - kininogens whereas hkh4 binds to the d1 domain . parasites were washed three times with pbs-1% bsa and incubated with secondary fluorescent antibody ( fitc1 : 50 ) for 30 min at 4c , protected from light . after washing , the samples were acquired by flow cytometry ( facscan ; bd biosciences ) , and data analyses were done with summit software ( dako colorado , inc ) . the activation of fxii / pk in human citrated ( platelet free ) plasma treated ( or not ) with dextran sulfate ( dxs ; 500 kda , tdb consultancy ) was monitored by spectrofluorimetry as previously described , using internally quenched fluorescent substrates whose sequences correspond to the c - terminal ( abz - gfspfrsvtvq - eddnp ) or n - terminal flanking region ( abz - mtemarrpq - eddnp m ) of bk of mouse kininogen . the hydrolysis of the cleaved substrate abz - peptidyl - eddnp ( abz = o - aminobenzoyl and eddnp = ethylenediamine 2,4-dinitrophenyl ) was monitored by measuring the fluorescence at ex . the reaction was carried out in pbs , ph 7.4 , using citrated human plasma 1 : 20 , 4 m of the abz - peptidyl - eddnp substrate and 20 nm of the contact system activator dxs ( 500 kda ) . as internal controls , the plasma was pretreated with the synthetic pka inhibitor ( pksi-5275 m ) . assays with recombinant isp-2 ( kindly supplied by a. p. c. a. lima ) were performed at final concentrations of 142 , 177 , 240 , and 355 nm ; the neutrophil elastase ( ne ) inhibitor meosuc - aapv - cmk ( calbiochem ) was tested at 10 , 20 , and 30 m . pksi or recombinant isp2 and meosuc - aapv - cmk were preincubated with human plasma for 15 min , at 37c , prior to the addition of dxs and the substrate . plasma was prepared by centrifugation of blood samples at 2500 g for 20 min at 4c . comparisons of the means of the different groups were done by one - way analysis of variance ( anova ) . when the mean values of the groups showed a significant difference , pairwise comparison was performed with the tukey test . a p value of 0.05 or less was considered to indicate a statistically significant difference . for intravital experiments , we used anova or pairwise t - test , when appropriate . intravital microscopy in hcp has provided a wealth of information about the interplay between the kks and the topically applied pathogens because this method dispenses the use of needles , thus ruling out the influence of bleeding and collateral activation of the contact system in the analysis of microcirculatory parameters . as a starting point in this work , we asked whether the proinflammatory responses evoked by isp - expressing l. major promastigotes or isp - deficient parasites were comparable . our results ( figures 13 ) revealed that l. major ( wt ) promastigotes induced a very robust and reversible microvascular leakage that was detectable at 20 min and reached its maximal value 45 min after pathogen application ( figures 1(a)1(c ) ) . in 4 out of 15 experiments we measured leukocyte accumulation in and around postcapillary venules and noted that these circulating cells were promptly mobilized locally , the response being detectable up to 90 min after pathogen application ( figure 1(b ) ) . the temporal course and dynamics of the plasma leakage response evoked by wt promastigotes were quite different from the classical responses elicited by bk , histamine , or leukotrienes , all of which cause a maximal increase within 10 min and reversed to steady - state conditions within 30 min [ 39 , 40 ] . intriguingly , we found that the leakage responses evoked by l. major ( wt ) promastigotes were generally more robust than those induced by the same inoculum of l. donovani promastigotes ( figure 1(c ) ) or t. cruzi ( tissue culture trypomastigotes , dm28c strain ) . akin to the findings made in the above - mentioned studies , we found that topically applied hoe-140 ( b2r antagonist ) markedly reduced l. major ( wt- ) induced plasma leakage ( figure 1(a ) ) . considering that mast cells are innate sentinel cells strategically localized in the perivascular tissues , we next interrogated whether l. major promastigotes might evoke plasma leakage in mast cell - dependent manner . as shown in figure 1(a ) , the topical addition of mepyramine ( histamine-1-receptor blocker ) markedly inhibited the macromolecular leakage induced by l. major promastigotes . in a limited series of studies , we found that cromoglycate , a well - known mast cell stabilizer , abolished the l. major - induced leakage of plasma ( figure 1(b ) , lower panel ) . of further interest , cromoglycate prevented leukocyte accumulation in the parasite - laden microvascular beds , reducing this parameter to levels below controls ( figure 1(b ) , top panel ) . it is noteworthy that the doses of mepyramine and hoe-140 that were topically added to the hcp at the onset of infection were sufficient to block the leakage induced by standard solutions of histamine ( 4 m ) and bk ( 0.5 m ) . further expanding this investigation , we next explored the possibility that the microvascular leakage elicited by l. major promastigotes requires the participation of circulating neutrophils . to this end , we injected separate group of hamsters intravenously with dxs , a negatively charged polymer ( 500 kda ) and found that it profoundly inhibited plasma leakage induced by l. major promastigotes . although dxs was initially thought to inhibit neutrophil - dependent microvascular permeability by blocking endothelial interaction with neutrophil - derived cationic proteins [ 4144 ] , there is now awareness that these effects might result from dxs - mediated activation of the kks , a systemic reaction that leads to hypotension as result of excessive bk formation . finally , we sought to compare the microvascular responses elicited by wt l. major with their counterparts genetically deficient in isp-2/isp-3 . as shown in figure 1(d ) , the dynamics of plasma leakage induced by topically applied isp2/3 mutants was similar to that evoked by wt parasites , the peak response being observed at 4550 min after pathogen application . however , somewhat surprisingly , the isp2/3 mutants were 20% less effective in eliciting transendothelial leakage of plasma as compared to wt promastigotes ( figure 1(d ) ) ; the difference in their proinflammatory phenotypes ( p < 0.05 ) was already noticeable at 25 min after parasite application . studies in mice models of acute chagas disease and visceral leishmaniasis have recently showed that b2r - deficient mice exhibited impaired development of type-1 effector t cells , the immune dysfunction of the transgenic strain being ascribed to primary deficiency in the maturation of b2r dcs in chagasic mice . in a third study , we examined the role of the kinin pathway in the in vitro outcome of macrophage interactions with l. chagasi promastigotes . interestingly , these studies revealed that activation of the kinin / b2r pathway may either fuel intracellular parasite outgrowth in splenic macrophages from hamsters , a species that is susceptible to visceral leishmaniasis , or limit parasite survival in thioglycolate - elicited mouse peritoneal macrophages . motivated by this groundwork , in the next series of experiments we examined the outcome of macrophage interaction ( 3 h in the absence of serum ) with l. major promastigotes or isp2/3 mutants . , we found that the phagocytic uptake of these mutants was strongly upregulated as compared to wt promastigotes ( figure 2 ) . next , we asked whether b2r ( constitutively expressed ) or b1r ( nf-b inducible ; ) contributed to the phagocytic uptake of l. major . infection assays performed in the presence of hoe-140 or dal - bk ( b1r antagonist ) revealed that none of these gpcr antagonists inhibited macrophage uptake of isp - expressing ( wt ) l. major . in striking contrast , however , both gpcr antagonists efficiently reduced the phagocytic uptake of isp2/3 promastigotes by the phagocytes ( resp . , to 58% and 63% ; figure 2 ) . for reasons that are not clear , the b1r antagonist had a mild but significant stimulatory effect ( 39% increase compared with medium ) on the uptake of wt l. major . since the studies of macrophage infection by l. major promastigotes were routinely performed in the absence of serum , we reasoned that the kinin agonists should either originate from kininogens molecules bound to the surface of macrophages or alternatively from kininogen molecules eventually sequestered from serum by promastigotes . to test the latter possibility , we washed stationary phase l. major promastigotes extensively as described for infection assays and then stained the parasites with two different domain - specific mabs : ( i ) mbk3 , a monoclonal antibody that recognizes the bk epitope ( domain d4 ) of kininogens ( hk / lk ) , and ( ii ) hkh4 , a mab that recognizes domain d1 of hk / lk . facs analysis showed that almost 70% of isp2/3 promastigotes are positive stained for hkh4 , compared to less than 50% of the wt parasites ( figure 3(a ) ) . along similar lines , mbk3 antibody showed that the bk epitope of kininogens was present in a higher proportion of isp2/3 promastigotes as compared to wt promastigotes ( figure 3(b ) , 82,4% ) as compared to wt parasites ( figure 3(b ) , 73.1% ) . these results suggest that both isp - expressing l. major promastigotes and isp2/3 mutants are able to sequester kininogens ( retaining the intact bk molecule ) from fcs . according to our working hypothesis ( figure 5 ) , the kininogen opsonins tethered on isp - deficient parasites may be cleaved by pericellular serine proteases ( s1a family ) of macrophages , whereas the surface - bound kininogens associated to isp - expressing ( wt ) l. major should be protected from proteolytic cleavage . considering that isp-2 is hardly detected in the supernatants of l. major promastigotes ( a. p. c. a. lima , personal communication ) , we reasoned that we reasoned that this ecotin - like inhibitor may target s1a serine proteases within the secluded spaces formed by the juxtaposition of host cell / parasite plasma membranes . given the technical obstacles to monitor kks activation and kinin release in this intercellular compartment , we first asked whether soluble ( recombinant ) isp-2 or meosuc - aapv - cmk ( ne inhibitor ) could inhibit the activation of the human contact system by dxs . this was addressed using a novel enzymatic assay that we recently used to detect the p. duboscq sandfly protein inhibitor of the contact system . briefly , the addition of dxs ( 500 kda ) to human plasma induces the reciprocal activation of fxii / pk , leading to the accumulation of pka , the major kinin - releasing ( s1a family ) in the plasma . using as - read - outs synthetic substrates spanning the n - terminal or c - terminal flanking sequences of bk in the kininogen molecule , the kinetic measurements shown in our positive controls ( figure 4 ) reflect dxs - induced hydrolysis of the kininogen - like substrate by pka . internal controls run in the presence of the synthetic pka inhibitor ( pksi-257 ) show , as expected , pronounced inhibition of the contact phase enzyme by dxs . assays performed with soluble isp-2 revealed that the onset of hydrolysis was consistently delayed , in dose - dependent manner ( range 142355 nm ; figures 4(a ) and 4(b ) ) . a similar trend was observed when we added meosuc - aapv - cmk ( ne inhibitor ) to the citrated plasma ( range 1030 m ; figures 4(c ) and 4(d ) ) . collectively , these findings are consistent with the proposition that the activity of the contact phase enzyme complex ( fxiia / pka ) is at least partially inhibited by isp-2 ( soluble ) or by the synthetic ne inhibitor . in the current study , we used genetically modified isp2/3 mutants of l. major to determine whether these ecotin - like inhibitors regulate the proinflammatory activity of the kinin / b2r pathway in vivo and in vitro . in the first group of studies , we demonstrated that l. major promastigotes potently evoke plasma leakage and induce leukocyte accumulation in microvascular beds through mast cell - dependent activation of the kinin / b2r pathway , irrespective of the presence or absence of isp-2 . extending this analysis to in vitro infection models , we showed that antagonists of b2r ( hoe-140 ) or b1r ( dal - bk ) efficiently reversed the upregulated phagocytic uptake of l. major isp2/3 by tg - macrophages without interfering with the internalization of isp - expressing ( wt ) promastigotes . as discussed further below , these findings suggested that , upon attachment to the macrophage surface , isp - expressing promastigotes might suppress the activation of b2r / b1r - dependent proinflammatory responses by inhibiting the kinin - releasing activity of serine proteases ( s1a family ) . while studying the functional interplay between ne and isp-2 during macrophage infection by leishmania , faria et al . [ 31 , 32 ] demonstrated that two prominent phenotypes of isp2/3 mutants ( upregulated phagocytosis and induction of ros via the elastase / tlr4/pkr pathway ) were completely reversed in cultures supplemented with three different inhibitors of serine peptidases : aprotinin , a non - specific inhibitor of arg - hydrolyzers , meosuc - aapv - cmk ( ne inhibitor ) , and the l. major isp-2 ( soluble / recombinant ) . given the precedent that ne and mast cell tryptase ( acting cooperatively ) liberate bioactive kinin from oxidized kininogens , our findings that hoe-140 and dal - bk also reversed the phenotype of isp2/3 mutants suggest that isp - expressing l. major might inhibit the pericellular processing of surface - bound hk through the targeting of ne . alternatively , our finding that soluble isp-2 ( or the ne inhibitor meosuc - aapv - cmk ) partially inhibit dxs - induced activation of the contact system in human citrated plasma ( i.e. , pka - mediated hydrolysis of the flanking sites of bk in kininogen - like substrates ) suggests that isp - expressing promastigotes might rely on their surface - associated isp-2 to target the contact phase enzymatic complex ( fxiia / pka / hk ) assembled on macrophage surfaces . admittedly , genetic studies will be required to dissect whether isp-2 subverts innate immunity by protecting surface kininogens from the kinin - releasing activity of ne and/or by targeting surface assembled contact phase peptidases ( fxii / pk ) . although we have not systematically analyzed the impact of pharmacological blockade of b2r / b1r on the intracellular parasitism of tg - macrophages , preliminary results suggest that hoe-140 ( tested at 100 nm ) upregulates the outgrowth / survival of isp2/3 mutants in tg - macrophages . if confirmed by genetic studies , our results may imply that l. major promastigotes might limit ros formation via the ne / tlr4/pkr / tnf-/ifn- pathway originally described by faria et al . through isp-2-dependent targeting of kinin - releasing peptidases assembled at the surface of macrophages . a key event in many inflammatory processes is the adhesive interaction of circulating neutrophils and activated endothelial cells in postcapillary venules , a process that is often coupled to increased microvascular permeability , which in turn leads to the progressive accumulation of protein - rich edema fluid in interstitial tissues . although conceding that the dynamics of the inflammatory responses that sand - fly - transmitted leishmania induces in the injured dermis is far more complex than what is described in our intravital microscopy studies , the analysis of microvascular leakage and leukocyte accumulation in hcp topically sensitized with l. major promastigotes ( wt or isp2/3-deficient parasites ) revealed that these parasites are far more potent inducers of plasma leakage and leukocyte accumulation than l. donovani , l. chagasi promastigotes ( figure 1(c ) ) , or t. cruzi trypomastigotes . although the mechanisms underlying the discrepant phenotypes of l. major and l. chagasi or t. cruzi remain unknown , we were intrigued to find out that a 3x - fold higher dose of l. chagasi promastigotes did not evoke such a strong microvascular response , not even after treating the hcp with captopril , an inhibitor of kinin degradation by angiotensin - converting enzyme ( figure 1(c ) , black curve ) . in contrast , t. cruzi and l. chagasi are potentially lethal pathogens that disseminate systemically and preferentially target tissue in organs irrigated by fenestrated capillaries . under these circumstances , plasma - borne substrates , such as kininogens , diffuse freely into the visceral tissues invaded by these visceralizing species of pathogenic trypanosomatids , both of which were empowered with kinin - releasing cysteine proteases [ 15 , 4749 ] . it is noteworthy that b2r - deficient mice acutely infected by t. cruzi or l. chagasi display heightened disease susceptibility , implying that the activation of the kinin / b2r pathway may preferentially shift the host / parasite balance towards protective immunity , at least during the acute phase . since proboscis inevitably provokes some extent of bleeding , we may predict plasma proteins and anti - inflammatory substances derived from the insect saliva are rapidly mixed with metacyclic parasites . our studies in hcp topically sensitized with l. major promastigotes ( which prevents bleeding and kks activation due to pathogen inoculation through needles ) suggest that these parasites potently evoke plasma leakage and leukocyte accumulation in microvascular beds via the kinin / b2r pathway . for reasons that are unclear , we found that isp2/3 promastigotes evoked a somewhat milder inflammatory response ( 20% ) . have reported that mice subcutaneously infected with l. major promastigotes transiently displayed higher tissue burden of isp2/isp-3-deficient parasites as compared to wt parasites . lasting 3 days , the parasite burden subsequently equalized , implying that the selective advantage conferred to isp - deficient promastigotes has waned as the infection progressed . based on pharmacological approaches , we showed evidences that l. major activates the kks via mechanisms that involve transcellular cross - talk between neutrophils ( intravascularly ) and mast cells , a subset of innate sentinel cells that are mostly localized in perivascular tissues . beyond the vasoactive role of histamine , a potent inducer of vascular permeability , mast cells also release heparin and polyphosphates , both of which were recently characterized as endogenous activators of the contact system [ 50 , 51 ] . given the interdependent nature of inflammatory circuits , it is likely that mast cells and the kks / complement cascades are reciprocally activated and fueled in the hcp sensitized with l. major promastigotes . although we have not studied the impact of the influx of complement into peripheral sites of l. major infection , it is well - documented that leishmania lipophosphoglycan is opsonized by c3bi . in the absence of other potent inflammatory cues , the engagement of macrophage cr3 by isp - expressing l. major ( wt ) promastigotes may drive phagocytosis without necessarily stimulating the production of reactive oxygen intermediates , thereby creating a hospitable environment for the intracellular growth of l. major [ 31 , 32 ] . although studies in cd11b - deficient balb / c mice have recently confirmed that activation of the c3bi / cr3 pathway increases host susceptibility to l. major infection , it will be interesting to know whether cr3-dependent suppression of il-12 responses might depend on parasite - evoked extravasation of complement components to the extravascular compartment , as proposed here for plasma - borne kininogens . studies in the mouse ear model of sandfly - transmitted infection showed that l. major metacyclic promastigotes deposited in the dermis are engulfed by the infiltrating neutrophils within approximately 3 h [ 1921 ] . based on the results described in hcp topically sensitized with l. major promastigotes , it is conceivable that the proteolytic release of vasoactive kinins may further stimulate the transendothelial migration of neutrophils at very early stages of the infection . furthermore , given evidence that parasitized neutrophils expose the apoptotic markers required for efferocytosis , it will be interesting to know whether plasma leakage may contribute to dc efferocytosis and to the ensuing suppression of th1-inductive functions of dcs in the draining lymph nodes [ 1921 ] . beyond the impact on adaptive immunity , it is well established that efferocytosis of apoptotic neutrophils has profound effect on parasite survival in infected macrophages . in this context , our finding that kininogen epitopes ( n - terminal d1 and internal / bk / d4 domain ) are tethered at the surface of leishmania promastigotes is an intriguing finding because it raises the possibility that isp - expressing promastigotes might protect the integrity of surface bound kinin precursors from premature proteolytic degradation by host proteolytic enzymes . this hypothesis is worth exploring in light of recent studies showing that hk ( an abundant protein in the bloodstream ; 660 nm ) binds to ps exposed on apoptotic neutrophils before stimulating upar - dependent efferocytosis by macrophages via the p130cas - crkii - dock-180-rac1 pathway . in other words , isps may protect the integrity of hk opsonins eat me signals while at the same time preventing the liberation of proinflammatory kinins ( see scheme , figure 5 ) within sites of parasite attachment to phagocytes . although we have not explored the potential significance of l. major opsonization by kininogens , it will be interesting to know whetherparasite subsets bearing the upar ligand hk / hka eat me signal might render macrophage permissive to intracellular survival , perhaps reminiscent of the apoptotic mimicry paradigm originally described by barcinski and coworkers . extending the breadth of our previous investigations about the role of the kallikrein - kinin system in the immunopathogenesis of experimental chagas disease and visceral leishmaniasis , the studies reported in this paper suggest that ecotin - like inhibitors expressed by l. major promastigotes fine - tune phagocytosis and may limit amastigote survival by inhibiting the pericellular activity of kinin - releasing serine proteases ( s1a family ) of macrophages .

inhibitors of serine peptidases ( isps ) expressed by leishmania major enhance intracellular parasitism in macrophages by targeting neutrophil elastase ( ne ) , a serine protease that couples phagocytosis to the prooxidative tlr4/pkr pathway . here we investigated the functional interplay between isp - expressing l. major and the kallikrein - kinin system ( kks ) . enzymatic assays showed that ne inhibitor or recombinant isp-2 inhibited kks activation in human plasma activated by dextran sulfate . intravital microscopy in the hamster cheek pouch showed that topically applied l. major promastigotes ( wt and isp2/3 mutants ) potently induced plasma leakage through the activation of bradykinin b2 receptors ( b2r ) . next , using mabs against kininogen domains , we showed that these bk - precursor proteins are sequestered by l. major promastigotes , being expressed at higher % in the isp2/3 mutant population . strikingly , analysis of the role of kinin pathway in the phagocytic uptake of l. major revealed that antagonists of b2r or b1r reversed the upregulated uptake of isp2/3 mutants without inhibiting macrophage internalization of wt l. major . collectively , our results suggest that l. major isp-2 fine - tunes macrophage phagocytosis by inhibiting the pericellular release of proinflammatory kinins from surface bound kininogens . ongoing studies should clarify whether l. major isp-2 subverts tlr4/pkr - dependent prooxidative responses of macrophages by preventing activation of g - protein coupled b2r / b1r .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Conclusions

integrated by 3 serine proteases , factor xii ( fxii ) , factor xi ( fxi ) , and plasma prekallikrein ( pk ) and by one nonenzymatic cofactor , high molecular weight kininogen ( hk ) , the kallikrein - kinin system ( kks ) , also referred to as the plasma contact pathway of coagulation , is assembled and activated when the blood comes in contact with negatively charged polymers of endogenous origin or microbial surfaces [ 1 , 2 ] . once liberated , the short - lived bk induces vasodilation and increases microvascular permeability through the activation of bradykinin b2 receptors ( b2r ) expressed in the endothelium lining . it is noteworthy that oxidized forms of kininogens may release bioactive kinins as result of cooperation between neutrophil elastase ( ne ) and mast cell tryptase [ 7 , 8 ] . acting as paracrine hormones , the short - lived kinins ( bk or lbk ) swiftly activate g - protein coupled bradykinin b2 receptors ( b2r ) , a subtype of receptor constitutively expressed by endothelial cells , nociceptive neurons , macrophages , and dcs [ 1 , 911 ] . intravital microscopy studies conducted in the mouse ear model of l. major infection [ 1921 ] have shown that infiltrating neutrophils engulf the promastigotes before expressing the apoptotic markers required for efferocytosis by dermal dcs . although efferocytosis has strong impact on dc function and th development in l. major infection , independent studies showed that macrophage clearance of apoptotic neutrophils may either induce pro- or anti - inflammatory responses in ne - dependent manner , the intracellular fate of the parasite being influenced by the host genetic background [ 22 , 23 ] . considering that activation of the procoagulative contact system induces microvascular leakage through pka - mediated release of bk , it is conceivable that sandfly - transmitted leishmania promastigotes have evolved the means to subvert the innate effector function of the kinin pathway at early stages of infection . the current study was motivated by the recent discovery that leishmania has three genes encoding ecotin - like inhibitors of serine peptidases ( isps ) . previous studies with the archetype of the family escherichia coli ecotin showed that this inhibitor targets neutrophil elastase ( ne ) a member of the trypsin - fold serine peptidases of clan pa / family s1a . predicted that l. major isps might target s1a - family serine peptidases expressed by cells of the innate immune system , such as ne , tryptase , and cathepsin g . after studying the outcome of interactions between l. major promastigotes and elicited macrophages , these authors found that these phagocytes internalized the isp2/3 promastigotes far more efficiently than isp - expressing wild - type ( wt ) parasites [ 27 , 31 ] and linked the upregulated cr3-dependent phagocytosis of the isp2/3 l. major mutants to ne - dependent activation of innate immunity via the tlr4/pkr / tnf-/ifn- , a prooxidative pathway that limits intracellular parasite survival [ 31 , 32 ] . based on these collective findings , these authors suggested that isp-2 expressing l. major promastigotes might downmodulate phagocytosis and limit microbicidal responses of macrophages by preventing ne - dependent activation of tlr4 [ 31 , 32 ] . intrigued by the similarities that exist between the phenotype of the l. major isp2/3 mutant and l. chagasi promastigotes and t. cruzi trypomastigotes ( dm28 strain ) [ 33 , 34 ] , in the current work we interrogated whether isp - expressing l. major and the isp-2 isp2/3 mutants differ in their ability to activate the kks in vivo and in vitro . using intravital microscopy , we first showed that l. major promastigotes topically applied to the hamster cheek pouch potently activate the kks extravascularly , irrespective of presence / absence of isp . in the second part of this study , we present evidence indicating that isp - expressing l. major may subvert innate immunity by targeting kinin - releasing serine proteases ( s1a family ) exposed at the cell - surface of macrophages . the hamster cheek pouch ( hcp ) was prepared and used for intravital microscopy as previously reported [ 34 , 35 ] . following 30 min control period after fitc - dextran injection hcps were topically exposed to wt l. major promastigotes or isp2/3 ( 7.5 10/500 l ) during interruption of the superfusion for 10 min . the activation of fxii / pk in human citrated ( platelet free ) plasma treated ( or not ) with dextran sulfate ( dxs ; 500 kda , tdb consultancy ) was monitored by spectrofluorimetry as previously described , using internally quenched fluorescent substrates whose sequences correspond to the c - terminal ( abz - gfspfrsvtvq - eddnp ) or n - terminal flanking region ( abz - mtemarrpq - eddnp m ) of bk of mouse kininogen . assays with recombinant isp-2 ( kindly supplied by a. p. c. a. lima ) were performed at final concentrations of 142 , 177 , 240 , and 355 nm ; the neutrophil elastase ( ne ) inhibitor meosuc - aapv - cmk ( calbiochem ) was tested at 10 , 20 , and 30 m . pksi or recombinant isp2 and meosuc - aapv - cmk were preincubated with human plasma for 15 min , at 37c , prior to the addition of dxs and the substrate . intravital microscopy in hcp has provided a wealth of information about the interplay between the kks and the topically applied pathogens because this method dispenses the use of needles , thus ruling out the influence of bleeding and collateral activation of the contact system in the analysis of microcirculatory parameters . as a starting point in this work , we asked whether the proinflammatory responses evoked by isp - expressing l. major promastigotes or isp - deficient parasites were comparable . our results ( figures 13 ) revealed that l. major ( wt ) promastigotes induced a very robust and reversible microvascular leakage that was detectable at 20 min and reached its maximal value 45 min after pathogen application ( figures 1(a)1(c ) ) . intriguingly , we found that the leakage responses evoked by l. major ( wt ) promastigotes were generally more robust than those induced by the same inoculum of l. donovani promastigotes ( figure 1(c ) ) or t. cruzi ( tissue culture trypomastigotes , dm28c strain ) . akin to the findings made in the above - mentioned studies , we found that topically applied hoe-140 ( b2r antagonist ) markedly reduced l. major ( wt- ) induced plasma leakage ( figure 1(a ) ) . considering that mast cells are innate sentinel cells strategically localized in the perivascular tissues , we next interrogated whether l. major promastigotes might evoke plasma leakage in mast cell - dependent manner . as shown in figure 1(a ) , the topical addition of mepyramine ( histamine-1-receptor blocker ) markedly inhibited the macromolecular leakage induced by l. major promastigotes . in a limited series of studies , we found that cromoglycate , a well - known mast cell stabilizer , abolished the l. major - induced leakage of plasma ( figure 1(b ) , lower panel ) . further expanding this investigation , we next explored the possibility that the microvascular leakage elicited by l. major promastigotes requires the participation of circulating neutrophils . to this end , we injected separate group of hamsters intravenously with dxs , a negatively charged polymer ( 500 kda ) and found that it profoundly inhibited plasma leakage induced by l. major promastigotes . although dxs was initially thought to inhibit neutrophil - dependent microvascular permeability by blocking endothelial interaction with neutrophil - derived cationic proteins [ 4144 ] , there is now awareness that these effects might result from dxs - mediated activation of the kks , a systemic reaction that leads to hypotension as result of excessive bk formation . as shown in figure 1(d ) , the dynamics of plasma leakage induced by topically applied isp2/3 mutants was similar to that evoked by wt parasites , the peak response being observed at 4550 min after pathogen application . studies in mice models of acute chagas disease and visceral leishmaniasis have recently showed that b2r - deficient mice exhibited impaired development of type-1 effector t cells , the immune dysfunction of the transgenic strain being ascribed to primary deficiency in the maturation of b2r dcs in chagasic mice . in a third study , we examined the role of the kinin pathway in the in vitro outcome of macrophage interactions with l. chagasi promastigotes . , we found that the phagocytic uptake of these mutants was strongly upregulated as compared to wt promastigotes ( figure 2 ) . next , we asked whether b2r ( constitutively expressed ) or b1r ( nf-b inducible ; ) contributed to the phagocytic uptake of l. major . infection assays performed in the presence of hoe-140 or dal - bk ( b1r antagonist ) revealed that none of these gpcr antagonists inhibited macrophage uptake of isp - expressing ( wt ) l. major . in striking contrast , however , both gpcr antagonists efficiently reduced the phagocytic uptake of isp2/3 promastigotes by the phagocytes ( resp . for reasons that are not clear , the b1r antagonist had a mild but significant stimulatory effect ( 39% increase compared with medium ) on the uptake of wt l. major . since the studies of macrophage infection by l. major promastigotes were routinely performed in the absence of serum , we reasoned that the kinin agonists should either originate from kininogens molecules bound to the surface of macrophages or alternatively from kininogen molecules eventually sequestered from serum by promastigotes . to test the latter possibility , we washed stationary phase l. major promastigotes extensively as described for infection assays and then stained the parasites with two different domain - specific mabs : ( i ) mbk3 , a monoclonal antibody that recognizes the bk epitope ( domain d4 ) of kininogens ( hk / lk ) , and ( ii ) hkh4 , a mab that recognizes domain d1 of hk / lk . these results suggest that both isp - expressing l. major promastigotes and isp2/3 mutants are able to sequester kininogens ( retaining the intact bk molecule ) from fcs . according to our working hypothesis ( figure 5 ) , the kininogen opsonins tethered on isp - deficient parasites may be cleaved by pericellular serine proteases ( s1a family ) of macrophages , whereas the surface - bound kininogens associated to isp - expressing ( wt ) l. major should be protected from proteolytic cleavage . considering that isp-2 is hardly detected in the supernatants of l. major promastigotes ( a. p. c. a. lima , personal communication ) , we reasoned that we reasoned that this ecotin - like inhibitor may target s1a serine proteases within the secluded spaces formed by the juxtaposition of host cell / parasite plasma membranes . given the technical obstacles to monitor kks activation and kinin release in this intercellular compartment , we first asked whether soluble ( recombinant ) isp-2 or meosuc - aapv - cmk ( ne inhibitor ) could inhibit the activation of the human contact system by dxs . briefly , the addition of dxs ( 500 kda ) to human plasma induces the reciprocal activation of fxii / pk , leading to the accumulation of pka , the major kinin - releasing ( s1a family ) in the plasma . in the current study , we used genetically modified isp2/3 mutants of l. major to determine whether these ecotin - like inhibitors regulate the proinflammatory activity of the kinin / b2r pathway in vivo and in vitro . in the first group of studies , we demonstrated that l. major promastigotes potently evoke plasma leakage and induce leukocyte accumulation in microvascular beds through mast cell - dependent activation of the kinin / b2r pathway , irrespective of the presence or absence of isp-2 . extending this analysis to in vitro infection models , we showed that antagonists of b2r ( hoe-140 ) or b1r ( dal - bk ) efficiently reversed the upregulated phagocytic uptake of l. major isp2/3 by tg - macrophages without interfering with the internalization of isp - expressing ( wt ) promastigotes . as discussed further below , these findings suggested that , upon attachment to the macrophage surface , isp - expressing promastigotes might suppress the activation of b2r / b1r - dependent proinflammatory responses by inhibiting the kinin - releasing activity of serine proteases ( s1a family ) . [ 31 , 32 ] demonstrated that two prominent phenotypes of isp2/3 mutants ( upregulated phagocytosis and induction of ros via the elastase / tlr4/pkr pathway ) were completely reversed in cultures supplemented with three different inhibitors of serine peptidases : aprotinin , a non - specific inhibitor of arg - hydrolyzers , meosuc - aapv - cmk ( ne inhibitor ) , and the l. major isp-2 ( soluble / recombinant ) . given the precedent that ne and mast cell tryptase ( acting cooperatively ) liberate bioactive kinin from oxidized kininogens , our findings that hoe-140 and dal - bk also reversed the phenotype of isp2/3 mutants suggest that isp - expressing l. major might inhibit the pericellular processing of surface - bound hk through the targeting of ne . alternatively , our finding that soluble isp-2 ( or the ne inhibitor meosuc - aapv - cmk ) partially inhibit dxs - induced activation of the contact system in human citrated plasma ( i.e. although we have not systematically analyzed the impact of pharmacological blockade of b2r / b1r on the intracellular parasitism of tg - macrophages , preliminary results suggest that hoe-140 ( tested at 100 nm ) upregulates the outgrowth / survival of isp2/3 mutants in tg - macrophages . if confirmed by genetic studies , our results may imply that l. major promastigotes might limit ros formation via the ne / tlr4/pkr / tnf-/ifn- pathway originally described by faria et al . although conceding that the dynamics of the inflammatory responses that sand - fly - transmitted leishmania induces in the injured dermis is far more complex than what is described in our intravital microscopy studies , the analysis of microvascular leakage and leukocyte accumulation in hcp topically sensitized with l. major promastigotes ( wt or isp2/3-deficient parasites ) revealed that these parasites are far more potent inducers of plasma leakage and leukocyte accumulation than l. donovani , l. chagasi promastigotes ( figure 1(c ) ) , or t. cruzi trypomastigotes . although the mechanisms underlying the discrepant phenotypes of l. major and l. chagasi or t. cruzi remain unknown , we were intrigued to find out that a 3x - fold higher dose of l. chagasi promastigotes did not evoke such a strong microvascular response , not even after treating the hcp with captopril , an inhibitor of kinin degradation by angiotensin - converting enzyme ( figure 1(c ) , black curve ) . our studies in hcp topically sensitized with l. major promastigotes ( which prevents bleeding and kks activation due to pathogen inoculation through needles ) suggest that these parasites potently evoke plasma leakage and leukocyte accumulation in microvascular beds via the kinin / b2r pathway . based on pharmacological approaches , we showed evidences that l. major activates the kks via mechanisms that involve transcellular cross - talk between neutrophils ( intravascularly ) and mast cells , a subset of innate sentinel cells that are mostly localized in perivascular tissues . given the interdependent nature of inflammatory circuits , it is likely that mast cells and the kks / complement cascades are reciprocally activated and fueled in the hcp sensitized with l. major promastigotes . in the absence of other potent inflammatory cues , the engagement of macrophage cr3 by isp - expressing l. major ( wt ) promastigotes may drive phagocytosis without necessarily stimulating the production of reactive oxygen intermediates , thereby creating a hospitable environment for the intracellular growth of l. major [ 31 , 32 ] . studies in the mouse ear model of sandfly - transmitted infection showed that l. major metacyclic promastigotes deposited in the dermis are engulfed by the infiltrating neutrophils within approximately 3 h [ 1921 ] . based on the results described in hcp topically sensitized with l. major promastigotes , it is conceivable that the proteolytic release of vasoactive kinins may further stimulate the transendothelial migration of neutrophils at very early stages of the infection . in this context , our finding that kininogen epitopes ( n - terminal d1 and internal / bk / d4 domain ) are tethered at the surface of leishmania promastigotes is an intriguing finding because it raises the possibility that isp - expressing promastigotes might protect the integrity of surface bound kinin precursors from premature proteolytic degradation by host proteolytic enzymes . extending the breadth of our previous investigations about the role of the kallikrein - kinin system in the immunopathogenesis of experimental chagas disease and visceral leishmaniasis , the studies reported in this paper suggest that ecotin - like inhibitors expressed by l. major promastigotes fine - tune phagocytosis and may limit amastigote survival by inhibiting the pericellular activity of kinin - releasing serine proteases ( s1a family ) of macrophages .

patients were eligible for inclusion when they had no vitreous surgery on the same eye previously and agreed to participate in this study , which was approved by our institutional ethics committee board and adhered to the declaration of helsinki . , diabetic retinopathy was evaluated according to the simplified international diabetic retinopathy classification ( 21 ) , made on the basis of clinical data , intraoperative assessment by the surgeon , and review of fundus photographs . baseline characteristics of the patients , ophthalmology features , and performed therapies data are n ( % ) unless otherwise indicated . * undiluted vitreous fluid samples ( 300400 l ) were collected from patients ' eyes at the start of a standard three - port pars plana vitrectomy for the treatment of retinal diseases . a central vitrectomy was performed after a 20- or 23-inch gauge sclerotomy at 4 mm from the limbus using a vitreotome ( accurus ) . vitreous samples were collected at the beginning of vitrectomy before opening the balanced salt solution infusion line to maintain intraocular pressure and homogenized by gently pipetting the suspension up and down several times . ninety - eight samples were collected from the eyes of 93 patients with proliferative diabetic retinopathy ( pdr ) ; in these cases , vitrectomy was performed for persistent vitreous hemorrhage , tractional retinal detachment , or macular edema . among the samples , 12 eyes had received an intravitreal injection of bevacizumab 1 week before surgery for pdr . in 11 additional diabetic patients , diabetic retinopathy was absent and vitrectomy the control group consisted of vitreous samples from 26 eyes of 26 nondiabetic patients with an idiopathic macular hole , an idiopathic epiretinal membrane , a rhegmatogenous retinal detachment , or age - related macular degeneration ( table 1 ) . vitreous was separated from cells and platelets after two centrifugations ( 500 g for 15 min and 13,500 g for 5 min ) . venous blood samples ( 10 ml ) were collected on citrated tubes before the eye surgery and platelet - free plasma ( pfp ) from 60 patients and was immediately prepared by successive centrifugations according to the methodology of amabile et al . ( 11 ) . for each included patient , pfp and vitreous were frozen and stored at 80c until subsequent use . monoclonal antibodies to vascular endothelial ( ve)-cadherin ( cd144 ) conjugated with phycoethrin , to human glycoprotein gpiib ( iiia ) ( cd41 ) conjugated with pc5 , and their corresponding isotype igg1 were from beckman coulter , france . human annexin v solution conjugated with fluorescein isothiocyanate ( fitc ) was from roche diagnostics , france . lectins from arachis hypogaea peanut agglutinin ( pna ) and from bandeiraea simplicifolia isolectin b4 ( ilb4 ) conjugated with fitc were from sigma aldrich , france . human umbilical vein endothelial cells ( huvecs ) ( passage 36 ) medium ( endothelium cell basal medium ) were obtained from promocell ( heidelberg , germany ) . cells were cultured in endothelial growth culture medium at 37c in a 95% air and 5% co2 atmosphere . matrigel basement membrane matrix was purchased from bd bioscience ( san jose , ca ) , and female c57/bl6 mice ( aged 7 weeks ) purchased from charles river labs were used for the matrigel plug assay . analyses were performed on a coulter epics xl flow cytometer ( beckman coulter ) by two independent examiners unaware of the subject status . human vitreous ( 60 l ) and pfp ( 20 l ) were incubated with anti - cd41-pc5 ( 10 l ) , anti - cd144-pe ( 20 l ) antibodies , or their respective isotypic immunoglobulins . microparticles expressing phosphatidylserine were labeled using fitc - conjugated annexin v solution in the presence of cacl2 ( 5 mmol / l final concentration ) according to the recommendation of the supplier . fitc - conjugated lectins were diluted in pbs to reach the final concentration of 100 g / ml . human vitreous ( 60 l ) was incubated with either lectin from pna fitc ( 20 g ) or lectin from ilb4 fitc ( 4 g ) ( 25,26 ) . their respective controls were preincubated with d - galactose ( 80 mmol / l for 30 min ) ( sigma - aldrich ) ( 2729 ) . microparticles were gated as events with a 0.1-to 1.0-m diameter identified in forward - scatter and side - scatter intensity dot - plot representation using standards synthetic beads of 1 m in diameter ( polyscience ) ( fig . microparticle concentration was assessed by comparison with calibrator flowcount beads ( 10 m diameter ; beckman coulter ) with a predetermined concentration . representative traces of flow cytometry analysis of annexin v , platelet , endothelial photoreceptor , and microglial microparticles in human vitreous samples . a e : size - selected events ( 0.11.0 m ) are plotted as a function of their fluorescence for specific annexin v fitc binding ( fl1 ) ( a ) , lectin pna - fitc labeling ( b ) , anti - human cd144-phycoethrin ( pe ) labeling ( c ) , anti - human cd41-pc5 labeling ( d ) , or lectin ilb4-fitc labeling ( e ) . gray - shaded areas represent unspecific labeling determined either in the absence of calcium ( a ) , fluorescent isotypic antibodies ( c and d ) , or d - galactose ( b and e ) . following samples embedding in epon 812 , thin sections were layed on a carbon - coated grid , stained with 7.6% uranyl acetate and 0.4% lead citrate , and then examined by electron microscopy ( phillips , eindhoven , the netherlands ; tecnai , fei , u.s . ) . fl1 , green fluorescence ; fl2 , orange fluorescence ; fl4 , infrared fluorescence . to study properties of vitreous microparticles on huvec proliferation and angiogenesis patients who previously had intravitreal anti - vegf therapy injection or complete panphotocoagulation were excluded . microparticles were pelleted and washed three times in endothelial growth culture medium medium ( 20,500 g for 150 min ) . at the end of the last centrifugation , the supernatant was filtered with 0.2 m and then 0.1 m acrodisc pf syringe filter ( pall life sciences ) and used as vehicle . levels of endothelial cd144 microparticles , the most abundant vitreous microparticle subpopulation in patients with pdr , were determined in each pellet using flow cytometry , and all proliferation experiments were performed at the final concentration of 65 cd144 microparticles/l . vitreous microparticles isolated using this procedure did not contain detectable levels of vegf when determined by immunoassay ( minimal detection limit of 3.3 pg / ml ; n = 6 ) . endothelial cell proliferation was evaluated by h - thymidine ( amersham ; biosciences ) incorporation in subconfluent quiescent huvecs . at 70% confluence , endothelial cells were rendered quiescent by replacing 10% fcs with 0.1% bsa for 24 h. endothelial cells were then incubated during 48 h with either 10% fcs ( used as a positive control ) , vitreous microparticles from patients with pdr ( corresponding to a final concentration of 65 microparticles/l of microparticles of endothelial origin ) , or vehicle . the concentration of microparticles used in these experiments was the highest concentration obtained after isolation of vitreous microparticles from patients with pdr . after 48 h , h - thymidine was added ( 1 ci / well ) for 16 h at 37c . endothelial cell proliferation was stopped by freezing ( 80c ) , and free thymidine was then separated by filtration on 1.2-m filters . the counting of the radioactivity remaining on the filters ( to determine thymidine incorporated in endothelial cells ) was then realized , after addition of a microscint solution ( 40 l / well ) , in a microplate luminescence counter ( perkin elmer , boston , ma ) . all animal experiments were performed in accordance with institutional animal care guidelines as previously described ( 30 ) . briefly , 500 l matrigel ( free of growth factors ) mixed with vitreous microparticles ( reaching a level of 5,000 cd144 microparticles of endothelial origin ) or vehicle was injected subcutaneously in the back of 16 7-week - old c57/bl6 mice . after 7 days , mice were killed by intraperitoneal injection of 0.1 ml pentobarbital ( ceva sante animale , libourne , france ) and matrigel plugs were excised . two pathologists graded the samples in a masked fashion and counted capillary structures ( considering each area surrounded by few contiguous cells forming a patent lumen as a capillary structure ) present on three different sections for every matrigel plug ( 10 , 20 , and 40 magnification , olympus bh-2 ; leica ) . microparticles levels are expressed as median and range and analyzed by kruskall - wallis nonparametric analysis to compare levels between more than two groups , followed by mann - whitney analysis of two groups when appropriate . other data are expressed as means sem according to the normality of distribution and analyzed by anova followed by appropriate bonferonni tests or by student 's t test . in addition , the test was performed for frequency analysis of matrigel angiogenesis assays . vitreous was separated from cells and platelets after two centrifugations ( 500 g for 15 min and 13,500 g for 5 min ) . venous blood samples ( 10 ml ) were collected on citrated tubes before the eye surgery and platelet - free plasma ( pfp ) from 60 patients and was immediately prepared by successive centrifugations according to the methodology of amabile et al . ( 11 ) . for each included patient , pfp and vitreous were frozen and stored at 80c until subsequent use . monoclonal antibodies to vascular endothelial ( ve)-cadherin ( cd144 ) conjugated with phycoethrin , to human glycoprotein gpiib ( iiia ) ( cd41 ) conjugated with pc5 , and their corresponding isotype igg1 were from beckman coulter , france . human annexin v solution conjugated with fluorescein isothiocyanate ( fitc ) was from roche diagnostics , france . lectins from arachis hypogaea peanut agglutinin ( pna ) and from bandeiraea simplicifolia isolectin b4 ( ilb4 ) conjugated with fitc were from sigma aldrich , france . human umbilical vein endothelial cells ( huvecs ) ( passage 36 ) medium ( endothelium cell basal medium ) were obtained from promocell ( heidelberg , germany ) . cells were cultured in endothelial growth culture medium at 37c in a 95% air and 5% co2 atmosphere . matrigel basement membrane matrix was purchased from bd bioscience ( san jose , ca ) , and female c57/bl6 mice ( aged 7 weeks ) purchased from charles river labs were used for the matrigel plug assay . analyses were performed on a coulter epics xl flow cytometer ( beckman coulter ) by two independent examiners unaware of the subject status . human vitreous ( 60 l ) and pfp ( 20 l ) were incubated with anti - cd41-pc5 ( 10 l ) , anti - cd144-pe ( 20 l ) antibodies , or their respective isotypic immunoglobulins . microparticles expressing phosphatidylserine were labeled using fitc - conjugated annexin v solution in the presence of cacl2 ( 5 mmol / l final concentration ) according to the recommendation of the supplier . fitc - conjugated lectins were diluted in pbs to reach the final concentration of 100 g / ml . human vitreous ( 60 l ) was incubated with either lectin from pna fitc ( 20 g ) or lectin from ilb4 fitc ( 4 g ) ( 25,26 ) . mmol / l for 30 min ) ( sigma - aldrich ) ( 2729 ) . microparticles were gated as events with a 0.1-to 1.0-m diameter identified in forward - scatter and side - scatter intensity dot - plot representation using standards synthetic beads of 1 m in diameter ( polyscience ) ( fig . microparticle concentration was assessed by comparison with calibrator flowcount beads ( 10 m diameter ; beckman coulter ) with a predetermined concentration . representative traces of flow cytometry analysis of annexin v , platelet , endothelial photoreceptor , and microglial microparticles in human vitreous samples . a e : size - selected events ( 0.11.0 m ) are plotted as a function of their fluorescence for specific annexin v fitc binding ( fl1 ) ( a ) , lectin pna - fitc labeling ( b ) , anti - human cd144-phycoethrin ( pe ) labeling ( c ) , anti - human cd41-pc5 labeling ( d ) , or lectin ilb4-fitc labeling ( e ) . gray - shaded areas represent unspecific labeling determined either in the absence of calcium ( a ) , fluorescent isotypic antibodies ( c and d ) , or d - galactose ( b and e ) . following samples embedding in epon 812 , thin sections were layed on a carbon - coated grid , stained with 7.6% uranyl acetate and 0.4% lead citrate , and then examined by electron microscopy ( phillips , eindhoven , the netherlands ; tecnai , fei , u.s . ) . to study properties of vitreous microparticles on huvec proliferation and angiogenesis , microparticles were isolated from vitreous samples pooled from six consecutive patients with pdr . patients who previously had intravitreal anti - vegf therapy injection or complete panphotocoagulation were excluded . microparticles were pelleted and washed three times in endothelial growth culture medium medium ( 20,500 g for 150 min ) . at the end of the last centrifugation , the supernatant was filtered with 0.2 m and then 0.1 m acrodisc pf syringe filter ( pall life sciences ) and used as vehicle . levels of endothelial cd144 microparticles , the most abundant vitreous microparticle subpopulation in patients with pdr , were determined in each pellet using flow cytometry , and all proliferation experiments were performed at the final concentration of 65 cd144 microparticles/l . vitreous microparticles isolated using this procedure did not contain detectable levels of vegf when determined by immunoassay ( minimal detection limit of 3.3 pg / ml ; n = 6 ) . endothelial cell proliferation was evaluated by h - thymidine ( amersham ; biosciences ) incorporation in subconfluent quiescent huvecs . at 70% confluence , endothelial cells were rendered quiescent by replacing 10% fcs with 0.1% bsa for 24 h. endothelial cells were then incubated during 48 h with either 10% fcs ( used as a positive control ) , vitreous microparticles from patients with pdr ( corresponding to a final concentration of 65 microparticles/l of microparticles of endothelial origin ) , or vehicle . the concentration of microparticles used in these experiments was the highest concentration obtained after isolation of vitreous microparticles from patients with pdr . after 48 h , h - thymidine was added ( 1 ci / well ) for 16 h at 37c . endothelial cell proliferation was stopped by freezing ( 80c ) , and free thymidine was then separated by filtration on 1.2-m filters . the counting of the radioactivity remaining on the filters ( to determine thymidine incorporated in endothelial cells ) was then realized , after addition of a microscint solution ( 40 l / well ) , in a microplate luminescence counter ( perkin elmer , boston , ma ) . all animal experiments were performed in accordance with institutional animal care guidelines as previously described ( 30 ) . briefly , 500 l matrigel ( free of growth factors ) mixed with vitreous microparticles ( reaching a level of 5,000 cd144 microparticles of endothelial origin ) or vehicle was injected subcutaneously in the back of 16 7-week - old c57/bl6 mice . after 7 days , mice were killed by intraperitoneal injection of 0.1 ml pentobarbital ( ceva sante animale , libourne , france ) and matrigel plugs were excised . two pathologists graded the samples in a masked fashion and counted capillary structures ( considering each area surrounded by few contiguous cells forming a patent lumen as a capillary structure ) present on three different sections for every matrigel plug ( 10 , 20 , and 40 magnification , olympus bh-2 ; leica ) . microparticles levels are expressed as median and range and analyzed by kruskall - wallis nonparametric analysis to compare levels between more than two groups , followed by mann - whitney analysis of two groups when appropriate . other data are expressed as means sem according to the normality of distribution and analyzed by anova followed by appropriate bonferonni tests or by student 's t test . in addition , the test was performed for frequency analysis of matrigel angiogenesis assays . microparticles were identified by flow cytometry analysis as events with 0.11 m positively labeled by specific antibodies ( fig . 1 ) : endothelium - derived microparticles ( emps ) ( cd144 or ve - cadherin positive ) and platelet - derived microparticles ( pmps ) ( cd41 or glycoprotein iib positive ) as previously described ( 11 ) . photoreceptor - derived microparticles ( pna positive ) and microglial - derived microparticles ( ilb4 positive ) were also identified . plasma levels of annexin v , platelet cd41 , and endothelial cd144 microparticles were not different in control and diabetic patients ( table 2 ) . microparticles from retinal origin ( pna or ilb4 microparticles ) were undetectable in plasma samples ( table 2 ) . vitreous levels of annexin v , platelet cd41 , and endothelial cd144 microparticles were all markedly increased in diabetic compared with control patients ( p = 0.035 , 0.018 , and < 0.001 , respectively ) ( fig . microparticles of endothelial origin , identified as expressing ve - cadherin ( cd144 ) , were the most abundant microparticle subpopulation in vitreous samples from diabetic patients . vitreous levels of annexin v , endothelial cd144 , and platelet cd41 microparticles were increased in association with pdr compared with nonpdr ( npdr ) ( p = 0.007 , 0.017 , and 0.018 , respectively ) ( table 2 ) . in addition , vitreous levels of annexin v , platelet cd41 , and endothelial cd144 microparticles were augmented in diabetic patients with pdr compared with control subjects ( p = 0.004 , 0.003 , and < 0.0001 , respectively ( fig . however , there were no differences between vitreous levels of retinal microparticles ( ilb4 and pna ) in diabetic patients and control patients . furthermore , circulating levels of endothelial - and platelet - derived microparticles were not different between control and diabetic patients with or without pdr ( fig . plasma levels of microparticles in control patients and in patients with or without pdr data are expressed as median ( range ) of microparticles/l . microparticles were identified of platelet ( cd41 ) and endothelial ( cd144 ) origin or from retinal origin ( pna and ilb4 ) . annexin v microparticles express phosphatidyserine and were positively labeled with annexin v. microparticles levels were analyzed by kruskall - wallis nonparametric analysis followed by mann - whitney analysis of two groups when appropriate . * significant difference between npdr and pdr or between nondiabetic patients and patients with pdr , respectively . vitreous levels of annexin v ( annv+ ) , platelet ( cd41 ) , endothelial ( cd144 ) , photoreceptor ( pna ) and microglial ( ilb4 ) microparticles ( mps ) in diabetic and control patients . data are expressed as microparticles/l and given as median ( horizontal bar ) , 25th and 75th percentile ( boxes ) , and 10th and 90th percentile ( error bar ) . * significantly different from control patients . the ratio of vitreous to plasma microparticles levels indicates the importance of intraocular formation versus potential plasma leakage of microparticles from microvessels in vitreous fluid ( fig . this ratio was markedly greater than unity for only endothelial cd144 microparticles in pdr ( p = 0.02 ) , indicating either an intraocular shedding or an abnormal clearance of endothelial microparticles . ratio of vitreous to plasma levels of annexin v ( annv+ ) , platelet ( cd41 ) , and endothelial ( cd144 ) microparticles ( mps ) in control and diabetic patients with either pdr or npdr . * ratio significantly greater than unity . in patients with pdr , complete panretinal photocoagulation ( prp ) significantly decreased vitreous endothelial microparticle levels ( p = 0.037 ) to reach values not different from those obtained in control patients . complete laser treatment tended to decrease vitreous levels of annexin v , cd41 , or pna microparticles but the change did not reach statistical significance ( fig . vitreous levels of annexin v ( annv+ ) ( a ) , platelet ( cd41 ) ( b ) , endothelial ( cd144 ) ( c ) , and retinal ( pna ) ( d ) microparticles in control subjects ( open bars ) and diabetic patients with pdr ( gray bars ) who underwent either no or incomplete laser panphotocoagulation ( prp ) , complete prp , or intravitreal injection of bevacizumab prior to vitrectomy . * one week prior to vitrectomy , 12 patients with pdr received an intravitreal injection of bevacizumab ( 50 l ; 25 g/l ) , a humanized recombinant antibody binding all isoforms of vegf - a , which causes at least short - term involution of retinal neovascularization , therefore reducing the risk of hemorrhage during surgery ( fig . 4 ) . bevacizumab treatment significantly decreased vitreous levels of annexin v microparticles and endothelial cd144 microparticles ( p = 0.04 and p = 0.02 , respectively ) and led to a complete disappearance of platelet - derived cd41 microparticles in pdr vitreous samples ( fig . 4 ) . however , anti - vegf treatment did not affect levels of photoreceptor microparticles ( pna ) . following bevacizumab treatment , vitreous levels of endothelial cd144 microparticles were no longer different from those found in control patients . vitreous microparticles or supernatant , as previously prepared , were incubated with subconfluent quiescent huvecs during 48 h ( n = 4 ) . vitreous microparticles significantly increased h - thymidine incorporation , by 1.6-fold , compared with microparticle vehicle ( vitreous supernatant ; p = 0.029 ) . fcs ( 10% ; used here as a positive control ) increased h - thymidine incorporation by 2.4-fold ( p = 0.003 ) ( fig . vitreous microparticles did not contain detectable levels of vegf when determined by immunoassay with a minimal detection limit of 3.3 pg / ml ( n = 6 ) . in the in vivo matrigel plug assay , vitreous microparticles significantly induced endothelial cell migration ( score 1 ) and new vessels formation ( score 2 ) compared with vehicle ( vitreous supernatant ; score 0 ) ( p = 0.001 ) ( fig . h - thymidine incorporation with the vehicle ( vitreous supernatant ) was used as the baseline ( 100% ) . levels of h - thymidine incorporation are represented with endothelium cell basal medium ( dulbecco 's modified eagle 's medium [ dmem ] ) , fcs ( 2% ) , and vitreous microparticles ( mps ) . vitreous microparticles ( estimated as 65 cd144 microparticles/l for the endothelial subpopulation ) increased h - thymidine incorporation by 1.6-fold compared with vehicle ( vitreous supernatant without cd144 microparticles ) ( * p = 0.029 ) . fcs ( 10% ) increased it by 2.4-fold ( * * p = 0.003 ) . the presence of capillary structures with massive cells invasion was noticed ( 2 ) . in some plugs low concentration of vitreous microparticles ( 10 emp/l ; 5,000 emps added to 500 l matrigel ) induced endothelial cell migration ( score 1 ; n = 6 ) and new vessel formation ( score 2 ; n = 3 ) compared with vitreous supernatant ( score 0 ; n = 12 ) ( p = 0.001 ) . ( a high - quality digital representation of this figure is available in the online issue . ) microparticles were identified by flow cytometry analysis as events with 0.11 m positively labeled by specific antibodies ( fig . 1 ) : endothelium - derived microparticles ( emps ) ( cd144 or ve - cadherin positive ) and platelet - derived microparticles ( pmps ) ( cd41 or glycoprotein iib positive ) as previously described ( 11 ) . photoreceptor - derived microparticles ( pna positive ) and microglial - derived microparticles ( ilb4 positive ) were also identified . plasma levels of annexin v , platelet cd41 , and endothelial cd144 microparticles were not different in control and diabetic patients ( table 2 ) . microparticles from retinal origin ( pna or ilb4 microparticles ) were undetectable in plasma samples ( table 2 ) . vitreous levels of annexin v , platelet cd41 , and endothelial cd144 microparticles were all markedly increased in diabetic compared with control patients ( p = 0.035 , 0.018 , and < 0.001 , respectively ) ( fig . microparticles of endothelial origin , identified as expressing ve - cadherin ( cd144 ) , were the most abundant microparticle subpopulation in vitreous samples from diabetic patients . vitreous levels of annexin v , endothelial cd144 , and platelet cd41 microparticles were increased in association with pdr compared with nonpdr ( npdr ) ( p = 0.007 , 0.017 , and 0.018 , respectively ) ( table 2 ) . in addition , vitreous levels of annexin v , platelet cd41 , and endothelial cd144 microparticles were augmented in diabetic patients with pdr compared with control subjects ( p = 0.004 , 0.003 , and < 0.0001 , respectively ( fig . however , there were no differences between vitreous levels of retinal microparticles ( ilb4 and pna ) in diabetic patients and control patients . furthermore , circulating levels of endothelial - and platelet - derived microparticles were not different between control and diabetic patients with or without pdr ( fig . plasma levels of microparticles in control patients and in patients with or without pdr data are expressed as median ( range ) of microparticles/l . microparticles were identified of platelet ( cd41 ) and endothelial ( cd144 ) origin or from retinal origin ( pna and ilb4 ) . annexin v microparticles express phosphatidyserine and were positively labeled with annexin v. microparticles levels were analyzed by kruskall - wallis nonparametric analysis followed by mann - whitney analysis of two groups when appropriate . * significant difference between npdr and pdr or between nondiabetic patients and patients with pdr , respectively . vitreous levels of annexin v ( annv+ ) , platelet ( cd41 ) , endothelial ( cd144 ) , photoreceptor ( pna ) and microglial ( ilb4 ) microparticles ( mps ) in diabetic and control patients . data are expressed as microparticles/l and given as median ( horizontal bar ) , 25th and 75th percentile ( boxes ) , and 10th and 90th percentile ( error bar ) . * significantly different from control patients . the ratio of vitreous to plasma microparticles levels indicates the importance of intraocular formation versus potential plasma leakage of microparticles from microvessels in vitreous fluid ( fig . this ratio was markedly greater than unity for only endothelial cd144 microparticles in pdr ( p = 0.02 ) , indicating either an intraocular shedding or an abnormal clearance of endothelial microparticles . ratio of vitreous to plasma levels of annexin v ( annv+ ) , platelet ( cd41 ) , and endothelial ( cd144 ) microparticles ( mps ) in control and diabetic patients with either pdr or npdr . in patients with pdr , complete panretinal photocoagulation ( prp ) significantly decreased vitreous endothelial microparticle levels ( p = 0.037 ) to reach values not different from those obtained in control patients . complete laser treatment tended to decrease vitreous levels of annexin v , cd41 , or pna microparticles but the change did not reach statistical significance ( fig . 4 ) . vitreous levels of annexin v ( annv+ ) ( a ) , platelet ( cd41 ) ( b ) , endothelial ( cd144 ) ( c ) , and retinal ( pna ) ( d ) microparticles in control subjects ( open bars ) and diabetic patients with pdr ( gray bars ) who underwent either no or incomplete laser panphotocoagulation ( prp ) , complete prp , or intravitreal injection of bevacizumab prior to vitrectomy . * one week prior to vitrectomy , 12 patients with pdr received an intravitreal injection of bevacizumab ( 50 l ; 25 g/l ) , a humanized recombinant antibody binding all isoforms of vegf - a , which causes at least short - term involution of retinal neovascularization , therefore reducing the risk of hemorrhage during surgery ( fig . 4 ) . bevacizumab treatment significantly decreased vitreous levels of annexin v microparticles and endothelial cd144 microparticles ( p = 0.04 and p = 0.02 , respectively ) and led to a complete disappearance of platelet - derived cd41 microparticles in pdr vitreous samples ( fig . however , anti - vegf treatment did not affect levels of photoreceptor microparticles ( pna ) . following bevacizumab treatment , vitreous levels of endothelial cd144 microparticles were no longer different from those found in control patients . vitreous microparticles or supernatant , as previously prepared , were incubated with subconfluent quiescent huvecs during 48 h ( n = 4 ) . vitreous microparticles significantly increased h - thymidine incorporation , by 1.6-fold , compared with microparticle vehicle ( vitreous supernatant ; p = 0.029 ) . fcs ( 10% ; used here as a positive control ) increased h - thymidine incorporation by 2.4-fold ( p = 0.003 ) ( fig . vitreous microparticles did not contain detectable levels of vegf when determined by immunoassay with a minimal detection limit of 3.3 pg / ml ( n = 6 ) . in the in vivo matrigel plug assay , vitreous microparticles significantly induced endothelial cell migration ( score 1 ) and new vessels formation ( score 2 ) compared with vehicle ( vitreous supernatant ; score 0 ) ( p = 0.001 ) ( fig . h - thymidine incorporation with the vehicle ( vitreous supernatant ) was used as the baseline ( 100% ) . levels of h - thymidine incorporation are represented with endothelium cell basal medium ( dulbecco 's modified eagle 's medium [ dmem ] ) , fcs ( 2% ) , and vitreous microparticles ( mps ) . vitreous microparticles ( estimated as 65 cd144 microparticles/l for the endothelial subpopulation ) increased h - thymidine incorporation by 1.6-fold compared with vehicle ( vitreous supernatant without cd144 microparticles ) ( * p = 0.029 ) . fcs ( 10% ) increased it by 2.4-fold ( * * p = 0.003 ) . b : matrigel scoring and angiogenesis assay . serial sections of matrigel were quantified by two independent pathologists . the presence of capillary structures with massive cells invasion was noticed ( 2 ) . in some plugs low concentration of vitreous microparticles ( 10 emp/l ; 5,000 emps added to 500 l matrigel ) induced endothelial cell migration ( score 1 ; n = 6 ) and new vessel formation ( score 2 ; n = 3 ) compared with vitreous supernatant ( score 0 ; n = 12 ) ( p = 0.001 ) . ( a high - quality digital representation of this figure is available in the online issue . ) this study reveals , for the first time , the presence of submicron membrane vesicles shed from platelet , endothelial , and retinal cells in human vitreous samples of patients undergoing vitrectomy and demonstrates the specific vitreous accumulation of endothelial microparticles in patients with pdr . in addition , we show that vitreous microparticles isolated from patients with diabetic retinopathy stimulate endothelial cell proliferation and formation of new vessels , suggesting that they could contribute to retinal angiogenesis . presence of shed - membrane microparticles has been reported in several human body fluids , such as plasma or synovial fluid ( 13,15,16 ) . plasma levels of microparticles of different cellular origins are increased in patients with high atherothrombotic risk or inflammatory diseases ( 15,16,31 ) . in this study , we observed no significant differences in circulating microparticle levels between diabetic patients and the control group . however , our diabetic group included mostly patients with type 2 diabetes , a disease for which conflicting results on changes in circulating microparticles have been reported ( 1720 ) . another potential confounding factor is that significant numbers of patients from the control group have cardiovascular risk factors such as hypertension or dislipidemia , which are known to be associated with increased circulating levels of microparticles ( 32,33 ) . pdr is associated with ocular increases in oxidative stress , protein glycation , growth factors , inflammatory cytokines , and cell apoptosis all of which stimulate the shedding of membrane microparticles from retinal or vascular cells ( 1,4,15,16,21,22,24,34,35 ) . we observed significant increases in the overall pool of vitreous annexin v microparticles in pdr compared with control patients or patients with nonproliferative diabetic retinopathy . in addition , we demonstrate the presence of microparticles positively labeled with either pna or ilb4 in vitreous samples but not in plasma , indicating their photoreceptor or microglial origin , respectively ( 2529 ) . if anything , levels of microparticles derived from photoreceptors or microglia tend to increase in patients with pdr , but the difference did not reach statistical significance . these microparticles originate from cells localized in deeper retinal layers and may be released in vitreous fluid following the tear of the retinal internal limiting membrane . microfracture of the internal limiting membrane is a likely hypothesis in our control patients , who were included for epimacular membrane or macular hole , and even more so in patients with pdr , where proliferating microvessels need to perforate the internal limiting membrane ( 36,37 ) . the present study also demonstrates increased vitreous levels of microparticles originating from vascular or circulating cells such as platelets . diabetic retinopathy is characterized by an augmented vascular permeability , and microparticles present in the blood could permeate through the leaky vascular wall into the vitreous fluid . by the same token , vitreous microparticles expressing endothelial ve - cadherin may originate from the circulating pool of microparticles , but they may be also locally generated from the retinal new vessel wall . to appreciate the relative importance of local formation versus potential leakage of microparticle from microvessels into the vitreous fluid , we determined the ratio of vitreous to plasma microparticles levels . this ratio was greater than unity only for endothelial cd144 microparticles in diabetic patients with pdr , suggesting that significant numbers of endothelial microparticles found in the vitreous fluid could be generated from local microvascular endothelial cells in pdr or that clearance of endothelial microparticles was abnormal . on the contrary , the ratio for platelet cd41 microparticles was lower than unity , favoring the interpretation that platelet microparticles present in pdr vitreous fluid likely originate from the plasma . complete laser photocoagulation causes regression of preretinal neovascularization by destroying the outer layers of the retina and improving the oxygen diffusion from the choroid to the inner retina ( 3841 ) , whereas intravitreal injection of the anti - vegf antibody bevacizumab leads to a complete resolution of angiographic leakage of neovascularization and to a rapid involution of retinal neovascularization ( 4245 ) . both treatments decreased levels of vitreous endothelial cd144 microparticles to values no longer different from those found in control patients . these observations further reinforce our findings that pdr is associated with a specific increase in local shedding of endothelial microparticles originating from new vessels . furthermore , bevacizumab decreased vitreous platelet cd41 microparticles to undetectable levels , which is a finding consistent with the decrease vascular leakage reported for the anti - vegf ocular therapy ( 42,46 ) . the present study shows that vitreous microparticles stimulate in vitro endothelial proliferation and in vivo new - vessel formation in a matrigel plug model . this finding is in agreement with previous studies showing that microparticles of different cellular origins ( platelet , leukocyte , or endothelial ) are proangiogenic ( 810,34,47 ) . however , the present data contrast with studies reporting the antiangiogenic effect of microparticles of endothelial or lymphocyte origin , which is associated with the stimulation of oxidative stress ( 48,49 ) . the reasons for this discrepancy are unknown but could result from the different nature or composition of microparticles . because of the paucity of microparticles in vitreous samples and their low recovery after isolation , we were unable to identify the cell origin of vitreous microparticles that affect endothelial cells . although the proangiogenic effect of platelet - derived microparticles is mediated in part by growth factors ( 8) , the mechanism of the proliferative effect of vitreous microparticles unlikely involves vegf because levels of this growth factor were below detection in isolated vitreous microparticles samples . because of the low recovery of the isolation procedure , the present data do not permit the examination of the proangiogenic effect of vitreous microparticles from nonproliferative diabetic retinopathy or from control patients . for similar reasons , we could not evaluate the biological effects of increasing concentrations of vitreous microparticles . in conclusion , we identified the presence in vitreous fluid of membrane microparticles shed from retinal , vascular , and circulating cells and their significant increase in patients with pdr , where they could contribute to disease progression .

objectivediabetic retinopathy is associated with progressive retinal capillary activation and proliferation , leading to vision impairment and blindness . microparticles are submicron membrane vesicles with biological activities , released following cell activation or apoptosis . we tested the hypothesis that proangiogenic microparticles accumulate in vitreous fluid in diabetic retinopathy.research design and methodslevels and cellular origin of vitreous and plasma microparticles from control ( n = 26 ) and diabetic ( n = 104 ) patients were analyzed by flow cytometry , and their proangiogenic activity was assessed by in vitro thymidine incorporation and neovessel formation in subcutaneous matrigel plugs in mice.resultsmicroparticles of endothelial , platelet , photoreceptor , and microglial origin were identified in vitreous samples . levels of photoreceptor and microglial microparticles were undetectable in plasmas but were comparable in diabetic and control vitreous samples . vitreous platelet and endothelial microparticles levels were increased in diabetic patients and decreased following panretinal laser photocoagulation or intravitreal antivascular endothelial growth factor injection in proliferative diabetic retinopathy ( pdr ) . the ratio of vitreous to plasma microparticle levels was calculated to estimate local formation versus potential plasma leakage . in pdr , the endothelial microparticles ratio but not that for platelet was greater than 1.0 , indicating local formation of endothelial microparticles from retinal vessels and permeation of platelet microparticles from plasma . isolated vitreous microparticles stimulated by 1.6-fold endothelial proliferation and increased new vessel formation in mice.conclusionsthe present study demonstrates that vitreous fluid contains shed membrane microparticles of endothelial , platelet , and retinal origin . vitreous microparticles levels are increased in patients with diabetic retinopathy , where they could contribute to disease progression .

RESEARCH DESIGN AND METHODS Microparticle isolation. Materials. Microparticle labeling and flow cytometry analysis. Isolation of vitreous microparticles for assessment of cell proliferation and angiogenesis. Assessment of cell proliferation. Matrigel angiogenesis assay. Statistical analysis. RESULTS Characterization of circulating and vitreous microparticles. Effect of therapies on vitreous microparticles levels in PDR. Vitreous microparticles induce endothelial cell proliferation and new vessel formation. DISCUSSION

ninety - eight samples were collected from the eyes of 93 patients with proliferative diabetic retinopathy ( pdr ) ; in these cases , vitrectomy was performed for persistent vitreous hemorrhage , tractional retinal detachment , or macular edema . in 11 additional diabetic patients , diabetic retinopathy was absent and vitrectomy the control group consisted of vitreous samples from 26 eyes of 26 nondiabetic patients with an idiopathic macular hole , an idiopathic epiretinal membrane , a rhegmatogenous retinal detachment , or age - related macular degeneration ( table 1 ) . representative traces of flow cytometry analysis of annexin v , platelet , endothelial photoreceptor , and microglial microparticles in human vitreous samples . levels of endothelial cd144 microparticles , the most abundant vitreous microparticle subpopulation in patients with pdr , were determined in each pellet using flow cytometry , and all proliferation experiments were performed at the final concentration of 65 cd144 microparticles/l . vitreous microparticles isolated using this procedure did not contain detectable levels of vegf when determined by immunoassay ( minimal detection limit of 3.3 pg / ml ; n = 6 ) . at 70% confluence , endothelial cells were rendered quiescent by replacing 10% fcs with 0.1% bsa for 24 h. endothelial cells were then incubated during 48 h with either 10% fcs ( used as a positive control ) , vitreous microparticles from patients with pdr ( corresponding to a final concentration of 65 microparticles/l of microparticles of endothelial origin ) , or vehicle . the concentration of microparticles used in these experiments was the highest concentration obtained after isolation of vitreous microparticles from patients with pdr . briefly , 500 l matrigel ( free of growth factors ) mixed with vitreous microparticles ( reaching a level of 5,000 cd144 microparticles of endothelial origin ) or vehicle was injected subcutaneously in the back of 16 7-week - old c57/bl6 mice . microparticles levels are expressed as median and range and analyzed by kruskall - wallis nonparametric analysis to compare levels between more than two groups , followed by mann - whitney analysis of two groups when appropriate . representative traces of flow cytometry analysis of annexin v , platelet , endothelial photoreceptor , and microglial microparticles in human vitreous samples . to study properties of vitreous microparticles on huvec proliferation and angiogenesis , microparticles were isolated from vitreous samples pooled from six consecutive patients with pdr . levels of endothelial cd144 microparticles , the most abundant vitreous microparticle subpopulation in patients with pdr , were determined in each pellet using flow cytometry , and all proliferation experiments were performed at the final concentration of 65 cd144 microparticles/l . vitreous microparticles isolated using this procedure did not contain detectable levels of vegf when determined by immunoassay ( minimal detection limit of 3.3 pg / ml ; n = 6 ) . at 70% confluence , endothelial cells were rendered quiescent by replacing 10% fcs with 0.1% bsa for 24 h. endothelial cells were then incubated during 48 h with either 10% fcs ( used as a positive control ) , vitreous microparticles from patients with pdr ( corresponding to a final concentration of 65 microparticles/l of microparticles of endothelial origin ) , or vehicle . the concentration of microparticles used in these experiments was the highest concentration obtained after isolation of vitreous microparticles from patients with pdr . microparticles were identified by flow cytometry analysis as events with 0.11 m positively labeled by specific antibodies ( fig . plasma levels of annexin v , platelet cd41 , and endothelial cd144 microparticles were not different in control and diabetic patients ( table 2 ) . microparticles from retinal origin ( pna or ilb4 microparticles ) were undetectable in plasma samples ( table 2 ) . vitreous levels of annexin v , platelet cd41 , and endothelial cd144 microparticles were all markedly increased in diabetic compared with control patients ( p = 0.035 , 0.018 , and < 0.001 , respectively ) ( fig . microparticles of endothelial origin , identified as expressing ve - cadherin ( cd144 ) , were the most abundant microparticle subpopulation in vitreous samples from diabetic patients . vitreous levels of annexin v , endothelial cd144 , and platelet cd41 microparticles were increased in association with pdr compared with nonpdr ( npdr ) ( p = 0.007 , 0.017 , and 0.018 , respectively ) ( table 2 ) . in addition , vitreous levels of annexin v , platelet cd41 , and endothelial cd144 microparticles were augmented in diabetic patients with pdr compared with control subjects ( p = 0.004 , 0.003 , and < 0.0001 , respectively ( fig . however , there were no differences between vitreous levels of retinal microparticles ( ilb4 and pna ) in diabetic patients and control patients . furthermore , circulating levels of endothelial - and platelet - derived microparticles were not different between control and diabetic patients with or without pdr ( fig . plasma levels of microparticles in control patients and in patients with or without pdr data are expressed as median ( range ) of microparticles/l . microparticles were identified of platelet ( cd41 ) and endothelial ( cd144 ) origin or from retinal origin ( pna and ilb4 ) . annexin v microparticles express phosphatidyserine and were positively labeled with annexin v. microparticles levels were analyzed by kruskall - wallis nonparametric analysis followed by mann - whitney analysis of two groups when appropriate . vitreous levels of annexin v ( annv+ ) , platelet ( cd41 ) , endothelial ( cd144 ) , photoreceptor ( pna ) and microglial ( ilb4 ) microparticles ( mps ) in diabetic and control patients . the ratio of vitreous to plasma microparticles levels indicates the importance of intraocular formation versus potential plasma leakage of microparticles from microvessels in vitreous fluid ( fig . this ratio was markedly greater than unity for only endothelial cd144 microparticles in pdr ( p = 0.02 ) , indicating either an intraocular shedding or an abnormal clearance of endothelial microparticles . ratio of vitreous to plasma levels of annexin v ( annv+ ) , platelet ( cd41 ) , and endothelial ( cd144 ) microparticles ( mps ) in control and diabetic patients with either pdr or npdr . in patients with pdr , complete panretinal photocoagulation ( prp ) significantly decreased vitreous endothelial microparticle levels ( p = 0.037 ) to reach values not different from those obtained in control patients . vitreous levels of annexin v ( annv+ ) ( a ) , platelet ( cd41 ) ( b ) , endothelial ( cd144 ) ( c ) , and retinal ( pna ) ( d ) microparticles in control subjects ( open bars ) and diabetic patients with pdr ( gray bars ) who underwent either no or incomplete laser panphotocoagulation ( prp ) , complete prp , or intravitreal injection of bevacizumab prior to vitrectomy . bevacizumab treatment significantly decreased vitreous levels of annexin v microparticles and endothelial cd144 microparticles ( p = 0.04 and p = 0.02 , respectively ) and led to a complete disappearance of platelet - derived cd41 microparticles in pdr vitreous samples ( fig . vitreous microparticles significantly increased h - thymidine incorporation , by 1.6-fold , compared with microparticle vehicle ( vitreous supernatant ; p = 0.029 ) . vitreous microparticles did not contain detectable levels of vegf when determined by immunoassay with a minimal detection limit of 3.3 pg / ml ( n = 6 ) . levels of h - thymidine incorporation are represented with endothelium cell basal medium ( dulbecco 's modified eagle 's medium [ dmem ] ) , fcs ( 2% ) , and vitreous microparticles ( mps ) . vitreous microparticles ( estimated as 65 cd144 microparticles/l for the endothelial subpopulation ) increased h - thymidine incorporation by 1.6-fold compared with vehicle ( vitreous supernatant without cd144 microparticles ) ( * p = 0.029 ) . in some plugs low concentration of vitreous microparticles ( 10 emp/l ; 5,000 emps added to 500 l matrigel ) induced endothelial cell migration ( score 1 ; n = 6 ) and new vessel formation ( score 2 ; n = 3 ) compared with vitreous supernatant ( score 0 ; n = 12 ) ( p = 0.001 ) . microparticles were identified by flow cytometry analysis as events with 0.11 m positively labeled by specific antibodies ( fig . plasma levels of annexin v , platelet cd41 , and endothelial cd144 microparticles were not different in control and diabetic patients ( table 2 ) . microparticles from retinal origin ( pna or ilb4 microparticles ) were undetectable in plasma samples ( table 2 ) . vitreous levels of annexin v , platelet cd41 , and endothelial cd144 microparticles were all markedly increased in diabetic compared with control patients ( p = 0.035 , 0.018 , and < 0.001 , respectively ) ( fig . microparticles of endothelial origin , identified as expressing ve - cadherin ( cd144 ) , were the most abundant microparticle subpopulation in vitreous samples from diabetic patients . vitreous levels of annexin v , endothelial cd144 , and platelet cd41 microparticles were increased in association with pdr compared with nonpdr ( npdr ) ( p = 0.007 , 0.017 , and 0.018 , respectively ) ( table 2 ) . in addition , vitreous levels of annexin v , platelet cd41 , and endothelial cd144 microparticles were augmented in diabetic patients with pdr compared with control subjects ( p = 0.004 , 0.003 , and < 0.0001 , respectively ( fig . however , there were no differences between vitreous levels of retinal microparticles ( ilb4 and pna ) in diabetic patients and control patients . furthermore , circulating levels of endothelial - and platelet - derived microparticles were not different between control and diabetic patients with or without pdr ( fig . plasma levels of microparticles in control patients and in patients with or without pdr data are expressed as median ( range ) of microparticles/l . microparticles were identified of platelet ( cd41 ) and endothelial ( cd144 ) origin or from retinal origin ( pna and ilb4 ) . annexin v microparticles express phosphatidyserine and were positively labeled with annexin v. microparticles levels were analyzed by kruskall - wallis nonparametric analysis followed by mann - whitney analysis of two groups when appropriate . * significant difference between npdr and pdr or between nondiabetic patients and patients with pdr , respectively . vitreous levels of annexin v ( annv+ ) , platelet ( cd41 ) , endothelial ( cd144 ) , photoreceptor ( pna ) and microglial ( ilb4 ) microparticles ( mps ) in diabetic and control patients . the ratio of vitreous to plasma microparticles levels indicates the importance of intraocular formation versus potential plasma leakage of microparticles from microvessels in vitreous fluid ( fig . this ratio was markedly greater than unity for only endothelial cd144 microparticles in pdr ( p = 0.02 ) , indicating either an intraocular shedding or an abnormal clearance of endothelial microparticles . ratio of vitreous to plasma levels of annexin v ( annv+ ) , platelet ( cd41 ) , and endothelial ( cd144 ) microparticles ( mps ) in control and diabetic patients with either pdr or npdr . in patients with pdr , complete panretinal photocoagulation ( prp ) significantly decreased vitreous endothelial microparticle levels ( p = 0.037 ) to reach values not different from those obtained in control patients . vitreous levels of annexin v ( annv+ ) ( a ) , platelet ( cd41 ) ( b ) , endothelial ( cd144 ) ( c ) , and retinal ( pna ) ( d ) microparticles in control subjects ( open bars ) and diabetic patients with pdr ( gray bars ) who underwent either no or incomplete laser panphotocoagulation ( prp ) , complete prp , or intravitreal injection of bevacizumab prior to vitrectomy . bevacizumab treatment significantly decreased vitreous levels of annexin v microparticles and endothelial cd144 microparticles ( p = 0.04 and p = 0.02 , respectively ) and led to a complete disappearance of platelet - derived cd41 microparticles in pdr vitreous samples ( fig . vitreous microparticles significantly increased h - thymidine incorporation , by 1.6-fold , compared with microparticle vehicle ( vitreous supernatant ; p = 0.029 ) . vitreous microparticles did not contain detectable levels of vegf when determined by immunoassay with a minimal detection limit of 3.3 pg / ml ( n = 6 ) . levels of h - thymidine incorporation are represented with endothelium cell basal medium ( dulbecco 's modified eagle 's medium [ dmem ] ) , fcs ( 2% ) , and vitreous microparticles ( mps ) . vitreous microparticles ( estimated as 65 cd144 microparticles/l for the endothelial subpopulation ) increased h - thymidine incorporation by 1.6-fold compared with vehicle ( vitreous supernatant without cd144 microparticles ) ( * p = 0.029 ) . in some plugs low concentration of vitreous microparticles ( 10 emp/l ; 5,000 emps added to 500 l matrigel ) induced endothelial cell migration ( score 1 ; n = 6 ) and new vessel formation ( score 2 ; n = 3 ) compared with vitreous supernatant ( score 0 ; n = 12 ) ( p = 0.001 ) . this study reveals , for the first time , the presence of submicron membrane vesicles shed from platelet , endothelial , and retinal cells in human vitreous samples of patients undergoing vitrectomy and demonstrates the specific vitreous accumulation of endothelial microparticles in patients with pdr . in addition , we show that vitreous microparticles isolated from patients with diabetic retinopathy stimulate endothelial cell proliferation and formation of new vessels , suggesting that they could contribute to retinal angiogenesis . plasma levels of microparticles of different cellular origins are increased in patients with high atherothrombotic risk or inflammatory diseases ( 15,16,31 ) . pdr is associated with ocular increases in oxidative stress , protein glycation , growth factors , inflammatory cytokines , and cell apoptosis all of which stimulate the shedding of membrane microparticles from retinal or vascular cells ( 1,4,15,16,21,22,24,34,35 ) . we observed significant increases in the overall pool of vitreous annexin v microparticles in pdr compared with control patients or patients with nonproliferative diabetic retinopathy . in addition , we demonstrate the presence of microparticles positively labeled with either pna or ilb4 in vitreous samples but not in plasma , indicating their photoreceptor or microglial origin , respectively ( 2529 ) . if anything , levels of microparticles derived from photoreceptors or microglia tend to increase in patients with pdr , but the difference did not reach statistical significance . microfracture of the internal limiting membrane is a likely hypothesis in our control patients , who were included for epimacular membrane or macular hole , and even more so in patients with pdr , where proliferating microvessels need to perforate the internal limiting membrane ( 36,37 ) . diabetic retinopathy is characterized by an augmented vascular permeability , and microparticles present in the blood could permeate through the leaky vascular wall into the vitreous fluid . to appreciate the relative importance of local formation versus potential leakage of microparticle from microvessels into the vitreous fluid , we determined the ratio of vitreous to plasma microparticles levels . this ratio was greater than unity only for endothelial cd144 microparticles in diabetic patients with pdr , suggesting that significant numbers of endothelial microparticles found in the vitreous fluid could be generated from local microvascular endothelial cells in pdr or that clearance of endothelial microparticles was abnormal . on the contrary , the ratio for platelet cd41 microparticles was lower than unity , favoring the interpretation that platelet microparticles present in pdr vitreous fluid likely originate from the plasma . these observations further reinforce our findings that pdr is associated with a specific increase in local shedding of endothelial microparticles originating from new vessels . the present study shows that vitreous microparticles stimulate in vitro endothelial proliferation and in vivo new - vessel formation in a matrigel plug model . however , the present data contrast with studies reporting the antiangiogenic effect of microparticles of endothelial or lymphocyte origin , which is associated with the stimulation of oxidative stress ( 48,49 ) . because of the paucity of microparticles in vitreous samples and their low recovery after isolation , we were unable to identify the cell origin of vitreous microparticles that affect endothelial cells . although the proangiogenic effect of platelet - derived microparticles is mediated in part by growth factors ( 8) , the mechanism of the proliferative effect of vitreous microparticles unlikely involves vegf because levels of this growth factor were below detection in isolated vitreous microparticles samples . because of the low recovery of the isolation procedure , the present data do not permit the examination of the proangiogenic effect of vitreous microparticles from nonproliferative diabetic retinopathy or from control patients . in conclusion , we identified the presence in vitreous fluid of membrane microparticles shed from retinal , vascular , and circulating cells and their significant increase in patients with pdr , where they could contribute to disease progression .

at least 44,000 people , and perhaps as many as 98,000 people , die in hospitals each year as a result of medical errors that could have been prevented . radio frequency identification ( rfid ) is a wireless technology capable of automatic and unambiguous identification without line of sight by extracting a unique identifier from microelectronic tags attached to objects . the rfid is a technology that uses radio waves to transfer data from an electronic tag , called rfid tag attached to an object , through a reader for the purpose of identifying and tracking the object . the rfid is already used to track and trace the victims in a disaster situation . data can be collected in real time and be immediately available to emergency personnel and saves time by the rfid . crisis management teams , hospitals and emergency personnel , have access to data through a computer database . the rfid was the first explored in the 1940s as a method to identify allied airplanes . today , the rfid system have been successfully applied to the areas of manufacturing , supply chain , agriculture , transportation , healthcare , and services to name a few . the rfid is valuable for quickly retrieving patient information and monitoring patient locations in the hospital . in the past decade the growing sophistication of computers and software should allow information technology to play a vital part in reducing that risk by streamlining care , catching and correcting errors , assisting with decisions , and providing feedback on performance . to our knowledge , there have been few previous reports based on on - site experiments showing that the rfid is suited for usage in a hospital , specifically for medication delivery and mapping nurse practice workflow . organizational resources and technical structures such as hardware and software chao et al , in a study titled improving patient safety with rfid and mobile technology announced these medical errors can be classified into five categories : poor decision making , poor communication , inadequate patient monitoring , patient misidentification , inability to respond rapidly and poor patient tracking . employing innovative information technologies in correcting these deficiencies and meeting the joint commission on accreditation of healthcare organization ( jcaho ) patient safety goals the result showed that the rfid adoption though assigned with low level of awareness ; adoption capital recompense and infrastructural challenges in the nigeria health sector in particular also has high and great tendencies to thrive . the result of the second research question revealed that the nigerian health care service delivery can harness the benefits of the information technology ( it ) solution system to function in its clientele servicing in forms like authentication and identification of personnel , patients data and blood verification ; drug dispensary among others . one study shows , an alarming statistic from an american healthcare organization shows that an average of 195000 people in the usa died in hospitals in each of the years 2000 , 2001 and 2002 as a result of potentially preventable , in - hospital medical errors asserts that the problem is not bad people in health care it is that good people are working in bad systems that need to be made safer . barriers and critical success factors towards rfid technology adoption in south - east asian healthcare industry showed that most respondents believed that barriers towards the rfid adoption are : ( 1 ) lack of information ; ( 2 ) insufficient budget available ; ( 3 ) complexity of technology and systems . critical success factors are : ( 1 ) top management support and the commitment of leadership ; ( 2 ) integrating the data collected ; ( 3 ) coordinating among departments ; and ( 4 ) starting with small the rfid project . the rfid technology offers healthcare practitioners advantages to improve patient safety , save time , and reduce costs but also causes critical issues for successful implementation . to increase the acceptance and wide use of rfid in healthcare , more customized the rfid systems , more institutional support , seamless integration with existing his , satisfactory security / privacy measures , and mature regulations to protect privacy are needed . the most rfid systems consist of tags that are attached to the objects to be identified . a typical configuration of this memory is to store product information , such as an object 's unique i d manufactured date , etc . the rfid reader generates magnetic fields that enable the rfid system to locate objects ( via the tags ) that are within its range . the high - frequency electromagnetic energy and query signal generated by the reader triggers the tags to reply to the query ; the query frequency could be up to 50 times per second . as a result communication between the main components of the system i.e. tags and reader supply chain industries control this problem by using filters that are routed to the backend information systems . in other words , in order to control this problem , software such as savant is used . the rfid system consists of various components which are integrated in a manner defined in the above section . this allows the rfid system to deduct the objects ( tag ) and perform various operations on it . the rfid system consists of following five components : tag ( attached to an object , unique identification).antenna ( tag detector , creates magnetic field).reader ( receiver of tag information , manipulator).communication infrastructure ( enable reader / rfid to work through it infrastructure).application software ( user database / application / interface ) . the role of the rfid technology in improving patient safety and increase the impact of this technology in the health care industry with was discussed . the most rfid systems consist of tags that are attached to the objects to be identified . a typical configuration of this memory is to store product information , such as an object 's unique i d manufactured date , etc . the rfid reader generates magnetic fields that enable the rfid system to locate objects ( via the tags ) that are within its range . the high - frequency electromagnetic energy and query signal generated by the reader triggers the tags to reply to the query ; the query frequency could be up to 50 times per second . as a result communication between the main components of the system i.e. tags and reader supply chain industries control this problem by using filters that are routed to the backend information systems . in other words , in order to control this problem , software such as savant is used . the rfid system consists of various components which are integrated in a manner defined in the above section . this allows the rfid system to deduct the objects ( tag ) and perform various operations on it . the rfid system consists of following five components : tag ( attached to an object , unique identification).antenna ( tag detector , creates magnetic field).reader ( receiver of tag information , manipulator).communication infrastructure ( enable reader / rfid to work through it infrastructure).application software ( user database / application / interface ) . the role of the rfid technology in improving patient safety and increase the impact of this technology in the health care industry with was discussed . this study was non - systematical review , which the literature search was conducted with the help of libraries , books , conference proceedings , pubmed databases and also search engines available at google , google scholar in which published between 2004 and 2013 during febuary 2013 . in our searches , we employed the following keywords and their combinations ; rfid , healthcare , patient safety , medical errors , and medication errors in the searching areas of title , keywords , abstract , and full text . we employed the following keywords and their combinations ; rfid , healthcare , patient safety , medical errors , and medication errors in the searching areas of titles , keywords , abstracts , and full texts . after a careful analysis of the content of each paper , a total of 33 papers was selected based on their relevancy . the rfid technology used for three purposes : tracking , inventory management , and validation . each of these characteristics could provide benefits that could yield cost savings and improved productivity . the rfid is used to follow a product through the supply chain and clinical workflow . it can be used to track a product to a particular patient and identify the clinician who used to say product to a patient . the rfid reduces the amount of time involved in locating or tracking , thereby making the process less cumbersome . it is a complex process that asks what ( what is in stock ) , how ( how much is it ) , who ( who has it ) , where ( where is it ) , and when ( when to reorder ) . the rfid helps manage patient inventories so that the right assets are available when and where they are needed . validation assures that an action has taken place or that the desired item is on hand . like tracking , the ability to validate through the rfid technology can reduce medical errors , check productivity , and help construct necessary documentation for administrative and audit purposes . the most important validating function is to verify that the patient being treated is , in fact , the right patient and that the treatment that is about to occur is appropriate . studies examining the relationship between the use of rfid and patient safety have been expressed as follows : patient safety will be improved and costs as well as medication errors will be reduced considerably . the rfid can help nurses to quickly identify patients and their corresponding medicine . using the rfid technology , such administration system provides automated medication verification for inpatient 's medicine doses and generates corresponding medication evidence , which may be audited later for medical dispute . the rfid had great potential for reducing medical errors and nurse workload with high efficiency . the information technology has to support healthcare in developing practices and nursing patients without confronting any complications or errors . one critical and important part of healthcare is medication care , which is very vulnerable for different kind of errors , even on fatal errors . thus , medication care needs new methods for avoiding errors in different situations during medication administration . this poster represents an rfid - based automated identification system for medication care in a hospital environment . the rfid makes the prevention about human error of medicine , easy and fast access to medical staff , equipment , medicine . all of these factors cause to increase the quality and quantity of care in the healthcare sector . in a study that its problem was mislabeling of tissue specimens in their gastrointestinal and colorectal surgery endoscopy units and most labeling errors have been due to either the wrong patient label or no label being affixed to a specimen bottle . as a result , this initiative involved the application of radiofrequency identification ( rfid ) technology to specimen bottles , moving to a paperless pathology requisition system and confirmation of the correct site and correct patient by both the endoscopy nursing staff and the endoscopist for each specimen bottle . their results confirmed that these data confirm that the initiation of a new specimen - labeling system that uses rfid technology , a paperless requisition process , and confirmation of the correct site and correct patient by two healthcare providers significantly decreased specimen - labeling errors at every level in a high - volume endoscopy center . implementation of a practical combined the rfid system that is fully integrated with the clinical system , which one day could be employed in large scale for improving clinical workflow and minimizing medical errors . wisely aware rfid dosage ( ward ) system , which based on an integration of barcodes and rfid tags , to demonstrate effective and safe patient care environment , for preventing the risk of medication error . the application of rfid can bring hospitals towards the integration of technology benefits and improved medical safety . the rfid by removing the human factor and would improve patient safety by eliminating the current surgical sponge count protocol . table 1 shows some examples of promising completed or on- going the rfid trials , pilots and applications by country . the rfid trials , pilots and applications by country most rfid applications in health care organizations in the above table , ultimately , patient safety has expressed that safety by reducing medical errors , and increase the quality of pharmaceutical and diagnostic services is achieved . the mobile nursing care system using rfid technology strengthens the capabilities of staff to track patient 's vital signs across various locations and in different medical facilities . the mobile intelligent medical system ( mims ) includes vital sign monitoring and alarming services , mobile nursing applications , and rule - based clinical decision support in a mobile nursing environment . the system continuously monitors critically ill patients with the objective of reducing the risk of serious harm resulting from the slow provision of health care . further , we also believe the system can be extended to most medical domains and integrated with other hospital information systems . with more medical centers linked into the system , the mims will bring better and safer medical services to the healthcare industry . the rfid technology has tangible benefits such as reduced cost , time , human resources , and prevention theft . the rfid is used to follow a product through the supply chain and clinical workflow . it can be used to track a product to a particular patient and identify the clinician who used to say product to a patient . the rfid reduces the amount of time involved in locating or tracking , thereby making the process less cumbersome . it is a complex process that asks what ( what is in stock ) , how ( how much is it ) , who ( who has it ) , where ( where is it ) , and when ( when to reorder ) . the rfid helps manage patient inventories so that the right assets are available when and where they are needed . validation assures that an action has taken place or that the desired item is on hand . like tracking , the ability to validate through the rfid technology can reduce medical errors , check productivity , and help construct necessary documentation for administrative and audit purposes . the most important validating function is to verify that the patient being treated is , in fact , the right patient and that the treatment that is about to occur is appropriate . studies examining the relationship between the use of rfid and patient safety have been expressed as follows : patient safety will be improved and costs as well as medication errors will be reduced considerably . the rfid can help nurses to quickly identify patients and their corresponding medicine . using the rfid technology , such administration system provides automated medication verification for inpatient 's medicine doses and generates corresponding medication evidence , which may be audited later for medical dispute . the rfid had great potential for reducing medical errors and nurse workload with high efficiency . the information technology has to support healthcare in developing practices and nursing patients without confronting any complications or errors . one critical and important part of healthcare is medication care , which is very vulnerable for different kind of errors , even on fatal errors . thus , medication care needs new methods for avoiding errors in different situations during medication administration . this poster represents an rfid - based automated identification system for medication care in a hospital environment . the rfid makes the prevention about human error of medicine , easy and fast access to medical staff , equipment , medicine . all of these factors cause to increase the quality and quantity of care in the healthcare sector . in a study that its problem was mislabeling of tissue specimens in their gastrointestinal and colorectal surgery endoscopy units and most labeling errors have been due to either the wrong patient label or no label being affixed to a specimen bottle . as a result , this initiative involved the application of radiofrequency identification ( rfid ) technology to specimen bottles , moving to a paperless pathology requisition system and confirmation of the correct site and correct patient by both the endoscopy nursing staff and the endoscopist for each specimen bottle . their results confirmed that these data confirm that the initiation of a new specimen - labeling system that uses rfid technology , a paperless requisition process , and confirmation of the correct site and correct patient by two healthcare providers significantly decreased specimen - labeling errors at every level in a high - volume endoscopy center . implementation of a practical combined the rfid system that is fully integrated with the clinical system , which one day could be employed in large scale for improving clinical workflow and minimizing medical errors . wisely aware rfid dosage ( ward ) system , which based on an integration of barcodes and rfid tags , to demonstrate effective and safe patient care environment , for preventing the risk of medication error . the application of rfid can bring hospitals towards the integration of technology benefits and improved medical safety . the rfid by removing the human factor and would improve patient safety by eliminating the current surgical sponge count protocol . table 1 shows some examples of promising completed or on- going the rfid trials , pilots and applications by country . the rfid trials , pilots and applications by country most rfid applications in health care organizations in the above table , ultimately , patient safety has expressed that safety by reducing medical errors , and increase the quality of pharmaceutical and diagnostic services is achieved . the mobile nursing care system using rfid technology strengthens the capabilities of staff to track patient 's vital signs across various locations and in different medical facilities . the mobile intelligent medical system ( mims ) includes vital sign monitoring and alarming services , mobile nursing applications , and rule - based clinical decision support in a mobile nursing environment . the system continuously monitors critically ill patients with the objective of reducing the risk of serious harm resulting from the slow provision of health care . further , we also believe the system can be extended to most medical domains and integrated with other hospital information systems . with more medical centers linked into the system , the mims will bring better and safer medical services to the healthcare industry . the rfid technology has tangible benefits such as reduced cost , time , human resources , and prevention theft . in the healthcare industry , there are other advantages for example ; increase accuracy tasks , reduce human errors , improve safety , and patient satisfaction . no doubt , in coming years , using of the rfid will be a requirement in the healthcare industry . although , the sole usage of rfid will not be able meet expectation of health care organizations . if it is used alone , health care organizations will be faced with numerous challenges . but , if we integrate it with hospital information systems ( his ) and electronic health records ( ehrs ) and support it by clinical decision support systems ( cdss ) , it facilitates magically processes and reduce medical , medication and diagnosis errors . what was already known on this topic ? there are critical successes in improving health services for patients through tracking ( staff , patients , and properties ) , inventory management , and validation ( medication , documents , treatment , and specimen).there are barriers to adopt the rfid that can classify into ; lack of information , insufficient budget available , and the complexity of technology and systems . there are critical successes in improving health services for patients through tracking ( staff , patients , and properties ) , inventory management , and validation ( medication , documents , treatment , and specimen ) . there are barriers to adopt the rfid that can classify into ; lack of information , insufficient budget available , and the complexity of technology and systems . this technology can facilitate to access individuals ( medical staff and patients ) and shorten wait time of care processes.the rfid technology has tangible benefits such as reduced cost and time , reduced human resources , theft prevention , improve productivity.in addition , in the healthcare industry , there are also intangible benefits include increasing accuracy tasks , refining business processes and reduce human errors , which will ultimately improve safety and patient satisfaction.use of the rfid in health care industry alone will be faced with numerous challenges . this technology can facilitate to access individuals ( medical staff and patients ) and shorten wait time of care processes . the rfid technology has tangible benefits such as reduced cost and time , reduced human resources , theft prevention , improve productivity . in addition , in the healthcare industry , there are also intangible benefits include increasing accuracy tasks , refining business processes and reduce human errors , which will ultimately improve safety and patient satisfaction . use of the rfid in health care industry alone will be faced with numerous challenges . to minimize medical errors and improve clinical workflow , we can implement the rfid system with the clinical system.if the rfid integrate with hospital information systems ( his ) and electronic health records ( ehrs ) and supported by clinical decision support systems ( cdss ) , it can facilitate processes , reduce medication and diagnosis errors , and increase patient safety . to minimize medical errors and improve clinical workflow , we can implement the rfid system with the clinical system . if the rfid integrate with hospital information systems ( his ) and electronic health records ( ehrs ) and supported by clinical decision support systems ( cdss ) , it can facilitate processes , reduce medication and diagnosis errors , and increase patient safety . what was already known on this topic ? there are critical successes in improving health services for patients through tracking ( staff , patients , and properties ) , inventory management , and validation ( medication , documents , treatment , and specimen).there are barriers to adopt the rfid that can classify into ; lack of information , insufficient budget available , and the complexity of technology and systems . there are critical successes in improving health services for patients through tracking ( staff , patients , and properties ) , inventory management , and validation ( medication , documents , treatment , and specimen ) . there are barriers to adopt the rfid that can classify into ; lack of information , insufficient budget available , and the complexity of technology and systems . this technology can facilitate to access individuals ( medical staff and patients ) and shorten wait time of care processes.the rfid technology has tangible benefits such as reduced cost and time , reduced human resources , theft prevention , improve productivity.in addition , in the healthcare industry , there are also intangible benefits include increasing accuracy tasks , refining business processes and reduce human errors , which will ultimately improve safety and patient satisfaction.use of the rfid in health care industry alone will be faced with numerous challenges . this technology can facilitate to access individuals ( medical staff and patients ) and shorten wait time of care processes . the rfid technology has tangible benefits such as reduced cost and time , reduced human resources , theft prevention , improve productivity . in addition , in the healthcare industry , there are also intangible benefits include increasing accuracy tasks , refining business processes and reduce human errors , which will ultimately improve safety and patient satisfaction . use of the rfid in health care industry alone will be faced with numerous challenges . to minimize medical errors and improve clinical workflow , we can implement the rfid system with the clinical system.if the rfid integrate with hospital information systems ( his ) and electronic health records ( ehrs ) and supported by clinical decision support systems ( cdss ) , it can facilitate processes , reduce medication and diagnosis errors , and increase patient safety . to minimize medical errors and improve clinical workflow if the rfid integrate with hospital information systems ( his ) and electronic health records ( ehrs ) and supported by clinical decision support systems ( cdss ) , it can facilitate processes , reduce medication and diagnosis errors , and increase patient safety .

background : radio frequency identification ( rfid ) systems have been successfully applied in areas of manufacturing , supply chain , agriculture , transportation , healthcare , and services to name a few . however , the different advantages and disadvantages expressed in various studies of the challenges facing the technology of the use of the rfid technology have been met with skepticism by managers of healthcare organizations . the aim of this study was to express and display the role of rfid technology in improving patient safety and increasing the impact of it in healthcare.materials and methods : this study was non - systematical review , which the literature search was conducted with the help of libraries , books , conference proceedings , pubmed databases and also search engines available at google , google scholar in which published between 2004 and 2013 during febuary 2013 . we employed the following keywords and their combinations ; rfid , healthcare , patient safety , medical errors , and medication errors in the searching areas of title , keywords , abstract , and full text.results:the preliminary search resulted in 68 articles . after a careful analysis of the content of each paper , a total of 33 papers was selected based on their relevancy.conclusion:we should integrate rfid with hospital information systems ( his ) and electronic health records ( ehrs ) and support it by clinical decision support systems ( cdss ) , it facilitates processes and reduce medical , medication and diagnosis errors .

INTRODUCTION How the RFID system works Components of an RFID system MATERIALS AND METHODS RESULTS Tracking Inventory management Validation CONCLUSIONS Summary points Highlights

at least 44,000 people , and perhaps as many as 98,000 people , die in hospitals each year as a result of medical errors that could have been prevented . radio frequency identification ( rfid ) is a wireless technology capable of automatic and unambiguous identification without line of sight by extracting a unique identifier from microelectronic tags attached to objects . the rfid is a technology that uses radio waves to transfer data from an electronic tag , called rfid tag attached to an object , through a reader for the purpose of identifying and tracking the object . the rfid is already used to track and trace the victims in a disaster situation . data can be collected in real time and be immediately available to emergency personnel and saves time by the rfid . the rfid was the first explored in the 1940s as a method to identify allied airplanes . today , the rfid system have been successfully applied to the areas of manufacturing , supply chain , agriculture , transportation , healthcare , and services to name a few . the rfid is valuable for quickly retrieving patient information and monitoring patient locations in the hospital . in the past decade the growing sophistication of computers and software should allow information technology to play a vital part in reducing that risk by streamlining care , catching and correcting errors , assisting with decisions , and providing feedback on performance . to our knowledge , there have been few previous reports based on on - site experiments showing that the rfid is suited for usage in a hospital , specifically for medication delivery and mapping nurse practice workflow . organizational resources and technical structures such as hardware and software chao et al , in a study titled improving patient safety with rfid and mobile technology announced these medical errors can be classified into five categories : poor decision making , poor communication , inadequate patient monitoring , patient misidentification , inability to respond rapidly and poor patient tracking . employing innovative information technologies in correcting these deficiencies and meeting the joint commission on accreditation of healthcare organization ( jcaho ) patient safety goals the result showed that the rfid adoption though assigned with low level of awareness ; adoption capital recompense and infrastructural challenges in the nigeria health sector in particular also has high and great tendencies to thrive . the result of the second research question revealed that the nigerian health care service delivery can harness the benefits of the information technology ( it ) solution system to function in its clientele servicing in forms like authentication and identification of personnel , patients data and blood verification ; drug dispensary among others . one study shows , an alarming statistic from an american healthcare organization shows that an average of 195000 people in the usa died in hospitals in each of the years 2000 , 2001 and 2002 as a result of potentially preventable , in - hospital medical errors asserts that the problem is not bad people in health care it is that good people are working in bad systems that need to be made safer . barriers and critical success factors towards rfid technology adoption in south - east asian healthcare industry showed that most respondents believed that barriers towards the rfid adoption are : ( 1 ) lack of information ; ( 2 ) insufficient budget available ; ( 3 ) complexity of technology and systems . critical success factors are : ( 1 ) top management support and the commitment of leadership ; ( 2 ) integrating the data collected ; ( 3 ) coordinating among departments ; and ( 4 ) starting with small the rfid project . the rfid technology offers healthcare practitioners advantages to improve patient safety , save time , and reduce costs but also causes critical issues for successful implementation . to increase the acceptance and wide use of rfid in healthcare , more customized the rfid systems , more institutional support , seamless integration with existing his , satisfactory security / privacy measures , and mature regulations to protect privacy are needed . a typical configuration of this memory is to store product information , such as an object 's unique i d manufactured date , etc . the rfid reader generates magnetic fields that enable the rfid system to locate objects ( via the tags ) that are within its range . as a result communication between the main components of the system i.e. tags and reader supply chain industries control this problem by using filters that are routed to the backend information systems . the rfid system consists of various components which are integrated in a manner defined in the above section . this allows the rfid system to deduct the objects ( tag ) and perform various operations on it . the rfid system consists of following five components : tag ( attached to an object , unique identification).antenna ( tag detector , creates magnetic field).reader ( receiver of tag information , manipulator).communication infrastructure ( enable reader / rfid to work through it infrastructure).application software ( user database / application / interface ) . the role of the rfid technology in improving patient safety and increase the impact of this technology in the health care industry with was discussed . a typical configuration of this memory is to store product information , such as an object 's unique i d manufactured date , etc . the rfid reader generates magnetic fields that enable the rfid system to locate objects ( via the tags ) that are within its range . tags and reader supply chain industries control this problem by using filters that are routed to the backend information systems . the rfid system consists of various components which are integrated in a manner defined in the above section . this allows the rfid system to deduct the objects ( tag ) and perform various operations on it . the rfid system consists of following five components : tag ( attached to an object , unique identification).antenna ( tag detector , creates magnetic field).reader ( receiver of tag information , manipulator).communication infrastructure ( enable reader / rfid to work through it infrastructure).application software ( user database / application / interface ) . the role of the rfid technology in improving patient safety and increase the impact of this technology in the health care industry with was discussed . this study was non - systematical review , which the literature search was conducted with the help of libraries , books , conference proceedings , pubmed databases and also search engines available at google , google scholar in which published between 2004 and 2013 during febuary 2013 . in our searches , we employed the following keywords and their combinations ; rfid , healthcare , patient safety , medical errors , and medication errors in the searching areas of title , keywords , abstract , and full text . we employed the following keywords and their combinations ; rfid , healthcare , patient safety , medical errors , and medication errors in the searching areas of titles , keywords , abstracts , and full texts . after a careful analysis of the content of each paper , a total of 33 papers was selected based on their relevancy . the rfid technology used for three purposes : tracking , inventory management , and validation . the rfid is used to follow a product through the supply chain and clinical workflow . the rfid reduces the amount of time involved in locating or tracking , thereby making the process less cumbersome . it is a complex process that asks what ( what is in stock ) , how ( how much is it ) , who ( who has it ) , where ( where is it ) , and when ( when to reorder ) . the rfid helps manage patient inventories so that the right assets are available when and where they are needed . like tracking , the ability to validate through the rfid technology can reduce medical errors , check productivity , and help construct necessary documentation for administrative and audit purposes . the most important validating function is to verify that the patient being treated is , in fact , the right patient and that the treatment that is about to occur is appropriate . studies examining the relationship between the use of rfid and patient safety have been expressed as follows : patient safety will be improved and costs as well as medication errors will be reduced considerably . the rfid can help nurses to quickly identify patients and their corresponding medicine . using the rfid technology , such administration system provides automated medication verification for inpatient 's medicine doses and generates corresponding medication evidence , which may be audited later for medical dispute . the rfid had great potential for reducing medical errors and nurse workload with high efficiency . one critical and important part of healthcare is medication care , which is very vulnerable for different kind of errors , even on fatal errors . thus , medication care needs new methods for avoiding errors in different situations during medication administration . the rfid makes the prevention about human error of medicine , easy and fast access to medical staff , equipment , medicine . all of these factors cause to increase the quality and quantity of care in the healthcare sector . in a study that its problem was mislabeling of tissue specimens in their gastrointestinal and colorectal surgery endoscopy units and most labeling errors have been due to either the wrong patient label or no label being affixed to a specimen bottle . as a result , this initiative involved the application of radiofrequency identification ( rfid ) technology to specimen bottles , moving to a paperless pathology requisition system and confirmation of the correct site and correct patient by both the endoscopy nursing staff and the endoscopist for each specimen bottle . their results confirmed that these data confirm that the initiation of a new specimen - labeling system that uses rfid technology , a paperless requisition process , and confirmation of the correct site and correct patient by two healthcare providers significantly decreased specimen - labeling errors at every level in a high - volume endoscopy center . implementation of a practical combined the rfid system that is fully integrated with the clinical system , which one day could be employed in large scale for improving clinical workflow and minimizing medical errors . wisely aware rfid dosage ( ward ) system , which based on an integration of barcodes and rfid tags , to demonstrate effective and safe patient care environment , for preventing the risk of medication error . the application of rfid can bring hospitals towards the integration of technology benefits and improved medical safety . the rfid by removing the human factor and would improve patient safety by eliminating the current surgical sponge count protocol . table 1 shows some examples of promising completed or on- going the rfid trials , pilots and applications by country . the rfid trials , pilots and applications by country most rfid applications in health care organizations in the above table , ultimately , patient safety has expressed that safety by reducing medical errors , and increase the quality of pharmaceutical and diagnostic services is achieved . the mobile nursing care system using rfid technology strengthens the capabilities of staff to track patient 's vital signs across various locations and in different medical facilities . the mobile intelligent medical system ( mims ) includes vital sign monitoring and alarming services , mobile nursing applications , and rule - based clinical decision support in a mobile nursing environment . further , we also believe the system can be extended to most medical domains and integrated with other hospital information systems . with more medical centers linked into the system , the mims will bring better and safer medical services to the healthcare industry . the rfid technology has tangible benefits such as reduced cost , time , human resources , and prevention theft . the rfid is used to follow a product through the supply chain and clinical workflow . it is a complex process that asks what ( what is in stock ) , how ( how much is it ) , who ( who has it ) , where ( where is it ) , and when ( when to reorder ) . the rfid helps manage patient inventories so that the right assets are available when and where they are needed . like tracking , the ability to validate through the rfid technology can reduce medical errors , check productivity , and help construct necessary documentation for administrative and audit purposes . the most important validating function is to verify that the patient being treated is , in fact , the right patient and that the treatment that is about to occur is appropriate . studies examining the relationship between the use of rfid and patient safety have been expressed as follows : patient safety will be improved and costs as well as medication errors will be reduced considerably . the rfid can help nurses to quickly identify patients and their corresponding medicine . using the rfid technology , such administration system provides automated medication verification for inpatient 's medicine doses and generates corresponding medication evidence , which may be audited later for medical dispute . the rfid had great potential for reducing medical errors and nurse workload with high efficiency . one critical and important part of healthcare is medication care , which is very vulnerable for different kind of errors , even on fatal errors . thus , medication care needs new methods for avoiding errors in different situations during medication administration . the rfid makes the prevention about human error of medicine , easy and fast access to medical staff , equipment , medicine . all of these factors cause to increase the quality and quantity of care in the healthcare sector . as a result , this initiative involved the application of radiofrequency identification ( rfid ) technology to specimen bottles , moving to a paperless pathology requisition system and confirmation of the correct site and correct patient by both the endoscopy nursing staff and the endoscopist for each specimen bottle . their results confirmed that these data confirm that the initiation of a new specimen - labeling system that uses rfid technology , a paperless requisition process , and confirmation of the correct site and correct patient by two healthcare providers significantly decreased specimen - labeling errors at every level in a high - volume endoscopy center . implementation of a practical combined the rfid system that is fully integrated with the clinical system , which one day could be employed in large scale for improving clinical workflow and minimizing medical errors . wisely aware rfid dosage ( ward ) system , which based on an integration of barcodes and rfid tags , to demonstrate effective and safe patient care environment , for preventing the risk of medication error . the application of rfid can bring hospitals towards the integration of technology benefits and improved medical safety . the rfid by removing the human factor and would improve patient safety by eliminating the current surgical sponge count protocol . table 1 shows some examples of promising completed or on- going the rfid trials , pilots and applications by country . the rfid trials , pilots and applications by country most rfid applications in health care organizations in the above table , ultimately , patient safety has expressed that safety by reducing medical errors , and increase the quality of pharmaceutical and diagnostic services is achieved . the mobile nursing care system using rfid technology strengthens the capabilities of staff to track patient 's vital signs across various locations and in different medical facilities . the mobile intelligent medical system ( mims ) includes vital sign monitoring and alarming services , mobile nursing applications , and rule - based clinical decision support in a mobile nursing environment . further , we also believe the system can be extended to most medical domains and integrated with other hospital information systems . with more medical centers linked into the system , the mims will bring better and safer medical services to the healthcare industry . the rfid technology has tangible benefits such as reduced cost , time , human resources , and prevention theft . in the healthcare industry , there are other advantages for example ; increase accuracy tasks , reduce human errors , improve safety , and patient satisfaction . no doubt , in coming years , using of the rfid will be a requirement in the healthcare industry . although , the sole usage of rfid will not be able meet expectation of health care organizations . but , if we integrate it with hospital information systems ( his ) and electronic health records ( ehrs ) and support it by clinical decision support systems ( cdss ) , it facilitates magically processes and reduce medical , medication and diagnosis errors . there are critical successes in improving health services for patients through tracking ( staff , patients , and properties ) , inventory management , and validation ( medication , documents , treatment , and specimen).there are barriers to adopt the rfid that can classify into ; lack of information , insufficient budget available , and the complexity of technology and systems . there are critical successes in improving health services for patients through tracking ( staff , patients , and properties ) , inventory management , and validation ( medication , documents , treatment , and specimen ) . there are barriers to adopt the rfid that can classify into ; lack of information , insufficient budget available , and the complexity of technology and systems . this technology can facilitate to access individuals ( medical staff and patients ) and shorten wait time of care processes.the rfid technology has tangible benefits such as reduced cost and time , reduced human resources , theft prevention , improve productivity.in addition , in the healthcare industry , there are also intangible benefits include increasing accuracy tasks , refining business processes and reduce human errors , which will ultimately improve safety and patient satisfaction.use of the rfid in health care industry alone will be faced with numerous challenges . this technology can facilitate to access individuals ( medical staff and patients ) and shorten wait time of care processes . the rfid technology has tangible benefits such as reduced cost and time , reduced human resources , theft prevention , improve productivity . in addition , in the healthcare industry , there are also intangible benefits include increasing accuracy tasks , refining business processes and reduce human errors , which will ultimately improve safety and patient satisfaction . use of the rfid in health care industry alone will be faced with numerous challenges . to minimize medical errors and improve clinical workflow , we can implement the rfid system with the clinical system.if the rfid integrate with hospital information systems ( his ) and electronic health records ( ehrs ) and supported by clinical decision support systems ( cdss ) , it can facilitate processes , reduce medication and diagnosis errors , and increase patient safety . to minimize medical errors and improve clinical workflow , we can implement the rfid system with the clinical system . if the rfid integrate with hospital information systems ( his ) and electronic health records ( ehrs ) and supported by clinical decision support systems ( cdss ) , it can facilitate processes , reduce medication and diagnosis errors , and increase patient safety . there are critical successes in improving health services for patients through tracking ( staff , patients , and properties ) , inventory management , and validation ( medication , documents , treatment , and specimen).there are barriers to adopt the rfid that can classify into ; lack of information , insufficient budget available , and the complexity of technology and systems . there are critical successes in improving health services for patients through tracking ( staff , patients , and properties ) , inventory management , and validation ( medication , documents , treatment , and specimen ) . there are barriers to adopt the rfid that can classify into ; lack of information , insufficient budget available , and the complexity of technology and systems . this technology can facilitate to access individuals ( medical staff and patients ) and shorten wait time of care processes.the rfid technology has tangible benefits such as reduced cost and time , reduced human resources , theft prevention , improve productivity.in addition , in the healthcare industry , there are also intangible benefits include increasing accuracy tasks , refining business processes and reduce human errors , which will ultimately improve safety and patient satisfaction.use of the rfid in health care industry alone will be faced with numerous challenges . this technology can facilitate to access individuals ( medical staff and patients ) and shorten wait time of care processes . the rfid technology has tangible benefits such as reduced cost and time , reduced human resources , theft prevention , improve productivity . in addition , in the healthcare industry , there are also intangible benefits include increasing accuracy tasks , refining business processes and reduce human errors , which will ultimately improve safety and patient satisfaction . use of the rfid in health care industry alone will be faced with numerous challenges . to minimize medical errors and improve clinical workflow , we can implement the rfid system with the clinical system.if the rfid integrate with hospital information systems ( his ) and electronic health records ( ehrs ) and supported by clinical decision support systems ( cdss ) , it can facilitate processes , reduce medication and diagnosis errors , and increase patient safety . to minimize medical errors and improve clinical workflow if the rfid integrate with hospital information systems ( his ) and electronic health records ( ehrs ) and supported by clinical decision support systems ( cdss ) , it can facilitate processes , reduce medication and diagnosis errors , and increase patient safety .

in 1913 goodman levy [ 24 , 25 ] showed that chloroform - induced ventricular tachyarrhythmias were abolished by cardiac sympathetic denervation . additionally , sympathetic activation induced identical ventricular tachyarrhythmias , indicating a direct neurological rather than cardiac effect of chloroform . subsequently , beattie showed that these tachyarrhythmias were abolished by mid - collicular , but not higher , diencephalic sectioning . histological examination indicated degeneration of a pathway originating in the hypothalamus and terminating in the intermediolateral column ( close to the cells of origin of sympathetic preganglionic fibers ) . aschenbrenner reported seven young patients with cortical and diencephalic tumors who developed ventricular ectopy or nodal rhythm . these patients lacked cardiac history or post mortem ischemic cardiac pathology to explain their death . much of this was forgotten with the advent of world war ii . in the aftermath , single case reports of intriguing ecg repolarization changes after subarachnoid hemorrhage ( sah ) were reported : inverted t waves ( often tall , symmetrical downward peaking ) and prolonged qtc interval with st elevation or depression . cropp initially thought these changes indicated coronary artery disease and delayed surgery , with fatal consequences . no patient had a cardiac history , most were young and no autopsied case showed coronary artery pathology . aneurysm position led to implication of inferior frontal structures and possible vagal mechanisms in the etiology of these cardiac changes . recently , kono compared 7 sah patients with repolarization changes and 5 in whom no ecg abnormalities were detected . additional to subendocardial hemorrhages , scattered myocyte necrosis was observed surrounded by infiltrating monocytes and interstitial hemorrhages ( myocytolysis ) . electron microscopy showed clustering of cardiac pathology around nerves and indicated a neural rather than vascular origin . others investigated stress as a pointer to cerebral - cardiac interactions . in a holter study , stress induced ventricular ectopy when healthy individuals addressed an audience . in those with documented cardiac disease , frequency and severity of ventricular ectopy toivonen investigated arousal effects on the ambulatory ecg of 30 healthy physicians without known heart disease . recordings were compared just before and during emergency calls . the t wave inverted in 67 % subjects and asymptomatic st depression as rr interval shortened , the qt interval failed to show the expected decrease . thus , arousal can significantly affect ventricular repolarization even in putatively normal hearts , presumably through sympathetic mechanisms . coupled with the resemblance of myocytolysis to phaeochromocytoma cardiomyopathy , the cardiac pathology accompanying catecholamine infusion and prolonged stress , these observations strongly indicate that sympathetic neural activation can destabilize cardiac structure and function . much recent evidence indicates the arrhythmogenic potential of catecholamines . mid - myocardial cells have been identified in the dog and human ventricle sensitive to catecholamines which may underlie the pro - arrhythmic effects of cardiac sympathetic activation . in this regard , cao compared cardiac innervation density in patients with or without history of ventricular arrhythmias . increased sympathetic nerve density was found around blood vessels and areas of myocardial damage only in those with an arrhythmia history . they then infused nerve growth factor ( ngf ) into canine left stellate ganglia . when combined with av block and experimental mi , vt and sudden cardiac death occurred only in the ngf - treated animals . this suggested that sympathetic neural remodeling might facilitate ventricular arrhythmias and contribute to sudden cardiac death . conversely , manitius - robeck reported ictal sinus arrest during temporal lobe seizures . in one patient , cortical dysplasia was associated with a near - complete absence of mibg - spect determined cardiac sympathetic innervation . the bradyarrhythmias were abolished by surgery indicating a permissive interaction and developmental link between cortical dysplasia and reduced cardiac sympathetic innervation . stroke is associated with an adverse long - term cardiac outlook , possibly relating to altered sympathovagal balance . barron reported that compared with age / gender matched controls , total spectral power of rr intervals was reduced after stroke ; power associated with cardiac parasympathetic neural activity was particularly affected indicating a shift in sympathovagal balance . strittmatter reported that cardiac sympathetic tone was increased within 5 days of stroke and similar findings were reported by giubilei , who also showed persistence for at least 3 weeks . additionally , korpelainen et al . demonstrated that diurnal cardiac autonomic variability was disturbed after acute stroke with a striking loss of nocturnal vagal dominance . sander [ 42 , 44 ] reported that absence of circadian cardiovascular variation was particularly striking after insular infarction . these patients had higher plasma no - radrenaline levels , increased nocturnal blood pressure , a higher incidence of qtc interval prolongation and ventricular arrhythmias than patients with stroke in other locations . however , in these , and many other such studies the contribution of concomitant coronary artery disease and medications were not assessed . collectively these data strongly indicate that the brain has a major influence on cardiac structure and function and that this is likely mediated through alterations in patterning of sympathovagal relationships . in 1913 goodman levy [ 24 , 25 ] showed that chloroform - induced ventricular tachyarrhythmias were abolished by cardiac sympathetic denervation . additionally , sympathetic activation induced identical ventricular tachyarrhythmias , indicating a direct neurological rather than cardiac effect of chloroform . subsequently , beattie showed that these tachyarrhythmias were abolished by mid - collicular , but not higher , diencephalic sectioning . histological examination indicated degeneration of a pathway originating in the hypothalamus and terminating in the intermediolateral column ( close to the cells of origin of sympathetic preganglionic fibers ) . aschenbrenner reported seven young patients with cortical and diencephalic tumors who developed ventricular ectopy or nodal rhythm . these patients lacked cardiac history or post mortem ischemic cardiac pathology to explain their death . much of this was forgotten with the advent of world war ii . in the aftermath , single case reports of intriguing ecg repolarization changes after subarachnoid hemorrhage ( sah ) were reported : inverted t waves ( often tall , symmetrical downward peaking ) and prolonged qtc interval with st elevation or depression . cropp initially thought these changes indicated coronary artery disease and delayed surgery , with fatal consequences . no patient had a cardiac history , most were young and no autopsied case showed coronary artery pathology . aneurysm position led to implication of inferior frontal structures and possible vagal mechanisms in the etiology of these cardiac changes . recently , kono compared 7 sah patients with repolarization changes and 5 in whom no ecg abnormalities were detected . additional to subendocardial hemorrhages , scattered myocyte necrosis was observed surrounded by infiltrating monocytes and interstitial hemorrhages ( myocytolysis ) . electron microscopy showed clustering of cardiac pathology around nerves and indicated a neural rather than vascular origin . others investigated stress as a pointer to cerebral - cardiac interactions . in a holter study , stress induced ventricular ectopy when healthy individuals addressed an audience . in those with documented cardiac disease , frequency and severity of ventricular ectopy toivonen investigated arousal effects on the ambulatory ecg of 30 healthy physicians without known heart disease . recordings were compared just before and during emergency calls . the t wave inverted in 67 % subjects and asymptomatic st depression as rr interval shortened , the qt interval failed to show the expected decrease . thus , arousal can significantly affect ventricular repolarization even in putatively normal hearts , presumably through sympathetic mechanisms . coupled with the resemblance of myocytolysis to phaeochromocytoma cardiomyopathy , the cardiac pathology accompanying catecholamine infusion and prolonged stress , these observations strongly indicate that sympathetic neural activation can destabilize cardiac structure and function . much recent evidence indicates the arrhythmogenic potential of catecholamines . mid - myocardial cells have been identified in the dog and human ventricle sensitive to catecholamines which may underlie the pro - arrhythmic effects of cardiac sympathetic activation . in this regard , cao compared cardiac innervation density in patients with or without history of ventricular arrhythmias . increased sympathetic nerve density was found around blood vessels and areas of myocardial damage only in those with an arrhythmia history . they then infused nerve growth factor ( ngf ) into canine left stellate ganglia . when combined with av block and experimental mi , vt and sudden cardiac death occurred only in the ngf - treated animals . this suggested that sympathetic neural remodeling might facilitate ventricular arrhythmias and contribute to sudden cardiac death . conversely , manitius - robeck reported ictal sinus arrest during temporal lobe seizures . in one patient , cortical dysplasia was associated with a near - complete absence of mibg - spect determined cardiac sympathetic innervation . the bradyarrhythmias were abolished by surgery indicating a permissive interaction and developmental link between cortical dysplasia and reduced cardiac sympathetic innervation . stroke is associated with an adverse long - term cardiac outlook , possibly relating to altered sympathovagal balance . barron reported that compared with age / gender matched controls , total spectral power of rr intervals was reduced after stroke ; power associated with cardiac parasympathetic neural activity was particularly affected indicating a shift in sympathovagal balance . strittmatter reported that cardiac sympathetic tone was increased within 5 days of stroke and similar findings were reported by giubilei , who also showed persistence for at least 3 weeks . additionally , korpelainen et al . demonstrated that diurnal cardiac autonomic variability was disturbed after acute stroke with a striking loss of nocturnal vagal dominance . sander [ 42 , 44 ] reported that absence of circadian cardiovascular variation was particularly striking after insular infarction . these patients had higher plasma no - radrenaline levels , increased nocturnal blood pressure , a higher incidence of qtc interval prolongation and ventricular arrhythmias than patients with stroke in other locations . however , in these , and many other such studies the contribution of concomitant coronary artery disease and medications were not assessed . collectively these data strongly indicate that the brain has a major influence on cardiac structure and function and that this is likely mediated through alterations in patterning of sympathovagal relationships . while evidence for cerebral involvement in cardiac dysfunction is persuasive , frequency , predisposing circumstances and relevance for outcome are unclear . regard , early traces of ( non - ischemic ) myocytolysis were found in 26% of patients dying of non - ischemic causes ( pneumonia , sepsis ) and in 89% of sah , 71% of intracerebral hemorrhage , and 52% of ischemic stroke deaths . while indicative , these data come from a select group of patients for whom post mortem data were available . stroke may provide a good model to investigate neurocardiac influences : lesion location may be identified with precision , and onset categorized with some accuracy . however few studies detail arrhythmia incidence after stroke , and even fewer have controlled for concomitant coronary artery disease . comparing ecgs before and after stroke , lavy indicated a 39 % incidence of new onset cardiac arrhythmias in patients without cardiac history . goldstein reviewed 53 acute stroke patients ( including sah ) ecgs obtained within 24 hours of admission and recordings taken an average of 4 months earlier . qt prolongation of new onset was seen in 32% of strokes ( against 2% of controls ) ; t wave inversion and u waves were present in 15% of stroke patients and in none of the controls . new onset cardiac arrhythmias ( of which atrial fibrillation was the most frequent ) were present in 25% of all stroke patients , and 3% of controls . curiously , there was no difference in ventricular arrhythmia incidence between the two groups . however , this may not be too surprising since the ecg underestimates arrhythmia incidence . in other studies , where holter recording has been used , ventricular arrhythmia incidence after stroke was higher , varying from 2575% without control for co - existing cardiac disorders or for whether these were new or pre - existing . in yamour s uncontrolled study of intracerebral hemorrhage patients , comparison with pre - event ecg status indicated a new onset ventricular arrhythmia in 10% of patients and interestingly correlated with a temporoparietal location . in our recent unpublished observations , ventricular tachyarrhythmias ventricular premature beats occurred in 2.4% of strokes , and in 2% of 37 patients admitted with transient ischemic attacks ( tia ) . tia patients served as controls with similar demographics , incidence of coronary artery disease ( 70% ) , atherosclerotic risk factors and medication experience . however , holter monitoring revealed a ventricular tachyarrhythmia incidence of 12% in stroke , and 3% in tia patients , bigeminy in 21% and 5% respectively , ventricular ectopy in 71% and 73% respectively , and atrial fibrillation in 9% and 3% respectively . atrial fibrillation is commonly found in ecg studies of stroke [ 14 , 23 ] . however , when pre - event ecgs are used for comparison , it is unclear whether paroxysmal arrhythmia preceded and in some cases , caused stroke because of embolic potential . additionally , most reports do not control for cardiac concomitants ( indeed , 40% of acute stroke patients without cardiac symptoms are reported to have > 70% stenosis on coronary angiography ) . cardiac arrhythmia incidence is reportedly highest after sah ( 98 % ) , of which multifocal ventricular beats occur on cardiac monitoring in 54% , couplets in 40% and unsustained ventricular tachycardia in 29 % . cardiac arrhythmias may be associated with adverse outcome : 80% mortality in acute stroke patients with ventricular tachyarrhythmias , compared to 23% in those without . similarly , an association was documented between poor outcome ( including death ) in patients with arrhythmias or ecg changes after sah . wong reported that after adjusting for overt cardiac disease and traditional ischemic risk factors , stroke patients with qtc prolongation in lead v6 had a 2.8 relative risk of cardiac death . if the qtc exceeded 480ms then the specificity was 94% for cardiac death prediction within five years . eckhardt showed that insular strokes were particularly associated with qt dispersion lengthening compared with those in other locations . this is most pronounced after subarachnoid hemorrhage , but may also accompany intracerebral hemorrhage and ischemia . in many reported series , precision is reduced by failure to account for concomitant ischemic cardiac disorders as a confounding variable , and by the use of ecg rather than holter data . additionally , most studies are of short duration , performed in the acute phase and do not investigate the longer - term cardiovascular consequences of the lesion . it is this latter which may prove to be of importance in determining survival , and , as yet , definitive data on this point are lacking . while evidence for cerebral involvement in cardiac dysfunction is persuasive , frequency , predisposing circumstances and relevance for outcome are unclear . regard , early traces of ( non - ischemic ) myocytolysis were found in 26% of patients dying of non - ischemic causes ( pneumonia , sepsis ) and in 89% of sah , 71% of intracerebral hemorrhage , and 52% of ischemic stroke deaths . while indicative , these data come from a select group of patients for whom post mortem data were available . stroke may provide a good model to investigate neurocardiac influences : lesion location may be identified with precision , and onset categorized with some accuracy . however few studies detail arrhythmia incidence after stroke , and even fewer have controlled for concomitant coronary artery disease . comparing ecgs before and after stroke , lavy indicated a 39 % incidence of new onset cardiac arrhythmias in patients without cardiac history . goldstein reviewed 53 acute stroke patients ( including sah ) ecgs obtained within 24 hours of admission and recordings taken an average of 4 months earlier . qt prolongation of new onset was seen in 32% of strokes ( against 2% of controls ) ; t wave inversion and u waves were present in 15% of stroke patients and in none of the controls . new onset cardiac arrhythmias ( of which atrial fibrillation was the most frequent ) were present in 25% of all stroke patients , and 3% of controls . curiously , there was no difference in ventricular arrhythmia incidence between the two groups . however , this may not be too surprising since the ecg underestimates arrhythmia incidence . in other studies , where holter recording has been used , ventricular arrhythmia incidence after stroke was higher , varying from 2575% without control for co - existing cardiac disorders or for whether these were new or pre - existing . in yamour s uncontrolled study of intracerebral hemorrhage patients , comparison with pre - event ecg status indicated a new onset ventricular arrhythmia in 10% of patients and interestingly correlated with a temporoparietal location . in our recent unpublished observations , ventricular tachyarrhythmias ventricular premature beats occurred in 2.4% of strokes , and in 2% of 37 patients admitted with transient ischemic attacks ( tia ) . tia patients served as controls with similar demographics , incidence of coronary artery disease ( 70% ) , atherosclerotic risk factors and medication experience . however , holter monitoring revealed a ventricular tachyarrhythmia incidence of 12% in stroke , and 3% in tia patients , bigeminy in 21% and 5% respectively , ventricular ectopy in 71% and 73% respectively , and atrial fibrillation in 9% and 3% respectively . atrial fibrillation is commonly found in ecg studies of stroke [ 14 , 23 ] . however , when pre - event ecgs are used for comparison , it is unclear whether paroxysmal arrhythmia preceded and in some cases , caused stroke because of embolic potential . additionally , most reports do not control for cardiac concomitants ( indeed , 40% of acute stroke patients without cardiac symptoms are reported to have > 70% stenosis on coronary angiography ) . cardiac arrhythmia incidence is reportedly highest after sah ( 98 % ) , of which multifocal ventricular beats occur on cardiac monitoring in 54% , couplets in 40% and unsustained ventricular tachycardia in 29 % . cardiac arrhythmias may be associated with adverse outcome : 80% mortality in acute stroke patients with ventricular tachyarrhythmias , compared to 23% in those without . similarly , an association was documented between poor outcome ( including death ) in patients with arrhythmias or ecg changes after sah . wong reported that after adjusting for overt cardiac disease and traditional ischemic risk factors , stroke patients with qtc prolongation in lead v6 had a 2.8 relative risk of cardiac death . if the qtc exceeded 480ms then the specificity was 94% for cardiac death prediction within five years . eckhardt showed that insular strokes were particularly associated with qt dispersion lengthening compared with those in other locations . this is most pronounced after subarachnoid hemorrhage , but may also accompany intracerebral hemorrhage and ischemia . in many reported series , precision is reduced by failure to account for concomitant ischemic cardiac disorders as a confounding variable , and by the use of ecg rather than holter data . additionally , most studies are of short duration , performed in the acute phase and do not investigate the longer - term cardiovascular consequences of the lesion . it is this latter which may prove to be of importance in determining survival , and , as yet , definitive data on this point are lacking . in many species the insular cortex plays a pivotal role in the integration of autonomic function . phasic microstimulation of the rat left insula resulted in qt prolongation , st depression , pronounced bradycardia , complete heart block and idioventricular rhythm ending in asystole . myocytolysis was apparent on cardiac examination and plasma noradrenaline levels were elevated , without a change in adrenaline ( which in the rat indicates neural rather than adrenal origin).neither was provoked by stimulation of peri - insular sensory cortex.thus , cerebral stimulation can reproducibly generate lethal cardiac arrhythmias and pathology resembling changes seen after stroke or sudden unexpected death in epilepsy . lesions confined mainly to the right posterior insula of the rat increase blood pressure and heart rate without altering baroreceptor sensitivity . conversely , left posterior insular lesions do not alter cardiovascular variables , but increase baroreceptor sensitivity . previously , we had shown that damage to the right hemisphere in a rat middle cerebral artery occlusion model increased both qt interval and plasma norepinephrine . direct recording from cardiac sympathetic nerves is difficult ; however surrogate measures of sympathovagal balance may be assessed from spectral analysis of heart rate.we have shown in the rat that right posterior insular stimulation increases cardiac sympathetic tone in the absence of heart rate , blood pressure or respiration changes . interestingly , baroreceptor sensitivity decreased , a finding also linked to increased mortality after stroke . these laterality issues are of interest because of previous observations that in the human , right carotid amylobarbital infusion produces bradycardia , and left carotid infusion is accompanied by tachycardia .additionally , an increased incidence of supraventricular tachycardia was reported in patients with right middle cerebral artery stroke . our investigations in the human indicate that left caudal anterior insular stimulation during surgery for intractable epilepsy increases the frequency of bradycardia and depressor responses , whereas stimulation of a similar region of the right anterior insula is associated with heart rate and diastolic blood pressure elevation .although both types of response were elicitable from either insula , the proportion varied , and the degree of bradycardia was greater on left insular stimulation . these data indicate that in the human at least , some lateralization of cardiovascular representation may exist with sympathetic predominance of cardiovascular regulation being a right insular function , and parasympathetic cardiac neural regulation relating to the left insula . consistent with the hypothesis of left insular cardioinhibitory representation , these patients had a higher basal heart rate than age - gender matched controls . cardiac autonomic balance was shifted towards sympathetic predominance and mirrored the effects of rising from sitting to standing in the control group . interestingly 40% of these patients developed ecg changes not seen on premorbid traces , comprising tachycardia , t wave inversion , prominent u waves and qtc prolongation . a similar case has also been reported of an arrhythmia resolving after evacuation of a left insular hematoma .we have also encountered a patient who developed transient st depression 3 days after left insular infarction in the absence of cardiac history , coronary artery disease , or echocardiographic abnormalities . others have indicated a significant role for the right insular cortex in cardiovascular decompensation after stroke . tokgozoglu showed that compared to age - gender matched controls total spectral energy was reduced after ischemic stroke as noted previously by others . however , this was particularly marked in patients with stroke involving the right middle cerebral artery , or the insular cortex . sudden death was observed in 11% , but occurred more often after right mca lesions . naver indicated that heart rate variability entrained to deep breathing ( a parasympathetic relationship ) was reduced with right sided stroke , whereas peripheral sympathetic influences were equally distributed between the two sides . sympathetic skin response and pulse rate variation ( parasympathetic measure ) were suppressed in patients with both right or left hemisphere lesions compared with controls . this effect was more marked with right hemisphere lesions , although the authors failed to show a statistical difference when right and left sides were compared . on the other hand , li indicated that supraventricular arrhythmias were more frequently encountered after right insular infarction compared with strokes in other locations . interestingly , st abnormalities were more frequent after left insular involvement in comparison with controls or strokes in other locations . sander showed that right hemisphere infarction reduced circadian blood pressure variability and increased nocturnal blood pressure compared to left hemisphere infarcts . additionally , higher serum noradrenaline levels , longer qtc prolongation and more cardiac arrhythmias were observed after right hemisphere infarction . in general , changes were greatest when there was insular involvement , under which circumstances no laterality effects were observed . whereas these changes indicate that left insular lesions may disrupt interactions of central oscillators regulating cardiac rhythmicity , there is some inconsistency in the data . certainly , right rostral posterior insular stimulation in the rat increases cardiac sympathetic tone ; however , lesions involving this region also increase heart rate and blood pressure . these seemingly incompatible effects may relate to anesthetic effects on descending inhibitory and excitatory pathways . under baseline anesthesia conditions inhibitory pathways ( probably insular efferents to the lateral hypothalamic area ) may be maximally operational , and this inhibition is released by insular lesioning . on the other hand , stimulation recruits excitatory pathways , possibly quiescent under basal anesthetic conditions . human investigations were conducted in minimally anesthetized individuals and so the relationships may differ . in the rat , the major cardiac effect , however , appears related to parasympathetic upregulation ( bradyarrhythmias and complete heart block ) . in many species the insular cortex plays a pivotal role in the integration of autonomic function . phasic microstimulation of the rat left insula resulted in qt prolongation , st depression , pronounced bradycardia , complete heart block and idioventricular rhythm ending in asystole . myocytolysis was apparent on cardiac examination and plasma noradrenaline levels were elevated , without a change in adrenaline ( which in the rat indicates neural rather than adrenal origin).neither was provoked by stimulation of peri - insular sensory cortex.thus , cerebral stimulation can reproducibly generate lethal cardiac arrhythmias and pathology resembling changes seen after stroke or sudden unexpected death in epilepsy . lesions confined mainly to the right posterior insula of the rat increase blood pressure and heart rate without altering baroreceptor sensitivity . conversely , left posterior insular lesions do not alter cardiovascular variables , but increase baroreceptor sensitivity . previously , we had shown that damage to the right hemisphere in a rat middle cerebral artery occlusion model increased both qt interval and plasma norepinephrine . direct recording from cardiac sympathetic nerves is difficult ; however surrogate measures of sympathovagal balance may be assessed from spectral analysis of heart rate.we have shown in the rat that right posterior insular stimulation increases cardiac sympathetic tone in the absence of heart rate , blood pressure or respiration changes . interestingly , baroreceptor sensitivity decreased , a finding also linked to increased mortality after stroke . these laterality issues are of interest because of previous observations that in the human , right carotid amylobarbital infusion produces bradycardia , and left carotid infusion is accompanied by tachycardia .additionally , an increased incidence of supraventricular tachycardia was reported in patients with right middle cerebral artery stroke . our investigations in the human indicate that left caudal anterior insular stimulation during surgery for intractable epilepsy increases the frequency of bradycardia and depressor responses , whereas stimulation of a similar region of the right anterior insula is associated with heart rate and diastolic blood pressure elevation .although both types of response were elicitable from either insula , the proportion varied , and the degree of bradycardia was greater on left insular stimulation . these data indicate that in the human at least , some lateralization of cardiovascular representation may exist with sympathetic predominance of cardiovascular regulation being a right insular function , and parasympathetic cardiac neural regulation relating to the left insula . consistent with the hypothesis of left insular cardioinhibitory representation , these patients had a higher basal heart rate than age - gender matched controls . cardiac autonomic balance was shifted towards sympathetic predominance and mirrored the effects of rising from sitting to standing in the control group . interestingly 40% of these patients developed ecg changes not seen on premorbid traces , comprising tachycardia , t wave inversion , prominent u waves and qtc prolongation . a similar case has also been reported of an arrhythmia resolving after evacuation of a left insular hematoma .we have also encountered a patient who developed transient st depression 3 days after left insular infarction in the absence of cardiac history , coronary artery disease , or echocardiographic abnormalities . others have indicated a significant role for the right insular cortex in cardiovascular decompensation after stroke . tokgozoglu showed that compared to age - gender matched controls total spectral energy was reduced after ischemic stroke as noted previously by others . however , this was particularly marked in patients with stroke involving the right middle cerebral artery , or the insular cortex . sudden death was observed in 11% , but occurred more often after right mca lesions . naver indicated that heart rate variability entrained to deep breathing ( a parasympathetic relationship ) was reduced with right sided stroke , whereas peripheral sympathetic influences were equally distributed between the two sides . sympathetic skin response and pulse rate variation ( parasympathetic measure ) were suppressed in patients with both right or left hemisphere lesions compared with controls . this effect was more marked with right hemisphere lesions , although the authors failed to show a statistical difference when right and left sides were compared . on the other hand , li indicated that supraventricular arrhythmias were more frequently encountered after right insular infarction compared with strokes in other locations . interestingly , st abnormalities were more frequent after left insular involvement in comparison with controls or strokes in other locations . sander showed that right hemisphere infarction reduced circadian blood pressure variability and increased nocturnal blood pressure compared to left hemisphere infarcts . additionally , higher serum noradrenaline levels , longer qtc prolongation and more cardiac arrhythmias were observed after right hemisphere infarction . in general , changes were greatest when there was insular involvement , under which circumstances no laterality effects were observed . whereas these changes indicate that left insular lesions may disrupt interactions of central oscillators regulating cardiac rhythmicity , there is some inconsistency in the data . certainly , right rostral posterior insular stimulation in the rat increases cardiac sympathetic tone ; however , lesions involving this region also increase heart rate and blood pressure . these seemingly incompatible effects may relate to anesthetic effects on descending inhibitory and excitatory pathways . under baseline anesthesia conditions inhibitory pathways ( probably insular efferents to the lateral hypothalamic area ) may be maximally operational , and this inhibition is released by insular lesioning . on the other hand , stimulation recruits excitatory pathways , possibly quiescent under basal anesthetic conditions . human investigations were conducted in minimally anesthetized individuals and so the relationships may differ . in the rat , the major cardiac effect , however , appears related to parasympathetic upregulation ( bradyarrhythmias and complete heart block ) . considerable evidence for the role of the cortex in the regulation of cardiac rate and rhythm exists from historical sources , contemporary clinical observation and animal experimentation . there is a large body of evidence indicating that insular involvement may adversely affect cardiac prognosis after stroke . whereas there is little doubt now that lateralization of cardiovascular function occurs within the cortex of many species , there is some discrepancy regarding the evidence as to whether lesions of the left or right insula may be more clinically significant . in some part , this may reflect interspecies extrapolations , state dependency of the observed responses , whether or not clinical data refer to lesions confined solely to the insular cortex ( as in our reports ) or merely involve the insula in addition to adjacent regions ( as in the case of others ) . for example , we have identified an inhibitory input to the insula from adjacent regions ; when damaged , but sparing the insula itself , this may result in cardiovascular disinhibition , an effect differing from involvement of the insula alone . on balance however , it is most likely that in the human , lesions ablating part or all of the left anterior insula and its efferent connections and ablation of inhibitory circuminsular efferents to the right insular cortex are of consequence with regard to determining cardiovascular outcomes after neurological damage . temporal dephasing of the relationships between the neuraxially dispersed oscillators controlling sympathovagal activity is the likely underlying mechanism which , interacting with concomitant ischemic cardiac and vascular disease if present , poses a potential threat to the integrity and function of the heart . these effects may not be manifest in the acute phase , but may become so chronically , especially with central synaptic reorganization after stroke . environmental , and especially psychological , stressors may activate a reorganized system leading to upregulation and dysfunction of sympathetic neural input with resultant cardiac arrhythmia generation , cardiac structural damage and decompensation . clearly , long - term follow - up studies of stroke patients could be exceptionally useful to determine the validity of this argument .

abstractthat the brain may be involved in cardiovascular regulation has been acknowledged for over a century . that cardiac arrhythmias may result from cortical derangement has been less well recognized . that cortical cardiac representation may be lateralized is even more controversial . recent evidence implicates several cortical structures , especially the insula , in cardiac rate and rhythm control . experimental models indicate that insular lesions may be arrhythmogenic . accumulating data show similar lesion effects in humans . in the rat , monkey and man sympathetic cardiovascular control is generally represented in the right insula , although pronounced insulo - insular connectivity has been demonstrated . proarrhythmic shifts in cardiac sympathovagal balance occur after human stroke , including left insular lesions . this evidence implicates the cortex in the promotion and even generation of cardiovascular dysfunction under appropriate circumstances .

Perspective Evidence for sympathetic upregulation as a mechanism for cerebral influences on cardiac structure and function Clinical implications of cerebrocardiac interactions Frequency and consequences of cerebrally induced cardiac dysfunction Cerebral lateralization of cardioregulatory function Clinical and physiological considerations Conclusions

in 1913 goodman levy [ 24 , 25 ] showed that chloroform - induced ventricular tachyarrhythmias were abolished by cardiac sympathetic denervation . histological examination indicated degeneration of a pathway originating in the hypothalamus and terminating in the intermediolateral column ( close to the cells of origin of sympathetic preganglionic fibers ) . aschenbrenner reported seven young patients with cortical and diencephalic tumors who developed ventricular ectopy or nodal rhythm . much of this was forgotten with the advent of world war ii . in the aftermath , single case reports of intriguing ecg repolarization changes after subarachnoid hemorrhage ( sah ) were reported : inverted t waves ( often tall , symmetrical downward peaking ) and prolonged qtc interval with st elevation or depression . cropp initially thought these changes indicated coronary artery disease and delayed surgery , with fatal consequences . aneurysm position led to implication of inferior frontal structures and possible vagal mechanisms in the etiology of these cardiac changes . coupled with the resemblance of myocytolysis to phaeochromocytoma cardiomyopathy , the cardiac pathology accompanying catecholamine infusion and prolonged stress , these observations strongly indicate that sympathetic neural activation can destabilize cardiac structure and function . much recent evidence indicates the arrhythmogenic potential of catecholamines . mid - myocardial cells have been identified in the dog and human ventricle sensitive to catecholamines which may underlie the pro - arrhythmic effects of cardiac sympathetic activation . when combined with av block and experimental mi , vt and sudden cardiac death occurred only in the ngf - treated animals . stroke is associated with an adverse long - term cardiac outlook , possibly relating to altered sympathovagal balance . barron reported that compared with age / gender matched controls , total spectral power of rr intervals was reduced after stroke ; power associated with cardiac parasympathetic neural activity was particularly affected indicating a shift in sympathovagal balance . strittmatter reported that cardiac sympathetic tone was increased within 5 days of stroke and similar findings were reported by giubilei , who also showed persistence for at least 3 weeks . demonstrated that diurnal cardiac autonomic variability was disturbed after acute stroke with a striking loss of nocturnal vagal dominance . however , in these , and many other such studies the contribution of concomitant coronary artery disease and medications were not assessed . collectively these data strongly indicate that the brain has a major influence on cardiac structure and function and that this is likely mediated through alterations in patterning of sympathovagal relationships . histological examination indicated degeneration of a pathway originating in the hypothalamus and terminating in the intermediolateral column ( close to the cells of origin of sympathetic preganglionic fibers ) . these patients lacked cardiac history or post mortem ischemic cardiac pathology to explain their death . in the aftermath , single case reports of intriguing ecg repolarization changes after subarachnoid hemorrhage ( sah ) were reported : inverted t waves ( often tall , symmetrical downward peaking ) and prolonged qtc interval with st elevation or depression . no patient had a cardiac history , most were young and no autopsied case showed coronary artery pathology . aneurysm position led to implication of inferior frontal structures and possible vagal mechanisms in the etiology of these cardiac changes . coupled with the resemblance of myocytolysis to phaeochromocytoma cardiomyopathy , the cardiac pathology accompanying catecholamine infusion and prolonged stress , these observations strongly indicate that sympathetic neural activation can destabilize cardiac structure and function . much recent evidence indicates the arrhythmogenic potential of catecholamines . mid - myocardial cells have been identified in the dog and human ventricle sensitive to catecholamines which may underlie the pro - arrhythmic effects of cardiac sympathetic activation . when combined with av block and experimental mi , vt and sudden cardiac death occurred only in the ngf - treated animals . this suggested that sympathetic neural remodeling might facilitate ventricular arrhythmias and contribute to sudden cardiac death . conversely , manitius - robeck reported ictal sinus arrest during temporal lobe seizures . stroke is associated with an adverse long - term cardiac outlook , possibly relating to altered sympathovagal balance . barron reported that compared with age / gender matched controls , total spectral power of rr intervals was reduced after stroke ; power associated with cardiac parasympathetic neural activity was particularly affected indicating a shift in sympathovagal balance . strittmatter reported that cardiac sympathetic tone was increased within 5 days of stroke and similar findings were reported by giubilei , who also showed persistence for at least 3 weeks . demonstrated that diurnal cardiac autonomic variability was disturbed after acute stroke with a striking loss of nocturnal vagal dominance . sander [ 42 , 44 ] reported that absence of circadian cardiovascular variation was particularly striking after insular infarction . however , in these , and many other such studies the contribution of concomitant coronary artery disease and medications were not assessed . collectively these data strongly indicate that the brain has a major influence on cardiac structure and function and that this is likely mediated through alterations in patterning of sympathovagal relationships . while evidence for cerebral involvement in cardiac dysfunction is persuasive , frequency , predisposing circumstances and relevance for outcome are unclear . stroke may provide a good model to investigate neurocardiac influences : lesion location may be identified with precision , and onset categorized with some accuracy . however few studies detail arrhythmia incidence after stroke , and even fewer have controlled for concomitant coronary artery disease . comparing ecgs before and after stroke , lavy indicated a 39 % incidence of new onset cardiac arrhythmias in patients without cardiac history . new onset cardiac arrhythmias ( of which atrial fibrillation was the most frequent ) were present in 25% of all stroke patients , and 3% of controls . in other studies , where holter recording has been used , ventricular arrhythmia incidence after stroke was higher , varying from 2575% without control for co - existing cardiac disorders or for whether these were new or pre - existing . however , holter monitoring revealed a ventricular tachyarrhythmia incidence of 12% in stroke , and 3% in tia patients , bigeminy in 21% and 5% respectively , ventricular ectopy in 71% and 73% respectively , and atrial fibrillation in 9% and 3% respectively . cardiac arrhythmias may be associated with adverse outcome : 80% mortality in acute stroke patients with ventricular tachyarrhythmias , compared to 23% in those without . similarly , an association was documented between poor outcome ( including death ) in patients with arrhythmias or ecg changes after sah . if the qtc exceeded 480ms then the specificity was 94% for cardiac death prediction within five years . eckhardt showed that insular strokes were particularly associated with qt dispersion lengthening compared with those in other locations . additionally , most studies are of short duration , performed in the acute phase and do not investigate the longer - term cardiovascular consequences of the lesion . while evidence for cerebral involvement in cardiac dysfunction is persuasive , frequency , predisposing circumstances and relevance for outcome are unclear . stroke may provide a good model to investigate neurocardiac influences : lesion location may be identified with precision , and onset categorized with some accuracy . however few studies detail arrhythmia incidence after stroke , and even fewer have controlled for concomitant coronary artery disease . comparing ecgs before and after stroke , lavy indicated a 39 % incidence of new onset cardiac arrhythmias in patients without cardiac history . new onset cardiac arrhythmias ( of which atrial fibrillation was the most frequent ) were present in 25% of all stroke patients , and 3% of controls . in other studies , where holter recording has been used , ventricular arrhythmia incidence after stroke was higher , varying from 2575% without control for co - existing cardiac disorders or for whether these were new or pre - existing . however , holter monitoring revealed a ventricular tachyarrhythmia incidence of 12% in stroke , and 3% in tia patients , bigeminy in 21% and 5% respectively , ventricular ectopy in 71% and 73% respectively , and atrial fibrillation in 9% and 3% respectively . cardiac arrhythmias may be associated with adverse outcome : 80% mortality in acute stroke patients with ventricular tachyarrhythmias , compared to 23% in those without . wong reported that after adjusting for overt cardiac disease and traditional ischemic risk factors , stroke patients with qtc prolongation in lead v6 had a 2.8 relative risk of cardiac death . eckhardt showed that insular strokes were particularly associated with qt dispersion lengthening compared with those in other locations . additionally , most studies are of short duration , performed in the acute phase and do not investigate the longer - term cardiovascular consequences of the lesion . in many species the insular cortex plays a pivotal role in the integration of autonomic function . phasic microstimulation of the rat left insula resulted in qt prolongation , st depression , pronounced bradycardia , complete heart block and idioventricular rhythm ending in asystole . myocytolysis was apparent on cardiac examination and plasma noradrenaline levels were elevated , without a change in adrenaline ( which in the rat indicates neural rather than adrenal origin).neither was provoked by stimulation of peri - insular sensory cortex.thus , cerebral stimulation can reproducibly generate lethal cardiac arrhythmias and pathology resembling changes seen after stroke or sudden unexpected death in epilepsy . lesions confined mainly to the right posterior insula of the rat increase blood pressure and heart rate without altering baroreceptor sensitivity . conversely , left posterior insular lesions do not alter cardiovascular variables , but increase baroreceptor sensitivity . previously , we had shown that damage to the right hemisphere in a rat middle cerebral artery occlusion model increased both qt interval and plasma norepinephrine . direct recording from cardiac sympathetic nerves is difficult ; however surrogate measures of sympathovagal balance may be assessed from spectral analysis of heart rate.we have shown in the rat that right posterior insular stimulation increases cardiac sympathetic tone in the absence of heart rate , blood pressure or respiration changes . these laterality issues are of interest because of previous observations that in the human , right carotid amylobarbital infusion produces bradycardia , and left carotid infusion is accompanied by tachycardia .additionally , an increased incidence of supraventricular tachycardia was reported in patients with right middle cerebral artery stroke . our investigations in the human indicate that left caudal anterior insular stimulation during surgery for intractable epilepsy increases the frequency of bradycardia and depressor responses , whereas stimulation of a similar region of the right anterior insula is associated with heart rate and diastolic blood pressure elevation .although both types of response were elicitable from either insula , the proportion varied , and the degree of bradycardia was greater on left insular stimulation . these data indicate that in the human at least , some lateralization of cardiovascular representation may exist with sympathetic predominance of cardiovascular regulation being a right insular function , and parasympathetic cardiac neural regulation relating to the left insula . consistent with the hypothesis of left insular cardioinhibitory representation , these patients had a higher basal heart rate than age - gender matched controls . cardiac autonomic balance was shifted towards sympathetic predominance and mirrored the effects of rising from sitting to standing in the control group . a similar case has also been reported of an arrhythmia resolving after evacuation of a left insular hematoma .we have also encountered a patient who developed transient st depression 3 days after left insular infarction in the absence of cardiac history , coronary artery disease , or echocardiographic abnormalities . others have indicated a significant role for the right insular cortex in cardiovascular decompensation after stroke . however , this was particularly marked in patients with stroke involving the right middle cerebral artery , or the insular cortex . naver indicated that heart rate variability entrained to deep breathing ( a parasympathetic relationship ) was reduced with right sided stroke , whereas peripheral sympathetic influences were equally distributed between the two sides . sympathetic skin response and pulse rate variation ( parasympathetic measure ) were suppressed in patients with both right or left hemisphere lesions compared with controls . this effect was more marked with right hemisphere lesions , although the authors failed to show a statistical difference when right and left sides were compared . interestingly , st abnormalities were more frequent after left insular involvement in comparison with controls or strokes in other locations . additionally , higher serum noradrenaline levels , longer qtc prolongation and more cardiac arrhythmias were observed after right hemisphere infarction . whereas these changes indicate that left insular lesions may disrupt interactions of central oscillators regulating cardiac rhythmicity , there is some inconsistency in the data . certainly , right rostral posterior insular stimulation in the rat increases cardiac sympathetic tone ; however , lesions involving this region also increase heart rate and blood pressure . under baseline anesthesia conditions inhibitory pathways ( probably insular efferents to the lateral hypothalamic area ) may be maximally operational , and this inhibition is released by insular lesioning . in the rat , the major cardiac effect , however , appears related to parasympathetic upregulation ( bradyarrhythmias and complete heart block ) . in many species the insular cortex plays a pivotal role in the integration of autonomic function . phasic microstimulation of the rat left insula resulted in qt prolongation , st depression , pronounced bradycardia , complete heart block and idioventricular rhythm ending in asystole . myocytolysis was apparent on cardiac examination and plasma noradrenaline levels were elevated , without a change in adrenaline ( which in the rat indicates neural rather than adrenal origin).neither was provoked by stimulation of peri - insular sensory cortex.thus , cerebral stimulation can reproducibly generate lethal cardiac arrhythmias and pathology resembling changes seen after stroke or sudden unexpected death in epilepsy . lesions confined mainly to the right posterior insula of the rat increase blood pressure and heart rate without altering baroreceptor sensitivity . conversely , left posterior insular lesions do not alter cardiovascular variables , but increase baroreceptor sensitivity . previously , we had shown that damage to the right hemisphere in a rat middle cerebral artery occlusion model increased both qt interval and plasma norepinephrine . direct recording from cardiac sympathetic nerves is difficult ; however surrogate measures of sympathovagal balance may be assessed from spectral analysis of heart rate.we have shown in the rat that right posterior insular stimulation increases cardiac sympathetic tone in the absence of heart rate , blood pressure or respiration changes . these laterality issues are of interest because of previous observations that in the human , right carotid amylobarbital infusion produces bradycardia , and left carotid infusion is accompanied by tachycardia .additionally , an increased incidence of supraventricular tachycardia was reported in patients with right middle cerebral artery stroke . our investigations in the human indicate that left caudal anterior insular stimulation during surgery for intractable epilepsy increases the frequency of bradycardia and depressor responses , whereas stimulation of a similar region of the right anterior insula is associated with heart rate and diastolic blood pressure elevation .although both types of response were elicitable from either insula , the proportion varied , and the degree of bradycardia was greater on left insular stimulation . these data indicate that in the human at least , some lateralization of cardiovascular representation may exist with sympathetic predominance of cardiovascular regulation being a right insular function , and parasympathetic cardiac neural regulation relating to the left insula . consistent with the hypothesis of left insular cardioinhibitory representation , these patients had a higher basal heart rate than age - gender matched controls . cardiac autonomic balance was shifted towards sympathetic predominance and mirrored the effects of rising from sitting to standing in the control group . a similar case has also been reported of an arrhythmia resolving after evacuation of a left insular hematoma .we have also encountered a patient who developed transient st depression 3 days after left insular infarction in the absence of cardiac history , coronary artery disease , or echocardiographic abnormalities . others have indicated a significant role for the right insular cortex in cardiovascular decompensation after stroke . however , this was particularly marked in patients with stroke involving the right middle cerebral artery , or the insular cortex . naver indicated that heart rate variability entrained to deep breathing ( a parasympathetic relationship ) was reduced with right sided stroke , whereas peripheral sympathetic influences were equally distributed between the two sides . this effect was more marked with right hemisphere lesions , although the authors failed to show a statistical difference when right and left sides were compared . interestingly , st abnormalities were more frequent after left insular involvement in comparison with controls or strokes in other locations . additionally , higher serum noradrenaline levels , longer qtc prolongation and more cardiac arrhythmias were observed after right hemisphere infarction . in general , changes were greatest when there was insular involvement , under which circumstances no laterality effects were observed . whereas these changes indicate that left insular lesions may disrupt interactions of central oscillators regulating cardiac rhythmicity , there is some inconsistency in the data . certainly , right rostral posterior insular stimulation in the rat increases cardiac sympathetic tone ; however , lesions involving this region also increase heart rate and blood pressure . under baseline anesthesia conditions inhibitory pathways ( probably insular efferents to the lateral hypothalamic area ) may be maximally operational , and this inhibition is released by insular lesioning . in the rat , the major cardiac effect , however , appears related to parasympathetic upregulation ( bradyarrhythmias and complete heart block ) . considerable evidence for the role of the cortex in the regulation of cardiac rate and rhythm exists from historical sources , contemporary clinical observation and animal experimentation . there is a large body of evidence indicating that insular involvement may adversely affect cardiac prognosis after stroke . whereas there is little doubt now that lateralization of cardiovascular function occurs within the cortex of many species , there is some discrepancy regarding the evidence as to whether lesions of the left or right insula may be more clinically significant . in some part , this may reflect interspecies extrapolations , state dependency of the observed responses , whether or not clinical data refer to lesions confined solely to the insular cortex ( as in our reports ) or merely involve the insula in addition to adjacent regions ( as in the case of others ) . for example , we have identified an inhibitory input to the insula from adjacent regions ; when damaged , but sparing the insula itself , this may result in cardiovascular disinhibition , an effect differing from involvement of the insula alone . on balance however , it is most likely that in the human , lesions ablating part or all of the left anterior insula and its efferent connections and ablation of inhibitory circuminsular efferents to the right insular cortex are of consequence with regard to determining cardiovascular outcomes after neurological damage . these effects may not be manifest in the acute phase , but may become so chronically , especially with central synaptic reorganization after stroke .

human prolactin ( hprl ) , a 23 kda polypeptide hormone with a single chain of 199 residues , is a member of the family of cytokines , which includes erythropoietin , interleukin-6 , and many others , but is most closely related , both evolutionarily and functionally , to human growth hormone and placental lactogen . this polypeptide is known to be involved in a variety of actions , more than all the other pituitary hormones combined , such as cell proliferation , growth and development , water and electrolyte balance , and several physiological and behavioral aspects of mammal , bird , and reptile reproduction . in humans , apart from the pituitary , many extrapituitary sites for hprl synthesis have been documented , including breast , prostate , and female reproductive tract , where it appears to act locally to regulate cellular growth and differentiation [ 1 , 4 , 5 ] . the hprl gene has been cloned and the authentic form of the hormone efficiently expressed in bacterial periplasm and in chinese hamster ovary ( cho ) cells in our laboratory [ 68 ] . since hprl is one of the hormones most frequently determined in routine clinical assays and several therapeutic applications are being considered , an increasing need for pure bioactive hprl can be anticipated [ 9 , 10 ] . sodium butyrate ( nabu ) is a short chain fatty acid , originally identified as a product of anaerobic bacterial fermentation , which has been shown to alter the structure of chromatin in the nucleus of mammalian cells by reducing the activity of histone deacetylase . one of the possible causes for improved gene expression by nabu cells is histone hyperacetylation , which facilitates the access of general transcription factors in eukaryotic cells [ 12 , 13 ] . however , nabu can also cause a cellular arrest , leading to increased apoptosis and resulting in an overall reduction in recombinant protein production over longer periods of time . nabu treatment has been shown , via immunoassay determination , to increase the expression levels of foreign proteins such as human thrombopoietin , interferon--1a , and chimeric igg3 antibodies in cho cell cultures [ 1517 ] by factors of 2- , 2.5- , and 3.6-fold , respectively , relative to nontreated cells.this led us to investigate , for the first time , via physicochemical techniques , whether addition of nabu would also increase hprl production in these cell cultures . in order to characterize the product obtained , we took advantage of a specific rp - hplc methodology previously developed in our laboratory , which permits qualitative and quantitative analysis of hprl directly in cho - conditioned medium . high - performance size - exclusion chromatography ( hpsec ) methodology was used for both analytical and preparative purposes and also allowed us to evaluated hprl activity directly in two bioassays , after its purification from cho - conditioned medium . the clone expressing hprl , derived from cho dhfr cells ( dukx - b11 ) that had been transfected with the vector peddc - hprl , was obtained in our laboratory . cells were thawed into t - flasks ( 75 cm , from corning costar corporation , cambridge , ma ) containing -minimal essential medium ( -mem , gibco - invitrogen corporation , grand island , ny , usa ) with l - glutamine and without ribonucleosides and deoxyribonucleosides , supplemented with 10% dialysed fetal bovine serum ( dfbs ) , 40 g / ml gentamycin , 4 g / l glucose , and 100 nm methotrexate . cultures were maintained in a humidified incubator at 37c in the presence of 5% co2 . 100 mm nabu was prepared in phosphate - buffered saline ( pbs ) ( 0.007 m na2hpo4 , 0.01 m nah2po4 , ph 7.4 and 0.15 m nacl ) , sterilized by passing through a 0.20 m syringe filter ( corning incorporated , corning , ny , usa ) , and stored in aliquots at 20c . to examine the effect of different concentrations of nabu on hprl production , 1 ml of cho cell suspension ( 5 10 cells / ml ) was seeded into each well of a 24-well plate to obtain a concentration of 2 10 cells / ml . cultures were then rinsed twice in pbs to remove serum and medium . fresh medium was then added containing nabu to a final concentration between 0 and 4 mm . cells were grown for 10 days and cho cell - conditioned medium was collected daily and stored at 80c for subsequent rp - hplc or western blot analysis . for product purification , cho cells were expanded into ten t - flasks , in -mem supplemented with dfbs , until they were confluent . when the cell density was ~2 10 cells / ml , fresh medium was then added to the t - flasks : five contained no nabu , while the chosen amounts of nabu were added to the other five . the hprl - secreting confluent cultures were then maintained for up to ten days by replacing the medium every 24 hours . the conditioned medium was collected and stored , as mentioned above , for sp - sepharose fast flow and hpsec purification . to examine the effect of different concentrations of nabu on cell density and viability , 1 ml of cho cell suspension ( 5 10 cells / ml ) was seeded into each well of a 24-well plate to obtain a concentration of 2 10 cells / ml . nabu was added to a final concentration between 0 and 4 mm and cells were grown for 10 days , as mentioned above . every 24 hours a sample for the different concentrations of nabu was detached from 24-well plate by treatment with 0.025% trypsin - edta in pbs . total cell number and viability were determined by using the trypan blue ( gibco - invitrogen corporation ) exclusion method via haemocytometry and phase contrast microscopy , to distinguish viable and nonviable cells on the basis of dye uptake . 5 10 cells / ml cells were seeded in 24-well plate , treated and detached at regular times , as mentioned above . 1 10 cells / ml per sample were lysed in 400 l of lysis buffer ( 50 mm tris , 10 mm edta ph 8.0 , 1% sds ) and 500 g / ml proteinase k ( fermentas , life sciences , so paulo , brazil ) at 37c for 16 hours . the lysates were extracted with phenol : clorophorm : isoamylalcohol ( 25 : 24 : 1 ) . the resulting pellets of dna were resuspended in te buffer ( ph 8.0 ) and treated with 10 g / ml rnase a ( fermentas , life sciences , brazil ) prior to electrophoresis on a 5% polyacrylamide gel at 70 v . discontinuous sds - page , based on 15% polyacrylamide gels , was carried out under nonreducing conditions as described by laemmli . proteins were revealed by silver or coomassie brilliant blue g-250 ( usb , cleveland , oh ) staining . western blot analysis was performed according to bannerman et al . , using i - labelled protein a and anti - hprl antiserum obtained in rabbit and validated in our laboratory against the niddk - anti - hprl-3 from national hormone and pituitary program , torrance , ca , usa [ 7 , 8 ] . total protein content was determinate by bca protein assay , using bicinchoninic acid ( micro bca protein assay kit , pierce , rockford , il , usa ) . pure bovine serum albumin ( bsa , sigma , so paulo , brazil ) was used as standard . after the treatment with different nabu concentrations , an aliquot of conditioned culture medium ( ~10 ml ) was concentrated ( ~10 fold ) by using a centrifugal membrane filter ultra 4 ( amicon , millipore corporation , billerica , ma , usa ) and applied to a rp - hplc column . a shimadzu model scl-10a hplc apparatus coupled to a spd-10av uv detector ( shimadzu , md , usa ) was used , employing the class vp software , also from shimadzu . the column was a c4 vydac 214tp54 ( 25 cm 4.6 mm i d , pore diameter of 300 and particle diameter of 5 m ) with a silica precolumn packed with lichrosorb si-60 , 7.912.4 m ( merck , darmstadt , germany ) located between the pump and the injector . all vydac columns were purchased from grace vydac ( hesperia , ca , usa ) . the mobile phase consisted of 71% tris - hcl buffer ( 50 mm , ph 7.5 ) and 29% n - propanol , as described by dalmora et al . , with a flow - rate of 0.5 ml / min , a detector wavelength of 220 nm , a column temperature maintained at 45c , and a sample volume of 25500 l . this methodology was used for analytical purposes to examine the effect of nabu on hprl production . the hpsec is an entropically controlled separation technique in which molecules are separated on the basis of their hydrodynamic molecular volume or size [ 25 , 26 ] . this methodology was used for both analytical and preparative purposes and allowed us to use purified hprl directly in bioassays , the n - propanol interference being absent due to the rp - hplc technique . hpsec was carried out with the same shimadzu apparatus , processing 25 to 500 l of sample on a tosohaas ( montgomeryville , pa , usa ) g2000 sw column ( 60 cm 7.5 mm i.d . , particle size of 10 m and pore size of 125 ) coupled to a 7.5 cm 7.5 mm i.d . the mobile phase was 0.025 m ammonium bicarbonate , ph 7.0 , with a flow - rate of 1.0 ml / min . the hprl present in conditioned culture medium was purified as described by soares et al . . a two - step purification process was used : sp - sepharose fast flow followed by a size exclusion chromatography employing hpsec for preparative purposes . the material was then applied onto the sp - sepharose fast flow column ( ge healthcare bio - science corp . , piscataway , nj , usa ) equilibrated in 50 mm sodium acetate , ph 5.0 . uv absorbance was monitored at 280 nm . after washing with the same buffer , the column was treated with 50 mm sodium acetate , ph 5.0 , 90 mm nacl buffer . the protein of interest was then eluted from the column with 25 mm hepes , ph 8.0 . different fractions containing hprl were analysed by hpsec and the most concentrated fractions were purified on the same hpsec column , working this time in a preparative mode . hprl in the final pool obtained via the hpsec purification was quantified for bioassay purposes by hpsec and stored at 80c . the biological activity of purified hprl was determined via the nb2 and ba / f3-llp lymphoma cell - proliferation assays , as previously described , against the first who reference reagent of recombinant hprl , lyophilized in ampoules coded 97/714 . ba / f3-llp cells were routinely maintained in suspension in rpmi-1640 medium supplemented with 10% heat - inactivated fbs , 2 mm glutamine , 50 u / ml of penicillin , 50 g / ml of streptomycin , 700 g / ml of geneticin and 1 ng / ml of hprl . before carrying out the proliferation assay , cells were starved for 6 hours in the rpmi-1640 medium , as mentioned above , without hprl . cells were then distributed in flat bottom 96-well plates at a density of 5 10 cells / well in a final volume of 200 l . after 72 hours at 37c and 5% co2 , the presence of viable cells was assessed using mts assay . briefly , 2 mg / ml mts dye [ 3(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulf - ophenyl)-2h - tetrazolin ( promega corp . , madison , wi , usa ) ] in pbs was mixed in a 20 : 1 ratio ( vol / vol ) with pms ( phenazine methosulphate ) ( sigma , st . twenty microliters of mixture were then added to each well , and 2 hours after incubation at 37c the absorbance at 490 nm was read in a microplate reader ( multiskan ex thermo electron corporation , vantaa , finland ) . nb2 cells also routinely maintained in suspension in rpmi-1640 medium supplemented with 10% heat - inactivated fbs , 2 mm glutamine , 50 u / ml of penicillin , and 50 g / ml of streptomycin mixed in a 50 : 1 ratio ( vol / vol ) with 5 mm 2- mercaptoethanol in pbs . before carrying out the proliferation assay , cells were maintained in the rpmi-1640 , as mentioned above , containing 1% heat - inactivated fbs for 8 hours , constituting the preassay . afterward , the cells were distributed in flat bottom 96-well plates at a density of 2 10 cells / well in a final volume of 200 l , with no heat - inactivated fbs addition . after 72 hours at 37c and 5% co2 , the presence of viable cells was assessed using mts assay , as mentioned before . the influence of different concentrations of nabu on hprl - secreting cho cellular growth and viability during a 10-day culture can be seen in figure 1 . the treatment with either 0.25 or 0.5 mm nabu did not result in significant growth inhibition ( p > 0.05 ) compared to the control without nabu ( figure 1(a ) ) . nonetheless , a dose response - effect on cellular growth was observed in the concentration range of 1 to 4 mm nabu , with a dramatic fall already occurring after day 2 at the concentrations of 2 mm and 4 mm nabu . the treatment with 1 mm nabu produced an apparent decline only 4 days after starting the treatment . with regard to cell viability , none of the concentrations except 4 mm nabu ( p < 0.05 ) showed any significant effect ( figure 1(b ) ) . the rate of apoptosis in nabu - treated and untreated cho cells was examined by polyacrylamide gel electrophoresis and revealed by silver staining . figure 2 shows that nabu - treated and untreated cho cells exhibit oligonucleosomal dna ladder which is the hallmark of apoptosis , resulting from endonuclease activity at internucleosomal sites in an apoptotic cell . the intensity of the oligonucleosomal dna ladder seems nabu dose - dependent . on the other hand , the progressive removal of dfbs from the culture medium could also be associated with apoptotic cell death in nabu - treated and untreated cho cells , leading to a decrease of cellular growth after day 5 . this can be due to the fact that serum contains growth factors that help the culture survival . a rp - hplc methodology , previously set up in our laboratory specifically for hprl qualitative and quantitative analysis , was then employed to quantify hprl production and detect the presence of possible alteration in hprl structure or hydrophobicity due to the prolonged presence of nabu . the data in table 1 and figure 3 show that 1 mm nabu is capable of greatly increasing hprl synthesis ( p < 0.02 ) , with no significant differences ( p > 0.05 ) in the retention time ( tr ) of the resultant hprl up to 4 mm nabu . human prolactin quantification carried out by rp - hplc in cho cell - conditioned medium ( table 1 ) indicated that the highest production was obtained with 1 mm nabu , which increased the secretion ~2-fold relative to control cultivation without nabu addition . despite an increase in protein expression , the concentrations of 0.5 mm and 2 mm nabu provided lower hprl volumetric yields than 1 mm nabu . the greatest inhibition of cell growth was produced by 4 mm nabu leading to lower hprl synthesis ( figure 1(a ) and table 1 ) . clearly , it will be important to determine the optimal nabu concentration in long - term cell cultures . to confirm the expression and purity level of hprl , equal volumes of cho - conditioned medium , collected on days 1 , 4 , 5 , and 6 , were analyzed by western blot ( figure 4 ) ; the same was done with pools obtained after 10 days of cultivation ( figure 5(a ) ) . these pools were also analyzed via sds - page followed by agno3 staining ( figure 5(b ) ) , which demonstrated the predominance , with 1 mm nabu , of hprl relative to other proteins that might be present in cho cell - conditioned medium . taken together , these rp - hplc , western blot , and sds - page analyses indicate that , under our conditions , 1 mm nabu was the ideal concentration , providing the highest hprl expression level , while still maintaining good cell viability and only a limited decrease in cell growth . sp - sepharose fast flow chromatographic purification of nabu - treated and untreated cells , together with sds - page analysis of applied and eluted fractions , is shown in figure 6 . this confirmed the presence of about twice as much hprl in the medium treated with 1 mm nabu and the efficient adsorption of both the 25 kda glycosylated ( g - hprl ) and the 23 kda nonglycosylated hprl ( ng - hprl ) isoforms by the cationic exchanger . the same partially purified products were also analyzed by hpsec before and after using this same column for preparative purposes ( figures 7(a ) , 7(b ) , and 7(c ) ) . the purity obtained for both products after this second chromatographic step is noteworthy ( > 97% ) ( figure 7(c ) and table 2 ) . from table 2 , on the basis of mass fractions , it is possible to calculate an overall purification factor of ~55 and 122-fold for nabu - treated and untreated cho cells , respectively . the ~2-fold increase in hprl production obtained in the presence of 1 mm nabu was also confirmed after purification . also remarkable are the perfectly coincident tr values of the two cho - derived hprl preparations and of the internal reference of e. coli - derived hprl ( 19.58 minutes < tr < 19.72 minutes ) , confirming their identical hydrodynamic properties in this long ( 60 cm ) hpsec column ( figure 7(c ) ) . it has been reported that these properties can be altered even by subtle modifications in the secondary and tertiary structure of the molecule [ 32 , 33 ] . finally , the two purified products were tested for their biological activity against the reference reagent for recombinant hprl ( who 97/714 ) in two different in vitro bioassays based on nb2 and ba / f3-llp cells . the data illustrated in figure 8 and reported in table 3 show that apparently there were no differences between the potencies of the purified products and that of the who reference reagent . previous studies have examined the production of recombinant proteins by nabu - treated cho cells . several of these have discussed the benefits or warned against the problems caused by the presence of nabu [ 1416 ] . nabu has , in fact , been used for increasing the expression of foreign proteins in animal cell cultures . it is recognized that nabu alters primarily chromatin structure through inhibition of histone deacetylases , resulting in hyperacetylation of histones and , consequently , alterations in dna transcription . therefore , these alterations might be correlated with the modulation of gene expression . on the other hand , hyperacetylation of histones in nabu - treated mammalian cancer cell lines has been shown to induce apoptosis mediated by activation of caspase-3 activity , leading to the translation of putative cell death proteins and resulting in an increased possibility of dna cleavage . in this study , we examined the expression level and quality of hprl directly in cho cell - conditioned medium , after adding different amounts of nabu . the different nabu concentrations ( 0.25 to 2 mm ) remarkably increased hprl expression . addition of 1 mm nabu to the culture medium resulted in a twofold increase in hprl expression , with only a relatively small decrease in cell growth compared with the untreated control ; this was confirmed by rp - hplc and by western blot and sds - page analysis . although 2 mm and 4 mm nabu apparently induced a higher synthesis of hprl in the first 24 hours of treatment , this synthesis exhibited a decrease in the following days of culture . a higher nabu concentration ( 2 or 4 mm ) may be more effective in inhibiting the activity of histone deacetylases and , consequently , increase the initial expression of proteins . however , a greater modification of the chromatin structure could make the dna more susceptible to apoptotic action of endonucleases , leading to decreased productivity of hprl . the addition of different nabu concentrations apparently did not affect the hydrophobicity or the hydrodynamic proprieties of the hprl molecule . this was inferred from the fact that the tr values determined by rp - hplc and hpsec were not significantly altered by the nabu treatment relative to the untreated control or the internal reference standard of periplasmic e. coli - derived hprl . our analytical determinations , as a difference from other authors that studied nabu influence on heterologous protein production with basis on immunological techniques [ 1517 ] , have been mostly based on physicochemical methodologies . the cell viability for different concentrations of nabu was similar to that of the untreated control except in the experiments with 4 mm added nabu . therefore , the arrest of cell growth after day 5 and similar decrease in cell viability between 02 mm nabu was probably caused by removal of dfbs from the culture medium and nabu treatment during the production process . this could make the cho cell more susceptible to apoptosis , leading to decreased cellular density and viability . finally , two different in vitro bioassays for purified hprl , based on nb2 and ba / f3-llp cells , were carried out . the first bioassay is the classical heterologous nb2 rat lymphoma cell proliferation assay , while the second is the recently developed homologous ba / f3-llp assay , based on murine pro - b lymphoma cells containing the sequence encoding the human prl receptor and previously utilized in our laboratory [ 9 , 27 ] . as described by crowell et al . , nabu treatment can potentially modify the oligosaccharide content of glycoproteins in various expression systems . although nabu apparently has no effect on the protein structure of hprl , there might be modifications of the oligosaccharide structure of g - hprl . in this regard , it is known that the oligosaccharide nature and content can influence the in vivo bioactivities of a glycoprotein [ 3942 ] . it is therefore important to note that the potency of hprl obtained in the presence of nabu was comparable to that of the reference reagent for recombinant hprl ( who 97/714 ) . in conclusion , for the first time recombinant hprl production from genetically modified cho cells has been significant increased ( ~2-fold ) via nabu addition , apparently without compromising either its structure or function ; the quantitative and qualitative effects of this addition have been determined via accurate physicochemical techniques . the same treatment can potentially be applied to other hormones , for example , human thyrotropin ( htsh ) , which is also produced in our laboratory . the qualitative and quantitative influence of this treatment on the carbohydrate moieties of g - hprl and possibly of other glycohormones requires further investigation .

sodium butyrate ( nabu ) has been used as a productivity enhancer for the synthesis of recombinant proteins in chinese hamster ovary ( cho ) cells . thus , the influence of nabu on the production of recombinant human prolactin ( hprl ) from cho cells was investigated for the first time . cho cell cultures were submitted to a treatment with different concentrations of nabu ( 0.25 to 4 mm ) . quantitative and qualitative analyses by reverse - phase high - performance liquid chromatography ( rp - hplc ) and western blot or sds - page , carried out directly on cho - conditioned medium , showed that the highest hprl expression was obtained with 1 mm nabu . in vitro biological assays based on noble rat lymphoma ( nb2 ) and mouse pro - b lymphoma ( ba / f3-llp ) cells were carried out on purified hprl . its bioactivity in the presence of nabu was not apparently different from that of the first international reference reagent of recombinant hprl ( who 97/714 ) . our results show that nabu increased the synthesis of recombinant hprl in cho cells , apparently without compromising either its structure or function .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion

human prolactin ( hprl ) , a 23 kda polypeptide hormone with a single chain of 199 residues , is a member of the family of cytokines , which includes erythropoietin , interleukin-6 , and many others , but is most closely related , both evolutionarily and functionally , to human growth hormone and placental lactogen . the hprl gene has been cloned and the authentic form of the hormone efficiently expressed in bacterial periplasm and in chinese hamster ovary ( cho ) cells in our laboratory [ 68 ] . since hprl is one of the hormones most frequently determined in routine clinical assays and several therapeutic applications are being considered , an increasing need for pure bioactive hprl can be anticipated [ 9 , 10 ] . sodium butyrate ( nabu ) is a short chain fatty acid , originally identified as a product of anaerobic bacterial fermentation , which has been shown to alter the structure of chromatin in the nucleus of mammalian cells by reducing the activity of histone deacetylase . one of the possible causes for improved gene expression by nabu cells is histone hyperacetylation , which facilitates the access of general transcription factors in eukaryotic cells [ 12 , 13 ] . nabu treatment has been shown , via immunoassay determination , to increase the expression levels of foreign proteins such as human thrombopoietin , interferon--1a , and chimeric igg3 antibodies in cho cell cultures [ 1517 ] by factors of 2- , 2.5- , and 3.6-fold , respectively , relative to nontreated cells.this led us to investigate , for the first time , via physicochemical techniques , whether addition of nabu would also increase hprl production in these cell cultures . in order to characterize the product obtained , we took advantage of a specific rp - hplc methodology previously developed in our laboratory , which permits qualitative and quantitative analysis of hprl directly in cho - conditioned medium . high - performance size - exclusion chromatography ( hpsec ) methodology was used for both analytical and preparative purposes and also allowed us to evaluated hprl activity directly in two bioassays , after its purification from cho - conditioned medium . the clone expressing hprl , derived from cho dhfr cells ( dukx - b11 ) that had been transfected with the vector peddc - hprl , was obtained in our laboratory . cells were thawed into t - flasks ( 75 cm , from corning costar corporation , cambridge , ma ) containing -minimal essential medium ( -mem , gibco - invitrogen corporation , grand island , ny , usa ) with l - glutamine and without ribonucleosides and deoxyribonucleosides , supplemented with 10% dialysed fetal bovine serum ( dfbs ) , 40 g / ml gentamycin , 4 g / l glucose , and 100 nm methotrexate . cultures were maintained in a humidified incubator at 37c in the presence of 5% co2 . 100 mm nabu was prepared in phosphate - buffered saline ( pbs ) ( 0.007 m na2hpo4 , 0.01 m nah2po4 , ph 7.4 and 0.15 m nacl ) , sterilized by passing through a 0.20 m syringe filter ( corning incorporated , corning , ny , usa ) , and stored in aliquots at 20c . to examine the effect of different concentrations of nabu on hprl production , 1 ml of cho cell suspension ( 5 10 cells / ml ) was seeded into each well of a 24-well plate to obtain a concentration of 2 10 cells / ml . fresh medium was then added containing nabu to a final concentration between 0 and 4 mm . cells were grown for 10 days and cho cell - conditioned medium was collected daily and stored at 80c for subsequent rp - hplc or western blot analysis . for product purification , cho cells were expanded into ten t - flasks , in -mem supplemented with dfbs , until they were confluent . when the cell density was ~2 10 cells / ml , fresh medium was then added to the t - flasks : five contained no nabu , while the chosen amounts of nabu were added to the other five . the hprl - secreting confluent cultures were then maintained for up to ten days by replacing the medium every 24 hours . the conditioned medium was collected and stored , as mentioned above , for sp - sepharose fast flow and hpsec purification . to examine the effect of different concentrations of nabu on cell density and viability , 1 ml of cho cell suspension ( 5 10 cells / ml ) was seeded into each well of a 24-well plate to obtain a concentration of 2 10 cells / ml . nabu was added to a final concentration between 0 and 4 mm and cells were grown for 10 days , as mentioned above . every 24 hours a sample for the different concentrations of nabu was detached from 24-well plate by treatment with 0.025% trypsin - edta in pbs . total cell number and viability were determined by using the trypan blue ( gibco - invitrogen corporation ) exclusion method via haemocytometry and phase contrast microscopy , to distinguish viable and nonviable cells on the basis of dye uptake . 5 10 cells / ml cells were seeded in 24-well plate , treated and detached at regular times , as mentioned above . 1 10 cells / ml per sample were lysed in 400 l of lysis buffer ( 50 mm tris , 10 mm edta ph 8.0 , 1% sds ) and 500 g / ml proteinase k ( fermentas , life sciences , so paulo , brazil ) at 37c for 16 hours . the resulting pellets of dna were resuspended in te buffer ( ph 8.0 ) and treated with 10 g / ml rnase a ( fermentas , life sciences , brazil ) prior to electrophoresis on a 5% polyacrylamide gel at 70 v . discontinuous sds - page , based on 15% polyacrylamide gels , was carried out under nonreducing conditions as described by laemmli . western blot analysis was performed according to bannerman et al . after the treatment with different nabu concentrations , an aliquot of conditioned culture medium ( ~10 ml ) was concentrated ( ~10 fold ) by using a centrifugal membrane filter ultra 4 ( amicon , millipore corporation , billerica , ma , usa ) and applied to a rp - hplc column . a shimadzu model scl-10a hplc apparatus coupled to a spd-10av uv detector ( shimadzu , md , usa ) was used , employing the class vp software , also from shimadzu . this methodology was used for analytical purposes to examine the effect of nabu on hprl production . this methodology was used for both analytical and preparative purposes and allowed us to use purified hprl directly in bioassays , the n - propanol interference being absent due to the rp - hplc technique . hpsec was carried out with the same shimadzu apparatus , processing 25 to 500 l of sample on a tosohaas ( montgomeryville , pa , usa ) g2000 sw column ( 60 cm 7.5 mm i.d . , particle size of 10 m and pore size of 125 ) coupled to a 7.5 cm 7.5 mm i.d . hprl in the final pool obtained via the hpsec purification was quantified for bioassay purposes by hpsec and stored at 80c . the biological activity of purified hprl was determined via the nb2 and ba / f3-llp lymphoma cell - proliferation assays , as previously described , against the first who reference reagent of recombinant hprl , lyophilized in ampoules coded 97/714 . ba / f3-llp cells were routinely maintained in suspension in rpmi-1640 medium supplemented with 10% heat - inactivated fbs , 2 mm glutamine , 50 u / ml of penicillin , 50 g / ml of streptomycin , 700 g / ml of geneticin and 1 ng / ml of hprl . before carrying out the proliferation assay , cells were starved for 6 hours in the rpmi-1640 medium , as mentioned above , without hprl . after 72 hours at 37c and 5% co2 , the presence of viable cells was assessed using mts assay . before carrying out the proliferation assay , cells were maintained in the rpmi-1640 , as mentioned above , containing 1% heat - inactivated fbs for 8 hours , constituting the preassay . afterward , the cells were distributed in flat bottom 96-well plates at a density of 2 10 cells / well in a final volume of 200 l , with no heat - inactivated fbs addition . after 72 hours at 37c and 5% co2 , the presence of viable cells was assessed using mts assay , as mentioned before . the influence of different concentrations of nabu on hprl - secreting cho cellular growth and viability during a 10-day culture can be seen in figure 1 . the treatment with either 0.25 or 0.5 mm nabu did not result in significant growth inhibition ( p > 0.05 ) compared to the control without nabu ( figure 1(a ) ) . nonetheless , a dose response - effect on cellular growth was observed in the concentration range of 1 to 4 mm nabu , with a dramatic fall already occurring after day 2 at the concentrations of 2 mm and 4 mm nabu . the treatment with 1 mm nabu produced an apparent decline only 4 days after starting the treatment . with regard to cell viability , none of the concentrations except 4 mm nabu ( p < 0.05 ) showed any significant effect ( figure 1(b ) ) . the rate of apoptosis in nabu - treated and untreated cho cells was examined by polyacrylamide gel electrophoresis and revealed by silver staining . figure 2 shows that nabu - treated and untreated cho cells exhibit oligonucleosomal dna ladder which is the hallmark of apoptosis , resulting from endonuclease activity at internucleosomal sites in an apoptotic cell . the intensity of the oligonucleosomal dna ladder seems nabu dose - dependent . on the other hand , the progressive removal of dfbs from the culture medium could also be associated with apoptotic cell death in nabu - treated and untreated cho cells , leading to a decrease of cellular growth after day 5 . a rp - hplc methodology , previously set up in our laboratory specifically for hprl qualitative and quantitative analysis , was then employed to quantify hprl production and detect the presence of possible alteration in hprl structure or hydrophobicity due to the prolonged presence of nabu . the data in table 1 and figure 3 show that 1 mm nabu is capable of greatly increasing hprl synthesis ( p < 0.02 ) , with no significant differences ( p > 0.05 ) in the retention time ( tr ) of the resultant hprl up to 4 mm nabu . human prolactin quantification carried out by rp - hplc in cho cell - conditioned medium ( table 1 ) indicated that the highest production was obtained with 1 mm nabu , which increased the secretion ~2-fold relative to control cultivation without nabu addition . despite an increase in protein expression , the concentrations of 0.5 mm and 2 mm nabu provided lower hprl volumetric yields than 1 mm nabu . the greatest inhibition of cell growth was produced by 4 mm nabu leading to lower hprl synthesis ( figure 1(a ) and table 1 ) . clearly , it will be important to determine the optimal nabu concentration in long - term cell cultures . to confirm the expression and purity level of hprl , equal volumes of cho - conditioned medium , collected on days 1 , 4 , 5 , and 6 , were analyzed by western blot ( figure 4 ) ; the same was done with pools obtained after 10 days of cultivation ( figure 5(a ) ) . these pools were also analyzed via sds - page followed by agno3 staining ( figure 5(b ) ) , which demonstrated the predominance , with 1 mm nabu , of hprl relative to other proteins that might be present in cho cell - conditioned medium . taken together , these rp - hplc , western blot , and sds - page analyses indicate that , under our conditions , 1 mm nabu was the ideal concentration , providing the highest hprl expression level , while still maintaining good cell viability and only a limited decrease in cell growth . sp - sepharose fast flow chromatographic purification of nabu - treated and untreated cells , together with sds - page analysis of applied and eluted fractions , is shown in figure 6 . this confirmed the presence of about twice as much hprl in the medium treated with 1 mm nabu and the efficient adsorption of both the 25 kda glycosylated ( g - hprl ) and the 23 kda nonglycosylated hprl ( ng - hprl ) isoforms by the cationic exchanger . the purity obtained for both products after this second chromatographic step is noteworthy ( > 97% ) ( figure 7(c ) and table 2 ) . from table 2 , on the basis of mass fractions , it is possible to calculate an overall purification factor of ~55 and 122-fold for nabu - treated and untreated cho cells , respectively . the ~2-fold increase in hprl production obtained in the presence of 1 mm nabu was also confirmed after purification . also remarkable are the perfectly coincident tr values of the two cho - derived hprl preparations and of the internal reference of e. coli - derived hprl ( 19.58 minutes < tr < 19.72 minutes ) , confirming their identical hydrodynamic properties in this long ( 60 cm ) hpsec column ( figure 7(c ) ) . it has been reported that these properties can be altered even by subtle modifications in the secondary and tertiary structure of the molecule [ 32 , 33 ] . finally , the two purified products were tested for their biological activity against the reference reagent for recombinant hprl ( who 97/714 ) in two different in vitro bioassays based on nb2 and ba / f3-llp cells . the data illustrated in figure 8 and reported in table 3 show that apparently there were no differences between the potencies of the purified products and that of the who reference reagent . previous studies have examined the production of recombinant proteins by nabu - treated cho cells . several of these have discussed the benefits or warned against the problems caused by the presence of nabu [ 1416 ] . nabu has , in fact , been used for increasing the expression of foreign proteins in animal cell cultures . it is recognized that nabu alters primarily chromatin structure through inhibition of histone deacetylases , resulting in hyperacetylation of histones and , consequently , alterations in dna transcription . on the other hand , hyperacetylation of histones in nabu - treated mammalian cancer cell lines has been shown to induce apoptosis mediated by activation of caspase-3 activity , leading to the translation of putative cell death proteins and resulting in an increased possibility of dna cleavage . in this study , we examined the expression level and quality of hprl directly in cho cell - conditioned medium , after adding different amounts of nabu . the different nabu concentrations ( 0.25 to 2 mm ) remarkably increased hprl expression . addition of 1 mm nabu to the culture medium resulted in a twofold increase in hprl expression , with only a relatively small decrease in cell growth compared with the untreated control ; this was confirmed by rp - hplc and by western blot and sds - page analysis . although 2 mm and 4 mm nabu apparently induced a higher synthesis of hprl in the first 24 hours of treatment , this synthesis exhibited a decrease in the following days of culture . a higher nabu concentration ( 2 or 4 mm ) may be more effective in inhibiting the activity of histone deacetylases and , consequently , increase the initial expression of proteins . this was inferred from the fact that the tr values determined by rp - hplc and hpsec were not significantly altered by the nabu treatment relative to the untreated control or the internal reference standard of periplasmic e. coli - derived hprl . our analytical determinations , as a difference from other authors that studied nabu influence on heterologous protein production with basis on immunological techniques [ 1517 ] , have been mostly based on physicochemical methodologies . the cell viability for different concentrations of nabu was similar to that of the untreated control except in the experiments with 4 mm added nabu . therefore , the arrest of cell growth after day 5 and similar decrease in cell viability between 02 mm nabu was probably caused by removal of dfbs from the culture medium and nabu treatment during the production process . this could make the cho cell more susceptible to apoptosis , leading to decreased cellular density and viability . finally , two different in vitro bioassays for purified hprl , based on nb2 and ba / f3-llp cells , were carried out . the first bioassay is the classical heterologous nb2 rat lymphoma cell proliferation assay , while the second is the recently developed homologous ba / f3-llp assay , based on murine pro - b lymphoma cells containing the sequence encoding the human prl receptor and previously utilized in our laboratory [ 9 , 27 ] . although nabu apparently has no effect on the protein structure of hprl , there might be modifications of the oligosaccharide structure of g - hprl . in this regard , it is known that the oligosaccharide nature and content can influence the in vivo bioactivities of a glycoprotein [ 3942 ] . it is therefore important to note that the potency of hprl obtained in the presence of nabu was comparable to that of the reference reagent for recombinant hprl ( who 97/714 ) . in conclusion , for the first time recombinant hprl production from genetically modified cho cells has been significant increased ( ~2-fold ) via nabu addition , apparently without compromising either its structure or function ; the quantitative and qualitative effects of this addition have been determined via accurate physicochemical techniques . the qualitative and quantitative influence of this treatment on the carbohydrate moieties of g - hprl and possibly of other glycohormones requires further investigation .

integrins are cell surface receptors that mediate cell adhesion processes , including cell cell , cell extracellular matrix , and cell pathogen interactions . in general , integrins are heterodimeric glycoproteins that are formed by a single and a single subunit . both the and subunits are single - pass transmembrane proteins , and such proteins are often involved in signal transduction processes . integrins are known to be involved in bidirectional signal transduction across the plasma membrane , commonly referred to as inside - out and outside - in signaling . both the and subunits are made up of a large extracellular ectomembrane domain , a single transmembrane helix , and a small cytoplasmic domain . it has been reported that the dissociation of the cytoplasmic domains of the and subunits is critical for bidirectional transmembrane signaling events . because of its role in the formation of blood clots and maintaining hemostasis , the structure of this complex is of particular interest . these two subunits associate in the membrane and in the cytoplasm with a low affinity . the interaction between these two parts of the dimeric integrin protein has been hard to detect in aqueous solution or studies resulted in different structures , especially for the cytoplasmic domains of iib3 , probably reflecting the dynamic nature of the cytoplasmic domain of iib3 . in the inside - out signaling pathway , the cytoskeletal protein talin has been reported to be able to bind specifically to the cytoplasmic domain of 3 and in turn activate integrin iib3 . the calcium- and integrin - binding protein 1 ( cib1 ) was originally discovered as a binding partner for the cytoplasmic domain of iib in a yeast two - hybrid screening study . cib1 is now known as a ubiquitous regulatory protein that is made up of four ef - hands with a molecular weight of 22 kda . cib1 can carry a myristoyl group at its n - terminus , but the function of this myristoyl group in vivo is not yet fully understood since it has been found that cib1 can interact with iib in vitro without the myristoyl group . the myristoyl group of cib1 appears to act like a membrane anchor , similar to what has been found for many related calcium - binding proteins . cib1 has four helix loop helix ef - hand motifs , but only ef - iii and ef - iv have the capacity to bind divalent metal ions . in the resting state of most cells , magnesium is present in the cytoplasm at 12 mm allowing the folded form of mg - cib1 to be present . when the cell is excited , ca enters the cytoplasm and its concentration rises to micromolar levels ; ca displaces mg and forms ca - cib1 in a well - folded conformation . interestingly , both ca - cib1 and mg - cib1 can interact with a synthetic peptide representing the iib cytoplasmic domain and part of its transmembrane domain , with similar micromolar affinities . solution nmr studies of cib1 require deuteration and methyl group labeling because of its relatively large molecular weight ( 24 kda including the purification - tag ) . it has been shown that ca - cib1 and mg - cib1 have a similar overall topology with mg - cib1 having a relatively more exposed hydrophobic pocket . nmr chemical shift perturbation studies concerning the interaction of iib with both ca- and mg - bound cib1 suggest that both the ca- and mg - forms interact with iib via a hydrophobic pocket in the c - terminal domain of cib1 . it has been reported previously that cib1 is capable of disrupting the association of iib and 3 by binding to iib and in turn activate the integrin iib3 . furthermore , blocking complex formation between the wiskott - aldrich syndrome protein ( wasp ) and cib1 has been shown to affect the conformational change of iib3 and further affects its interaction with fibrinogen . in contrast , cib1 has also been suggested to negatively regulate the activation of integrin iib3 by competing with talin for binding to iib3 . therefore , structural characterization of the cib1/iib complex is of considerable interest to provide further insight into the mechanism of the regulation of the activation of iib3 by cib1 or to determine the role of cib1 in collaborating with talin in the regulation of iib3 activation as suggested by yuan et al . even though the cib1/iib interaction has been suggested to be similar to that of the homologous protein calcineurin b ( cnb ) with calcineurin a ( cna ) in terms of the relative orientation of the proteins , no direct experimental results have been reported to date to support this notion . by determining a structure for the ca - cib1/iib complex , we hope to obtain insight into the inside - out signaling of integrin as well as to establish the connections between the ca - signaling pathway and the integrin - signaling pathways . in this work , an experimental approach of employing reverse cross - saturation nmr experiments using the labeled methyl groups of ile / leu / val ( i / l / v ) in an otherwise deuterated cib1 protein has been implemented to accurately identify the interface for the ca - cib1/iib complex . moreover , residual dipolar couplings ( rdcs ) as determined in multiple alignment media were used to provide additional information about the structure of ca - cib1 in complex with iib . the [ u h , n , c]-labeled sample was used to acquire backbone dcn and dnh residual dipolar couplings ( rdcs ) . the { [ u n , h , c ] ; ile1-[ch3 ] ; leu , val-[ch3,cd3]}-labeled sample was used for the methyl cross - saturation experiments to determine the interface for the interaction between cib1 and the human platelet iib subunit . the 26-residue iib synthetic peptide ( ac - l983vlamwkvgffkrnrppleeddeegq1008-oh ) as previously described was used ; this peptide corresponds to amino acids 9831008 of the iib cytoplasmic domain and part of the transmembrane domain ( hereafter referred to as iib - l , figure 1 ) . another truncated 15-residue synthetic peptide ( ac - l983vlamwkvgffkrnr997-nh2 ) ( iib - s , figure 1 ) was also studied , which corresponds to amino acids 983997 of the iib subunit . and they were more than 95% pure as determined by mass spectrometry and hplc . protein and peptide concentrations were determined by using the extinction coefficients : 280= 3040 for cib1 , 280= 5500 for iib - l , and 280= 5500 for iib - s . iib - l represents the human integrin iib 9831008 region , in which the l983 , w988 , f992 , and f993 have been previously identified to be critical for the interaction between ca - cib1 and iib . iib - l contains part of the transmembrane domain ( shaded in gray box ) and the full cytoplasmic domain of integrin iib . the r995 ( red ) has been reported to form a salt bridge with the integrin 3 subunit , which is critical for the association of the cytoplasmic domain of iib and 3 .. nmr spectra were recorded at 37 c on a bruker avance 500 mhz or a bruker avance 700 mhz nmr spectrometer each equipped with a triple resonance inverse cryoprobe with a single axis z - gradient . each ca - cib sample , with or without iib peptides , contained 0.50.7 mm cib1 in 50 mm hepes , 100 mm kcl , 0.1 mm 2,2-dimethyl-2-silapentane-5-sulfonic acid ( dss ) , 10 mm dtt , 10% or 99.9% d2o , ph 7.5 0.05 ; 2 mm cacl2 was added for the ca - bound sample and 3 mm mgcl2 for mg - bound sample . the [ u h , n , c]-labeled ca - cib1 sample complexed with iib - l was used to acquire the backbone rdcs ( dcn and dnh ) in different alignment media . one of the alignment media used was 14 mg / ml pf1 phage ( asla lab ) for partial alignment of protein molecules . a second medium used was the polyethylene glycol ether ( peg ) medium containing 5% c12e5/hexanol in 0.96/1 ratio . the dcn rdc were measured using the 3d ipap - j - hnco ( ca ) experiment , with 1024 128 40 complex points . a scale factor of 4 was used in the measurement of the dcn rdcs . the dnh rdcs were measured using the 2d ipap - hsqc experiment , with 1024 512 complex points , a digital resolution of 1.1 hz was obtained after linear prediction and zero filling . reverse methyl cross - saturation experiments were performed using 500 m samples of { [ u n , h , c ] ; ile1-[ch3 ] ; leu , val-[ch3,cd3]}-labeled cib1 in 100 mm kcl , 99.9% d2o , 10 mm dtt , 50 mm d18-hepes ( 98% pure ) with and without 600 m iib - l at pd 7.5 ( not corrected for isotope effects ) . four scans were used , resulting in a total measurement time of just 40 min . the methyl - based cross - saturation irradiation using an rf ( radio frequency ) field was applied as suggested , which covers from 3.5 to 8.5 ppm with the irradiation centered at 6.0 ppm , a region including the residual water protons , amide protons , aromatic protons , most of the -protons , and some of the other aliphatic ( and ) protons of the iib - l peptide . selective irradiation was done by using the adiabatic wurst-2 band with a strength up to 0.17 khz . the saturation time ( tsat ) was set at 1.5 s and the interscan delay was 2 s. on the basis of the spectra with ( tsat = 1.5 s ) and without irradiation ( tsat = 0 s ) , the signal loss was calculated using the intensity ( i ) ratio for each methyl group : signal loss = 1 [ i(tsat=1.5)/i(tsat=0 ) ] . a two - stage simulated annealing approach based on three sets of dnh , dcn rdcs measured in two alignment media ( phage pf1 and organic solvent peg ) was implemented for the structure determination of ca - cib1 in complex with iib - l using the program xplor - nih 2.18 . this calculation protocol is designed for studies of homologous proteins or the same protein under different conditions . briefly , for stage 1 , the temperature was cooled from 200 to 20 k , and a strong unramped force constant at 300 kcal mol rad for the dihedral angle ( derived from the chemical shifts of ca - cib1 in complex with aiib - l using the program talos , after correction for the deuterium isotope effect ) was used to ensure proper secondary structure of the resulting structure . also in stage 1 , the force constant of dipolar couplings was ramped from 0.05 to 5 kcal mol hz . the obtained structure still has a certain amount of rdc energy ; in stage 2 , the temperature was cooled from 20 to 2 k and the lowest rdc energy structure obtained from stage 1 was used as a starting model . in stage 2 , the force constant for the dihedral angle ( derived from the lowest energy structure from stage 1 ) was ramped down from 300 to 50 kcal mol rad while the force constant for rdc was kept static at 1 kcal mol hz . other force constants used in the calculation using xplor - nih were the same as previously described . the same approach has been used successfully to determine the structures of various other proteins . the structure of iib - l was generated based on a recent nmr structure for the transmembrane and cytoplasmic domains of iib3 ( pdb 2knc ) . in this study , the iib subunit adopts an -helical structure from i966 until n996 with an extended c - terminal tail at r997e1008 . we used the homology modeling program swiss - model web - server to generate the starting structure for iib - l , which has a q1008 residue instead of e1008 at the c - terminal end . the structure of iib - l ( l983-q1008 ) obtained from homology modeling is essentially the same as the published iib structure in the iib3 complex . the structure of iib - l generated in this manner was used in the subsequent docking model studies of the ca - cib1/iib - l complex . in doing so , we assume that the tail of iib does not undergo a large conformational change when it binds to ca - cib1 . using the refined solution structure of ca - cib1 in the complex and the structure of iib - l , a docking model for the ca - cib1/iib - l complex it is known that the c - terminal extension of ca - cib1 experiences on / off slow motions from the hydrophobic pocket in the c - lobe of ca - cib1 in solution , thereby blocking nonspecific interactions . hence , in our calculations , the structure of the complexed ca - cib1 protein was truncated in order to avoid blocking the access of iib - l to the hydrophobic pocket of ca - cib1 ; the truncated version of ca - cib1 contains residues 8178 with the c - terminal tail of the protein ( residues 179191 ) removed . in the setup , the significantly affected methyl groups of residues i73 , i114 , l131 , l135 , i153 , i168 , v176 , and i177 were set as the active residues , while the slightly affected methyl - containing residues i27 , l28 , l61 , v76 , v97 , v132 , l152 , and i156 were set as passive residues in the haddock calculations . residues l983 , w988 , f992 , and f993 in iib - l have been reported to be critical for the interaction between ca - cib1 and iib - l by a combined approach of site - directed mutagenesis and in vitro fluorescence studies . hence , these four residues were set as the active residues for iib - l . also in the setup , since iib - l residues l983r995 have been determined to be helical and the remainder ( n996q1008 ) to be an extended structure , fragment 9951008 of iib - l was set as fully flexible for the docking model generation . because haddock can automatically detect semiflexible fragments , cib1 was not set as fully flexible . rdc restrains that were used for the structure calculation for the ca - cib1 in complex with iib - l were also implemented in the docking model generation . in addition , dihedral angle restraints for cib1 ( 8178 ) and iib - l ( 983995 ) were created based on the lowest rdc energy structure determined for the ca - cib1 in protein complex with iib - l as well as the available iib structure ( pdb 2knc ) in complex with 3 . other parameters were standard default values except that 3000 structures were calculated for the stage of rigid body docking , 400 structures for the subsequent structural analysis stage , and 20 structures were set as the minimal number for clustering . the lowest interaction energy docking structure cluster was taken as the complex model for ca - cib1/iib - l . the lowest rdc energy structures of ca - cib1 ( residues 8191 ) in complex with iib - l were selected for further analysis . an in - house script was used to measure the interhelical angles for the helix pairs in each ef - hand . the solution structure ( figure 2 ) of the ca - cib1 protein in complex with iib - l was refined based on three sets of backbone rdcs obtained in two alignment media ( nh and cn rdcs in pf1 , and nh rdcs in peg ) using the solution structure of ca - cib1 ( pdb 2l4h ) as the starting model and a two - stage low - temperature simulated annealing protocol . the use of multiple sets of alignment media has been previously suggested because it can avoid the intrinsic degeneracy problem of using rdcs as well as avoid any preference for a minor conformation that could result from the use of a single alignment medium . some residues on the flexible loop of cib1 ( residues 137145 ) and the c - terminal end ( 180191 ) have reduced rdc values due to local mobility , and these residues were excluded from the structure calculation . a good structural precision has been obtained for the structure calculation ; the backbone rmsd of the 10 lowest energy structures is 0.46 ( figure 2a ) . the statistics of the structure determination of the ca - cib1 protein when complexed with iib - l have been summarized in supporting information table s1 . the solution structure of the ca - cib1 protein in the complex also shows a similar overall topology compared with the solution structure of ca - cib1 alone , with a backbone rmsd of 3.7 ( figure 2b ) . compared with the ca - cib1 structure , the n - terminal -helix ( helix 0 , h0 ) in the new structure has shifted to some extent . the n - terminal -helix is assumed to carry a myristoyl group in vivo which should associate with the cell membrane . therefore , binding of iib - l could affect the orientation of cib1 relative to the cell membrane . moreover , the interhelical angles measured using an in - house script suggest that the opening of the helix pairs in each ef hand changes upon binding aiib . the two ef - hands in the c - lobe have a bigger interhelical opening than free ca - cib1 by approximately 20 ( figure 2c ) . the resultant enlarged hydrophobic pocket in the c - lobe could readily adopt aiib . the q factors ( table s3 and figure s1 ) before and after structural refinement denote a significant improvement in the results of fitting rdcs to the obtained structure of ca - cib1 complexed with iib - l compared with the starting model , that is , the free ca - cib1 structure ( pdb 2l4h ) . the structure of ca - cib1 in complex with iib - l . ( a ) superposition of the best 10 structures of the ca - cib1 protein in complex with iib - l ; ( b ) superposition of the solution structures of ca - cib1 alone ( pdb 2l4h , cyan ) and in complex with iib - l ( pink ) ( backbone rmsd = 3.7 ) . ( c ) the superimposed structures of the c - lobe of ca - cib1 alone ( cyan ) and in complex with iib - l ( pink ) demonstrate the increased opening of helix pairs h6/h7 and h8/h9 . the two structures were superimposed based on h7 and h8 ; thus , the opening is highlighted by the altered orientations of h6 and h9 . cross - saturation nmr experiments employing backbone amide proton nh resonances were originally developed to detect the interface for protein protein interactions . compared with the commonly used nmr chemical shift perturbation ( csp ) approach , which is widely used for determining the interface of protein / peptide complexes , the cross - saturation method avoids possible artifacts arising from induced conformational changes , where residues that experienced a large chemical shift change can in fact be far away from the actual interface . a more recent version of the cross - saturation experiment uses isotope labeled methyl groups rather than backbone amides . it has several advantages over the traditional cross - saturation backbone n h method , such as high sensitivity , higher efficiency of magnetization transfer , and suitability for studying hydrophobic interactions that are often involved in protein / protein complex formation . traditionally , the cross - saturation approach has been used to detect the binding interface of the smaller partner by irradiating the larger partner because the magnetization will be more efficiently transferred by spin diffusion in a larger protein . in our work , we applied the selective irradiation in a reverse manner ( referred to as reverse cross - saturation ) , in a way that is somewhat similar to previous studies . we saturated the smaller binding partner ( iib - l , 3 kda ) to detect the interface on the larger binding partner ( ca - cib1 , 24 kda including the purification his - tag ) ( figure 3 ) . in our methyl - probe cross - saturation experiments , selective irradiation was employed to cover the spectral region from 3.5 to 8.5 ppm ( figure s2 ) . to avoid artifacts , 99.9% d2o was used as the solvent . in the spectral area from 3.5 to 8.5 ppm ( figure s2 ) , methyl labeled but otherwise perdeuterated ca - cib1 has no signals except for some peaks due to the d18-hepes buffer ( 98% pure ) used to dissolve the ca - cib1/iib - l complex , while iib - l contains aromatic protons , aliphatic proteins ( h , h , h , etc . ) and no amide protons ( nh ) because of the presence of the d2o solvent ( figure s2 ) . therefore , the irradiation selectively targets iib - l but not the deuterated cib1 . this saturation should then be transferred to the protonated methyl groups involved in the ca - cib1/iib - l interface by spin - diffusion ( figure 3 ) . the interface of cib1 in its complex with iib - l can therefore be accurately mapped using cross - saturation experiments . control experiments were carried out on an nmr sample containing only ca - cib1 , and the results show that all the methyl groups were evenly affected to a minimum level ( half of all methyls are below 0.1 and the other half are between 0.1 and 0.2 in terms of signal loss ) ( figure 4c ) . since the irradiation pulse was suggested to be adiabatic , the signal loss in the control experiments is probably due to the presence of residual protons of the d18-hepes buffer ( 50 mm , 98% pure ) ( figure s2 ; for further discussion see shimada ) . schematic diagram illustrating the mechanism of the reverse methyl cross - saturation for the ca - cib1/iib - l complex . selective rf ( radio frequency ) irradiation was applied to the h , h , and aromatic proton spectral region of iib - l , and the intensity change of the protonated methyl groups on deuterated ca - cib1 was monitored . ( a ) hsqc spectra of ca - cib1/iib - l showing the effects of saturation on the ile residues . the spectrum on the left is the h , c - hsqc with no saturation , and the spectrum on the right includes selective saturation . ( b ) signal loss ratio of the intensities of the methyl groups in ca - cib1 complexed with iib - l , with and without the selective irradiation . the signal losses over 30% and 20% were highlighted with gridlines and these results were further utilized in the generation of docking complex structural model for ca - cib1 and iib - l using the program haddock . ( c ) control experiment of methyl cross - saturation with the free ca - cib1 : only a minimum saturation effect was observed on nearly all methyl groups . to identify the interface between ca - cib1 and iib - l , an identical setup as the one used for the control experiments was used for a sample of ca - cib1 complexed with 1/1.2 ratio of iib - l . the results obtained are illustrated for the superimposed hsqc spectra of the ile region ( figure 4a1 ) . with irradiation , the i73 , i114 , and i177 residues show significant signal loss compared to the reference spectrum ( tsat = 0 s ) ( figure 4a2 and b ) , whereas other methyl protonated isoleucines ( e.g. , i58 , i106 , i162 , and i189 ) are relatively less affected by the cross - saturation . the significantly affected methyl - containing ile , leu , and val residues with a signal loss ratio above 0.3 include i73 , i114 , l131 , l135 , i153 , i168 , v176 , and i177 ( figure 4b ) , and these were classified as the active residues in the ca - cib1/iib - l interface that make direct contact with the binding partner iib - l . other slightly affected methyl - containing residues with signal loss ratio between 0.2 and 0.3 , which include i27 , l28 , l61 , v76 , v97 , v132 , l152 , and i156 ( figure 4b ) , have been classified as passive residues that are close to the interface , but lack direct contact with iib - l . the remaining peaks are the nonaffected or marginally affected methyl groups with a signal loss ratio less than 0.2 ( figure 4b ) , and these are considered not involved in the hydrophobic interactions between the protein and the peptide . the cross - saturation effects were mapped on the newly determined structure of ca - cib1 in complex with iib - l ( figure 5b ) . compared with methyl csp ( chemical shift perturbation ) effects ( figure 5a ) , cross - saturation experiments provide a similar interface for the interactions between ca - cib1 and iib - l , suggesting that the c - domain of ca - cib1 is the primary binding site for the cytoplasmic domain of iib . small differences between csp and cross - saturation were also observed ; the distribution of active interfacial residues from the cross - saturation experiments ( figure 5b ) seems to suggest one possible orientation for the iib - l peptide , whereas two possible orientations were suggested by csp ( figure 5a ) . clearly , a majority of the significantly affected methyl groups are found in the c - lobe of ca - cib1 , except for residue i73 , which is in the n - lobe . ( a ) mapping the methyl chemical shift perturbation ( csp ) on the structure of ca - cib1 in complex with iib ; the methyl csp values were previously reported ; ( b ) mapping the signal loss values from reverse cross - saturation experiments on the structure of ca - cib1 in complex with iib . in both panels , the dotted lines ( red ) resemble possible orientations of iib - l in its complex structure with ca - cib1 . the previously discussed c - terminal displacement mechanism for cib1 allows us to interpret our methyl cross - saturation results . the c - terminal extension of cib1 undergoes a conformational transition between free and bound states as suggested by our earlier backbone relaxation and relaxation dispersion measurements . the three assigned methyl groups on the c - terminal extension of ca - cib1 , that is , i1891 , and v1901 and 2 , are only marginally affected by the saturation from iib - l , which suggests that these three residues are not in direct contact with iib - l in the complex . thus , the cross - saturation results are consistent with the backbone relaxation and relaxation dispersion results regarding the flexibility of the c - terminal extension ; the extension remains flexible when the protein binds to iib - l . initial attempts of using isotope - filtered or transferred noesy experiments to determine the structure of iib - l in complex with ca - cib1 failed to provide sufficient intermolecular noes between iib - l and ca - cib1 . even though nmr titration experiments ( results not shown ) suggest that the interaction between these two is in slow exchange regime , previous studies suggested that iib interacts relatively weakly with ca - cib1 with a dissociation constant ( kd ) of 1.4 m at 37 c . this exchange behavior seems to have hindered the determination of the solution structure of iib - l in complex with ca - cib1 . isotope - labeled iib - l was then prepared through bacterial expression as a fusion protein but the solubility in aqueous solution became a problem , even though the n - terminal acetylated synthetic iib - l peptide was quite soluble . fortunately , a structure for the iib subunit ( including the transmembrane and cytoplasmic domains ) ( pdb 2knc ) , in complex with 3 , has recently been reported . this structure of iib ( pdb 2knc ) covers the fragment of iib - l used in our work ( residues 9831008 ) . it is strikingly similar to a structure previously determined in a 45% trifluoroethanol aqueous solution for the synthetic iib - l peptide , containing a typical -helical structure ( l983n996 ) followed by an extended tail ( r997q1008 ) . in addition , it should be noted that the c - terminal extension of cib1 in the crystal structure of ca - cib1 ( 1xo5 ) is folded back into the protein and adopts an -helical conformation ( figure s3 ) . this implies that the hydrophobic pocket in the c - domain of ca - cib1 can readily accommodate a binding partner in an -helical conformation . therefore , we suggest that it is reasonable to assume that the fragment l983r997 adopts an -helical conformation when interacting with ca - cib1 . the solution structure of the ca - cib1/iib - l complex has been determined by data - driven docking . the 20 lowest energy structures of the ca - cib1/iib - l complex are displayed in figure 6 in both ribbon and surface charge forms . in figure 6a , the n - terminal helical part of iib - l interacts with the c - domain of ca - cib1 , while the negatively charged c - terminal tail of iib - l remains unstructured ( figure 6a ) , possibly interacting with some positively charged residues , for example , r33 and k65 as was suggested by backbone csp measurements . in our docking experiment setup , we solely relied on the methyl cross - saturation results for ca - cib1 and on previously reported critical residues in iib - l ; thus , these restraints do not reflect any potential electrostatic interactions . therefore , the relative orientation of these two partners was mostly driven by the enthalpy of the hydrophobic interactions between the iib - l helix and the c - domain of ca - cib1 . a related situation is found for the structure of the homologous protein calcineurin b ( cnb ) complexed with calcineurin a ( cna ) ( pdb 1tco ) ( figure 6c ) , in which the c - terminal domain of cnb interacts with the n - terminal of the cnb binding domain of cna , with the cnb binding domain of cna adopting an -helical conformation . regarding the structure of the acidic tail ( p998pleeddeegq1008 ) of iib , we attempted to dock this portion better by adding r33 and k65 ( residues identified by backbone csp ) of cib1 as heavily affected residues but the tail still remained dynamic probably because no restraints were available for this portion of the iib tail . ( a ) the superposition of the 20 best structures of the ca - cib1/iib - l complex generated using haddock . ( b ) the ca - cib1 ( 8178 ) protein is represented with a surface structure , while aiib - l is represented as a ribbon structure . the surface polarity is scaled with colors , using blue for the positively charged surface and red for the negatively charged surface , while a white color indicates nonpolar patches . ( c ) the surface charge model for the complex of cnb / cna ( pdb 1tco ) , in which cnb is presented with a surface structure and cna ( residues 340373 ) is presented as a ribbon structure . a shorter version of the iib peptide ( iib - s , ac - l983vlamwkvgffkrnr997-nh2 ) was synthesized to examine the role of the acidic tail of iib in its interaction with ca - cib1 . specifically , the difference between the iib - l and iib - s peptides resides in the c - terminal fragment p998pleeddeegq1008 , which bears multiple negative charges , making this region of iib extremely acidic . in figure s4a , the superimposed hsqc spectra of ca - cib1/iib - l and ca - cib1/iib - s show small differences with a few methyl groups experiencing minor chemical shift changes . this suggests that the c - terminal fragment p998pleeddeegq1008 was not involved in specific interactions with ca - cib1 , which could affect the interactions between ca - cib1 and iib - l . the residues ( l8 , v45 , v76 , l92 , l94 , v97 , i 106 , and l170 ) with minor chemical shift changes are mainly located in the n - lobe of cib1 . on the other hand , these shifted methyl groups do not center at a specific location in the structure , implying a dynamic interaction between the n - lobe of cib1 and the acidic tail ( p998pleeddeegq1008 ) . a recent study shows that this part of the iib cytoplasmic domain can in fact play a role in platelet activation , but it only does so when it is anchored to the inner surface of the membrane . since mg ions are present in the cytoplasm with an almost invariant concentration of around 12 mm and the structure of mg - cib1 is overall similar to that of ca - cib1 , we were interested in the structural differences between the complexes of cib1/iib - l when bound to ca and mg , in order to further understand the role of ca in the regulation of the cib1/iib interaction . therefore , h , c - hsqc spectra for the methyl groups of cib1 complexed with iib - l were recorded in the presence of either ca or mg ( figure s4b ) . similar to the backbone nmr studies , methyl side chain h , c - hsqc spectra for ca- and mg - bound cib1/iib - l show nearly the same pattern with minor deviations except for two residues in the calcium / magnesium binding loop , i168 and l123 ( figure s4b ) . these results suggest that ca - cib and mg - cib1 interact with iib in nearly the same manner . in this study , we have obtained a solution structure for the isotope - labeled ca - cib1 protein , complexed with the synthetic iib - l peptide that encompasses the cytoplasmic domain and part of the transmembrane domain of the iib subunit of platelet integrin . the structure for the complexed protein could be determined based on rdc restraints . with the use of the available structure of iib ( pdb 2knc ) , a structural model for the ca - cib1/iib - l complex was obtained using data - driven docking based on reverse cross - saturation nmr experiments . the two binding partners in this complex structure adopt a relative orientation with the -helical n - terminal portion of iib - l buried into the c - lobe hydrophobic pocket of ca - cib1 and the negatively charged and extended c - terminal tail of iib - l pointing toward the n - lobe of ca - cib1 but being relatively dynamic . the superposition of the ordered structural fragment ( residues 983995 ) and the corresponding fragment of 2knc provides the orientation of the ca - cib1/iib - l complex relative to the cell membrane ( figure 7a ) . as expected , the n - terminal extension of ca - cib1(helix 0 , h0 ) points toward the cell membrane , which is consistent with its role in linking to a membrane - bound myristoyl group ( figure 7a ) . however , when modeled together with a membrane , a steric clash was observed between the transmembrane helix of iib and the eighth helix of ca - cib1 ( h8 ) ( figure 7a ) . moreover , cib1 would become associated directly with the membrane to a large extent if the iib subunit interacts with ca - cib1 with both of its transmembrane and cytoplasmic domains . since cib1 is mainly found as a myristoylated protein in the cytoplasm and the binding interface on iib primarily includes the fragment of 983995 , it seems unlikely that ca - cib1 will penetrate into the cell membrane or be engaged with a significantly large part of the iib transmembrane domain . therefore , a conformational change is likely to happen in iib upon binding to ca - cib1 . we propose that the transmembrane helix of iib will bend around w988 ( the membrane - border residue for iib - l ) when binding to ca - cib1 ( figure 7b , c ) . in this manner , the steric clash and the potential energy barrier caused by an intrusion of cib1 into the membrane would be avoided . ( a ) superposition of the ordered structural fragment ( 983995 ) of the averaged structure of iib - l complexed with ca - cib1 with the corresponding fragment of 2knc . a steric clash was observed between 2knc and the 8th helix of ca - cib1 ( h8 , brown color ) ; the n - terminal extension of ca - cib1 ( h0 , pink color ) points toward the cell membrane , which is consistent with its role of linking the myristoyl group and the ca - cib1 to a membrane . ( b ) the side view of ( a ) ; ( c ) a possible bending mechanism was proposed for the conformational change of iib upon binding of ca - cib1 , as indicated by the red dashed line with arrows . the letter n in italics indicates the n - terminal of ca - cib1 . recent structural studies of the transmembrane domains as well as the protein fragments including both the transmembrane and cytoplasmic domains of iib3 have shed some light on the molecular mechanism of the regulation of the activation of iib3 . for the structure of the iib3 transmembrane domains , various approaches gave rise to almost converged structures ( see detailed analysis in yang et al . and however , the structure of the cytoplasmic domains of iib3 was reported to be diverse . the importance of the previously suggested salt bridge between iib r995 and 3 d723 that was considered for a long time to be critical for the association of iib3 has recently been brought into question , consistent with the dynamic nature of the cytoplasmic domains of iib3 . the g991ffkr995 motif on iib is critical for the interactions between iib and 3 as revealed in the iib3 complex structure determined using cys - scanning - mutagenesis / disulfide - cross - linking / rosetta - modeling and another iib3 complex nmr structure that has been determined using lipid bicelles as the solvent ( pdb 2k9j ) . on the basis of a comparison with these two structures , ca - cib1 seems to dissociate the iib3 heterodimer , which would potentially activate the iib3 complex because the f992 and f993 side chains become buried in the hydrophobic pocket in the ca - cib1/iib - l complex structure ( figure s5 ) . it has also been reported that an f992a mutation in iib may partially impair the interaction with 3 although both f992 and f993 in iib point away from the 3 subunit in 2knc . moreover , a potential conformational change of iib upon binding to ca - cib1 ( figure 7 ) will also disturb the stability of the structure of the iib3 complex and cause the dissociation of these two subunits . on the basis of the classic studies indicating that the activation of integrin can be caused by the dissociation of the heterodimer , ca - cib1 is highly likely able to disrupt the association of iib and 3 . taken all data together , a mechanism for activation of iib3 upon interacting with ca - cib1 can be proposed , that is similar to the action of the cytoskeletal protein talin , where ca - cib1 dissociates the iib3 heterodimer and consequently activates integrin iib3 ( figure 8) . alternatively cib1 can maintain the activated state of iib3 by covering the binding site in iib and preventing its association with 3 . since nearly identical hsqc nmr patterns were observed for mg - cib1 and for ca - cib1 upon interaction with iib - l ( figure s4b ) , our data suggest that the same complex can form under various physiological conditions and is not calcium - dependent per se . this notion is supported by previous in vivo studies , in which iib was shown to interact with cib1 in both resting and stimulated platelets with no requirement for a rise in the intracellular calcium concentration . in addition , it is possible that protein phosphatase 1 could compete with cib1 for iib , as these two proteins share the same binding motif ( k989vgf992 ) on the iib cytoplasmic domain . a structural model for the mechanism of ca - cib1 regulation of integrin activation . ( ( b ) in the presence of ca - cib1 , the iib cytoplasmic subunit interacts with ca - cib1 , with its n - terminal -helix buried in the hydrophobic pocket of the c - lobe of cib1 and its c - terminal negatively charged tail being dynamic but likely being in contact with the n - lobe of cib1 . subsequently , iib and 3 are separated by ca - cib1 and this iib3 complex dissociates . the integrin iib3 dimer is activated by ca - cib1 , which is similar to the activation by talin , a cytoskeleton protein , which binds to the 3 cytoplasmic domain .. in this work , we have used a reverse cross - saturation nmr approach to detect the interface on ca - cib1 that recognizes the bound iib - l peptide . in these experiments , we selectively irradiated the smaller partner in the protein protein complex , and the results from our work ( figure 4 and figure 5 ) demonstrate that it is possible to identify the hydrophobic residues that are involved in the interactions between cib1 and iib . this represents a powerful way of using the nmr cross - saturation experiment , where normally the larger partner has been irradiated to achieve more effective spin - difussion . many protein complexes currently under investigation can be represented by studying a large protein and a synthetic peptide to represent the intact protein partner or a protein domain . provided that the proteins of interest can be bacterially expressed , deuterated , and selectively protonated on methyl groups , the reverse cross - saturation experiment with methyl detection should become a useful nmr approach for better understanding protein protein interactions in general .

calcium and integrin binding protein 1 ( cib1 ) is a specific binding partner for the cytoplasmic domain of the iib subunit of the highly abundant platelet integrin iib3 . this protein has been suggested to be involved in the regulation of the activation of iib3 , a process leading to platelet aggregation and blood coagulation . in this work , the solution structure of the deuterated ca2 + -cib1 protein complexed with an iib peptide was first determined through modern rdc - based nmr methods . next , we generated a complex structure for cib1 and the iib domain ( ca2 + -cib1/iib ) using the program haddock , which is based on experimental restraints obtained for the protein peptide interface from cross - saturation nmr experiments . in this data - driven complex structure , the n - terminal -helix of the cytoplasmic domain of iib is buried in the hydrophobic pocket of the c - lobe of ca2 + -cib1 . the c - terminal acidic tail of iib remains unstructured and likely interacts with several positively charged residues in the n - lobe of ca2 + -cib1 . a potential molecular mechanism for the cib1-mediated activation of the platelet integrin could be proposed on the basis of the model structure of this protein complex . another feature of this work is that , in the nmr cross - saturation experiments , we applied the selective radio frequency irradiation to the smaller binding partner ( the iib peptide ) , and successfully detected the binding interface on the larger binding partner ca2 + -cib1 through its selectively protonated methyl groups . this reverse methodology has a broad potential to be employed to many other complexes where synthetic peptides and a suitably isotope - labeled medium- to large - sized protein are used to study protein protein interactions .

Introduction Materials and Methods Results Discussion Conclusion

the interaction between these two parts of the dimeric integrin protein has been hard to detect in aqueous solution or studies resulted in different structures , especially for the cytoplasmic domains of iib3 , probably reflecting the dynamic nature of the cytoplasmic domain of iib3 . in the inside - out signaling pathway , the cytoskeletal protein talin has been reported to be able to bind specifically to the cytoplasmic domain of 3 and in turn activate integrin iib3 . the calcium- and integrin - binding protein 1 ( cib1 ) was originally discovered as a binding partner for the cytoplasmic domain of iib in a yeast two - hybrid screening study . nmr chemical shift perturbation studies concerning the interaction of iib with both ca- and mg - bound cib1 suggest that both the ca- and mg - forms interact with iib via a hydrophobic pocket in the c - terminal domain of cib1 . therefore , structural characterization of the cib1/iib complex is of considerable interest to provide further insight into the mechanism of the regulation of the activation of iib3 by cib1 or to determine the role of cib1 in collaborating with talin in the regulation of iib3 activation as suggested by yuan et al . in this work , an experimental approach of employing reverse cross - saturation nmr experiments using the labeled methyl groups of ile / leu / val ( i / l / v ) in an otherwise deuterated cib1 protein has been implemented to accurately identify the interface for the ca - cib1/iib complex . the { [ u n , h , c ] ; ile1-[ch3 ] ; leu , val-[ch3,cd3]}-labeled sample was used for the methyl cross - saturation experiments to determine the interface for the interaction between cib1 and the human platelet iib subunit . the r995 ( red ) has been reported to form a salt bridge with the integrin 3 subunit , which is critical for the association of the cytoplasmic domain of iib and 3 .. nmr spectra were recorded at 37 c on a bruker avance 500 mhz or a bruker avance 700 mhz nmr spectrometer each equipped with a triple resonance inverse cryoprobe with a single axis z - gradient . the methyl - based cross - saturation irradiation using an rf ( radio frequency ) field was applied as suggested , which covers from 3.5 to 8.5 ppm with the irradiation centered at 6.0 ppm , a region including the residual water protons , amide protons , aromatic protons , most of the -protons , and some of the other aliphatic ( and ) protons of the iib - l peptide . the saturation time ( tsat ) was set at 1.5 s and the interscan delay was 2 s. on the basis of the spectra with ( tsat = 1.5 s ) and without irradiation ( tsat = 0 s ) , the signal loss was calculated using the intensity ( i ) ratio for each methyl group : signal loss = 1 [ i(tsat=1.5)/i(tsat=0 ) ] . briefly , for stage 1 , the temperature was cooled from 200 to 20 k , and a strong unramped force constant at 300 kcal mol rad for the dihedral angle ( derived from the chemical shifts of ca - cib1 in complex with aiib - l using the program talos , after correction for the deuterium isotope effect ) was used to ensure proper secondary structure of the resulting structure . in this study , the iib subunit adopts an -helical structure from i966 until n996 with an extended c - terminal tail at r997e1008 . we used the homology modeling program swiss - model web - server to generate the starting structure for iib - l , which has a q1008 residue instead of e1008 at the c - terminal end . using the refined solution structure of ca - cib1 in the complex and the structure of iib - l , a docking model for the ca - cib1/iib - l complex it is known that the c - terminal extension of ca - cib1 experiences on / off slow motions from the hydrophobic pocket in the c - lobe of ca - cib1 in solution , thereby blocking nonspecific interactions . hence , in our calculations , the structure of the complexed ca - cib1 protein was truncated in order to avoid blocking the access of iib - l to the hydrophobic pocket of ca - cib1 ; the truncated version of ca - cib1 contains residues 8178 with the c - terminal tail of the protein ( residues 179191 ) removed . also in the setup , since iib - l residues l983r995 have been determined to be helical and the remainder ( n996q1008 ) to be an extended structure , fragment 9951008 of iib - l was set as fully flexible for the docking model generation . the solution structure ( figure 2 ) of the ca - cib1 protein in complex with iib - l was refined based on three sets of backbone rdcs obtained in two alignment media ( nh and cn rdcs in pf1 , and nh rdcs in peg ) using the solution structure of ca - cib1 ( pdb 2l4h ) as the starting model and a two - stage low - temperature simulated annealing protocol . some residues on the flexible loop of cib1 ( residues 137145 ) and the c - terminal end ( 180191 ) have reduced rdc values due to local mobility , and these residues were excluded from the structure calculation . compared with the ca - cib1 structure , the n - terminal -helix ( helix 0 , h0 ) in the new structure has shifted to some extent . cross - saturation nmr experiments employing backbone amide proton nh resonances were originally developed to detect the interface for protein protein interactions . traditionally , the cross - saturation approach has been used to detect the binding interface of the smaller partner by irradiating the larger partner because the magnetization will be more efficiently transferred by spin diffusion in a larger protein . in our work , we applied the selective irradiation in a reverse manner ( referred to as reverse cross - saturation ) , in a way that is somewhat similar to previous studies . we saturated the smaller binding partner ( iib - l , 3 kda ) to detect the interface on the larger binding partner ( ca - cib1 , 24 kda including the purification his - tag ) ( figure 3 ) . since the irradiation pulse was suggested to be adiabatic , the signal loss in the control experiments is probably due to the presence of residual protons of the d18-hepes buffer ( 50 mm , 98% pure ) ( figure s2 ; for further discussion see shimada ) . selective rf ( radio frequency ) irradiation was applied to the h , h , and aromatic proton spectral region of iib - l , and the intensity change of the protonated methyl groups on deuterated ca - cib1 was monitored . the significantly affected methyl - containing ile , leu , and val residues with a signal loss ratio above 0.3 include i73 , i114 , l131 , l135 , i153 , i168 , v176 , and i177 ( figure 4b ) , and these were classified as the active residues in the ca - cib1/iib - l interface that make direct contact with the binding partner iib - l . the remaining peaks are the nonaffected or marginally affected methyl groups with a signal loss ratio less than 0.2 ( figure 4b ) , and these are considered not involved in the hydrophobic interactions between the protein and the peptide . compared with methyl csp ( chemical shift perturbation ) effects ( figure 5a ) , cross - saturation experiments provide a similar interface for the interactions between ca - cib1 and iib - l , suggesting that the c - domain of ca - cib1 is the primary binding site for the cytoplasmic domain of iib . clearly , a majority of the significantly affected methyl groups are found in the c - lobe of ca - cib1 , except for residue i73 , which is in the n - lobe . the previously discussed c - terminal displacement mechanism for cib1 allows us to interpret our methyl cross - saturation results . the three assigned methyl groups on the c - terminal extension of ca - cib1 , that is , i1891 , and v1901 and 2 , are only marginally affected by the saturation from iib - l , which suggests that these three residues are not in direct contact with iib - l in the complex . thus , the cross - saturation results are consistent with the backbone relaxation and relaxation dispersion results regarding the flexibility of the c - terminal extension ; the extension remains flexible when the protein binds to iib - l . isotope - labeled iib - l was then prepared through bacterial expression as a fusion protein but the solubility in aqueous solution became a problem , even though the n - terminal acetylated synthetic iib - l peptide was quite soluble . fortunately , a structure for the iib subunit ( including the transmembrane and cytoplasmic domains ) ( pdb 2knc ) , in complex with 3 , has recently been reported . in addition , it should be noted that the c - terminal extension of cib1 in the crystal structure of ca - cib1 ( 1xo5 ) is folded back into the protein and adopts an -helical conformation ( figure s3 ) . this implies that the hydrophobic pocket in the c - domain of ca - cib1 can readily accommodate a binding partner in an -helical conformation . the solution structure of the ca - cib1/iib - l complex has been determined by data - driven docking . in figure 6a , the n - terminal helical part of iib - l interacts with the c - domain of ca - cib1 , while the negatively charged c - terminal tail of iib - l remains unstructured ( figure 6a ) , possibly interacting with some positively charged residues , for example , r33 and k65 as was suggested by backbone csp measurements . in our docking experiment setup , we solely relied on the methyl cross - saturation results for ca - cib1 and on previously reported critical residues in iib - l ; thus , these restraints do not reflect any potential electrostatic interactions . therefore , the relative orientation of these two partners was mostly driven by the enthalpy of the hydrophobic interactions between the iib - l helix and the c - domain of ca - cib1 . a related situation is found for the structure of the homologous protein calcineurin b ( cnb ) complexed with calcineurin a ( cna ) ( pdb 1tco ) ( figure 6c ) , in which the c - terminal domain of cnb interacts with the n - terminal of the cnb binding domain of cna , with the cnb binding domain of cna adopting an -helical conformation . regarding the structure of the acidic tail ( p998pleeddeegq1008 ) of iib , we attempted to dock this portion better by adding r33 and k65 ( residues identified by backbone csp ) of cib1 as heavily affected residues but the tail still remained dynamic probably because no restraints were available for this portion of the iib tail . specifically , the difference between the iib - l and iib - s peptides resides in the c - terminal fragment p998pleeddeegq1008 , which bears multiple negative charges , making this region of iib extremely acidic . this suggests that the c - terminal fragment p998pleeddeegq1008 was not involved in specific interactions with ca - cib1 , which could affect the interactions between ca - cib1 and iib - l . the residues ( l8 , v45 , v76 , l92 , l94 , v97 , i 106 , and l170 ) with minor chemical shift changes are mainly located in the n - lobe of cib1 . on the other hand , these shifted methyl groups do not center at a specific location in the structure , implying a dynamic interaction between the n - lobe of cib1 and the acidic tail ( p998pleeddeegq1008 ) . since mg ions are present in the cytoplasm with an almost invariant concentration of around 12 mm and the structure of mg - cib1 is overall similar to that of ca - cib1 , we were interested in the structural differences between the complexes of cib1/iib - l when bound to ca and mg , in order to further understand the role of ca in the regulation of the cib1/iib interaction . in this study , we have obtained a solution structure for the isotope - labeled ca - cib1 protein , complexed with the synthetic iib - l peptide that encompasses the cytoplasmic domain and part of the transmembrane domain of the iib subunit of platelet integrin . with the use of the available structure of iib ( pdb 2knc ) , a structural model for the ca - cib1/iib - l complex was obtained using data - driven docking based on reverse cross - saturation nmr experiments . the two binding partners in this complex structure adopt a relative orientation with the -helical n - terminal portion of iib - l buried into the c - lobe hydrophobic pocket of ca - cib1 and the negatively charged and extended c - terminal tail of iib - l pointing toward the n - lobe of ca - cib1 but being relatively dynamic . as expected , the n - terminal extension of ca - cib1(helix 0 , h0 ) points toward the cell membrane , which is consistent with its role in linking to a membrane - bound myristoyl group ( figure 7a ) . since cib1 is mainly found as a myristoylated protein in the cytoplasm and the binding interface on iib primarily includes the fragment of 983995 , it seems unlikely that ca - cib1 will penetrate into the cell membrane or be engaged with a significantly large part of the iib transmembrane domain . a steric clash was observed between 2knc and the 8th helix of ca - cib1 ( h8 , brown color ) ; the n - terminal extension of ca - cib1 ( h0 , pink color ) points toward the cell membrane , which is consistent with its role of linking the myristoyl group and the ca - cib1 to a membrane . recent structural studies of the transmembrane domains as well as the protein fragments including both the transmembrane and cytoplasmic domains of iib3 have shed some light on the molecular mechanism of the regulation of the activation of iib3 . the g991ffkr995 motif on iib is critical for the interactions between iib and 3 as revealed in the iib3 complex structure determined using cys - scanning - mutagenesis / disulfide - cross - linking / rosetta - modeling and another iib3 complex nmr structure that has been determined using lipid bicelles as the solvent ( pdb 2k9j ) . on the basis of a comparison with these two structures , ca - cib1 seems to dissociate the iib3 heterodimer , which would potentially activate the iib3 complex because the f992 and f993 side chains become buried in the hydrophobic pocket in the ca - cib1/iib - l complex structure ( figure s5 ) . on the basis of the classic studies indicating that the activation of integrin can be caused by the dissociation of the heterodimer , ca - cib1 is highly likely able to disrupt the association of iib and 3 . taken all data together , a mechanism for activation of iib3 upon interacting with ca - cib1 can be proposed , that is similar to the action of the cytoskeletal protein talin , where ca - cib1 dissociates the iib3 heterodimer and consequently activates integrin iib3 ( figure 8) . ( ( b ) in the presence of ca - cib1 , the iib cytoplasmic subunit interacts with ca - cib1 , with its n - terminal -helix buried in the hydrophobic pocket of the c - lobe of cib1 and its c - terminal negatively charged tail being dynamic but likely being in contact with the n - lobe of cib1 . the integrin iib3 dimer is activated by ca - cib1 , which is similar to the activation by talin , a cytoskeleton protein , which binds to the 3 cytoplasmic domain .. in this work , we have used a reverse cross - saturation nmr approach to detect the interface on ca - cib1 that recognizes the bound iib - l peptide . in these experiments , we selectively irradiated the smaller partner in the protein protein complex , and the results from our work ( figure 4 and figure 5 ) demonstrate that it is possible to identify the hydrophobic residues that are involved in the interactions between cib1 and iib . this represents a powerful way of using the nmr cross - saturation experiment , where normally the larger partner has been irradiated to achieve more effective spin - difussion . provided that the proteins of interest can be bacterially expressed , deuterated , and selectively protonated on methyl groups , the reverse cross - saturation experiment with methyl detection should become a useful nmr approach for better understanding protein protein interactions in general .

acute lymphoblastic leukemia ( all ) arises from immature hematopoietic progenitors that are destined to develop into lymphocytes but acquire somatic gene mutations , which results in altered proliferation and arrest of differentiation . although regulation of growth and differentiation is altered in these cells , they retain many of the features of their normal lymphoid counterparts . this includes rearrangement of their immunoglobulin ( ig ) and t - cell receptor ( tcr ) genes . similarly , cell surface antigens characteristic of normal b and t lymphocytes are expressed on the cell surface of the malignant lymphoid blast , and the pattern of this antigen expression can help delineate where in the maturation sequence the malignant transformation occurred . the clonal nature of the malignant lymphoblasts has been established by the demonstration of identical rearrangements of ig or tcr genes within the all cell population.1 all of b- or t - cell lineage can be further subcategorized immunophenotypically by the point in maturation when their development is interrupted and they become malignant . most cases of b - cell all have an immature immunophenotype and are designated as precursor lymphoid neoplasms or lymphoblastic leukemia / lymphoma . these cases can be identified by the cell surface expression of cluster of differentiation 19 ( cd19 ) and one other b - lineage - associated antigen , such as cd20 , cd21 , cd22 , cd24 , or cd79 . early b - cell blasts lack this expression but are cd10-positive , whereas the most immature subtype , pro - b , are cd10-negative . it is important to note that although leukemic lymphoblasts express antigens related to their stage of development , they may also have an aberrant immunophenotype with asynchronous gene expression related to their malignant transformation.1,2 similarly , an all of t - cell origin can be classified on the basis of the sequence of expression of t - cell - associated cell surface antigens that evolve during normal thymocyte development . the earliest t - cell precursors lack expression of cd4 and cd8 and are referred to as double - negative thymocytes . they progress through a series of stages of differentiation characterized by rearrangement of the tcr genes , lose expression of cd34 , and gain expression of cd1a.1 an early t - cell precursor phenotype has been identified that has a very high clinical risk and makes up 8%15% of t - all in children and a higher percentage in adults . this subtype has been shown to express activating mutations of ras , il-7r , and flt3 , along with pten deletions.3 all can also frequently express antigens associated with cells of myeloid origin ( eg , cd13 , cd14 , or cd33 ) . these patients were previously felt to have a poorer prognosis , but this has not been borne out with the use of chemotherapy regimens in the modern era.4 genetic abnormalities play a key pathogenic role in the origin and development of all . these were first identified by conventional cytogenetics and can be found in up to 75% of patients with all . recurring abnormalities have been identified , and the distribution of these abnormalities varies significantly between patients with pediatric all compared with those with adult all , with adult patients having a higher frequency of adverse cytogenetic abnormalities . the main adverse cytogenetic changes include the presence of t(9;22 ) ( bcr - abl1 or the philadelphia chromosome ) , t(4;11 ) , a complex karyotype ( five or more chromosomal abnormalities ) , or low hypodiploidy / near triploidy . in contrast , patients with a hyperdiploid karyotype or a t(12;21 ) ( tel - aml1 ) have a favorable prognosis and are much more frequently seen in pediatric all , where these latter two abnormalities make up more than 50% of cases.5 the molecular revolution has led to the ability to sequence the genome of patients with all and identified numerous recurring genetic mutations and other alterations in the genome of patients with all . some of the more common genetic alterations have included mutations in the paired box 5 ( pax5 ) gene.6 however , this has not been shown to have any prognostic significance . janus kinase ( jak ) 1 and 2 gene mutations are present in up to 35% of down syndrome - associated all and about 10% of bcr - abl1 all . in adults , jak1 mutations are more prevalent in t - cell all and are associated with a poor prognosis . the ikaros family zinc finger protein 1 ( ikzf1 ) has been associated with high - risk all and poor outcomes . mutations of ikzf1 are common in bcr - abl1-positive all and in the lymphoid blast phase of chronic myeloid leukemia . the cytokine receptor - like factor 2 ( crlf2 ) has alterations in about 5% of adult all.7 abnormal expression of crlf2 can be detected by immunohistochemistry , and crlf2 rearranged all was associated with mutant jak2 in about 50% of cases . in pediatric all , elevated crlf2 expression a new finding of great interest is the identification of a gene expression profile in bcr - abl1-negative all that is similar to that seen in patients with the bcr - abl1 translocation . these cases also commonly harbor mutations of ikzf1 and have a poor prognosis.8,9 this phenotype is seen with increasing frequency in childhood all patients ( 10%14% , and up to 26% in young adults aged 2139 years).10 in vitro studies suggest that these cells may also be sensitive to tyrosine kinase inhibitors similar to their bcr - abl1-positive counterparts . intrachromosomal amplification of chromosome 21 ( iamp-21 ) is defined as a gain of at least five copies of the runx1 region of chromosome 21 . in pediatrics , this abnormality has been associated with significantly inferior survival.11 increasing identification of genetic abnormalities in all brings the hope that these abnormalities can translate into new therapeutic targets.12 all represents a remarkable odyssey of success in the era of cancer treatment . from the first report by sidney farber13 of temporary remissions induced by aminopterin in five children with all this was followed by reports in the 1960s of combination chemotherapy , including mercaptopurine and methotrexate , leading to 2-year survival rates of 20%.14 this led to the concept of using combination chemotherapy to treat malignancy . a recognition that all also could frequently involve the central nervous system ( cns ) led to the recognition of a need for cns - directed therapy , including cranial radiation and intrathecal methotrexate . , which was pioneered by donald pinkel and colleagues at st jude s research hospital in memphis , tennessee . total therapy encompassed different phases of treatment , including remission induction , cns - directed therapy , intensification ( also referred to as consolidation ) therapy , and continuation ( or maintenance ) treatment.15 remarkably , these components of therapy remain the foundation of modern all therapy in both children and adults . as new chemotherapy agents were found to have activity in all , including anthracyclines , cytarabine , and asparaginase , they were incorporated into the treatment regimens . during this same time , it became apparent that radiation - induced complications could be severe and led to the use of triple intrathecal therapy with methotrexate , cytarabine , and hydrocortisone , along with higher doses of intravenous methotrexate to effectively prophylax the cns and replace prophylactic cranial irradiation . many of the advances in the treatment of childhood all during the 1980s and 1990s were related to optimizing the doses and schedules of existing agents , rather than simply the introduction of more new agents.14 these efforts led to remarkable improvement in survival for children with all , such that by the middle of the previous decade , 10-year survival estimates are at 91% ( figure 2).14 paralleling these advances in treatment were advances in the understanding of the biology of all , as outlined in the previous section . these advances led to the ability to risk - stratify patients and alter treatment intensity on the basis of prognosis . the identification of these cytogenetic and genetic markers has also begun to result in the development of new agents effective in the treatment of all , including the development of tyrosine kinase inhibitors such as imatinib , which in combination with chemotherapy , has significantly improved the outcome of children and adults with bcr - abl1 all ( philadelphia chromosome - positive).16,17 unfortunately , results of treatment in adults with all have not paralleled the success seen with children . these poorer results can be attributed to multiple factors including the inability of older adults to tolerate the intensive chemotherapy given to pediatric patients ; the relative rarity of all in adults compared with children , which makes it more challenging for adult oncologists to follow the complex treatment regimens developed for adult patients ; and most important , the different biology of the disease in adults compared with in children.18 in particular , adults with all more frequently harbor adverse cytogenetic abnormalities , including the t(9;22 ) ( philadelphia chromosome ) , t(4;11 ) , a complex karyotype ( five or more chromosomal abnormalities ) , or low hypodiploidy / near triploidy , and less frequently have favorable cytogenetic abnormalities such as the t(12;21 ) ( tel - aml1 ) or hyperdiploidy.5 thus , adults have increased rates of death from complications and more risk of relapse than children . survival rates in adults at 3 years are , therefore , only in the range of 30%40%.18 however , in the last 10 years , it has been increasingly recognized that adolescents and young adults ( aya ) fare differently if they are treated with a pediatric as opposed to an adult all chemotherapy regimen . this was first reported by stock et al19 in a retrospective comparison of outcome in 321 aya ( age , 1620 years ) who were treated on consecutive trials on either the children s cancer group or the cancer and leukemia group b from 19882001 . although complete remission rates were identical in the two cohorts of patients , the children s cancer group aya had a 67% overall survival at 7 years in contrast to the cancer and leukemia group b aya , for whom overall survival was 46% ( p0.001).19 multiple subsequent similar comparisons from other countries comparing aya treated on adult versus pediatric regimens showed similar results.20 the reasons for these differences in outcome are likely multiple , but an important factor is that pediatric regimens include much higher doses of nonmyelosuppressive chemotherapy drugs including corticosteroids , vincristine , and asparaginase . several studies have now prospectively assessed the use of pediatric - intensive regimens in adults . this was first reported by huguet et al,21 for the french group for research on adult acute lymphoblastic leukemia , who gave a pediatric intensive regimen to 225 adults with a median age of 31 years but a range of 1560 years . it was 95% in patients aged 1545 years and 87% in patients aged 4660 years . the overall complete response rate of 93.5% was superior to a previous trial ( leucmies aigus lymphoblastiques de ladulte-94 [ lala-94 ] ) , which used a traditional adult regimen and had a complete response rate of 88% ( p=0.02 ) . the overall survival rate was 66% in patients younger than 45 years , which compared favorably with the lala-94 trial , in which the overall survival rate was 44% at 42 months of follow - up . patients older than 45 years did not tolerate this pediatric - intensive regimen as well as patients younger than 45 years , as there was a higher cumulative incidence of chemotherapy - related deaths ( 23% versus 5% , respectively ; p0.001 ) and deaths in first complete remission ( 22% versus 5% , respectively ; p0.01).21 a recently published meta - analysis of trials with adult - intensive regimens has suggested that incorporation of allogeneic stem cell transplantation ( sct ) into the treatment of adults with all in first remission results in superior outcomes compared with chemotherapy or autologous sct.22 thus , the question has arisen as to whether a younger adult with all should be treated with a pediatric intensive regimen or directed to allogeneic transplant once they achieve first remission . many investigators feel that young adults without other high - risk features can be managed with a pediatric intensive chemotherapy regimen alone and only considered for transplant if they relapse.23 however , the therapy of older adults with all remains particularly challenging . they do not tolerate pediatric intensive regimens as well as younger adults but also do not tolerate intensive therapies such as allogeneic sct either . another option that is gaining increasing interest is the use of reduced - intensity conditioning allogenic sct . recent studies have suggested that the outcomes with reduced - intensity conditioning allogeneic sct may be comparable to myeloablative conditioning allogeneic sct , even though the patients who received reduced - intensity conditioning were older and likely had more comorbidities.24,25 the management of older adults with all has recently been summarized.26 these were first identified by conventional cytogenetics and can be found in up to 75% of patients with all . recurring abnormalities have been identified , and the distribution of these abnormalities varies significantly between patients with pediatric all compared with those with adult all , with adult patients having a higher frequency of adverse cytogenetic abnormalities . the main adverse cytogenetic changes include the presence of t(9;22 ) ( bcr - abl1 or the philadelphia chromosome ) , t(4;11 ) , a complex karyotype ( five or more chromosomal abnormalities ) , or low hypodiploidy / near triploidy . in contrast , patients with a hyperdiploid karyotype or a t(12;21 ) ( tel - aml1 ) have a favorable prognosis and are much more frequently seen in pediatric all , where these latter two abnormalities make up more than 50% of cases.5 the molecular revolution has led to the ability to sequence the genome of patients with all and identified numerous recurring genetic mutations and other alterations in the genome of patients with all . some of the more common genetic alterations have included mutations in the paired box 5 ( pax5 ) gene.6 however , this has not been shown to have any prognostic significance . janus kinase ( jak ) 1 and 2 gene mutations are present in up to 35% of down syndrome - associated all and about 10% of bcr - abl1 all . in adults , jak1 mutations are more prevalent in t - cell all and are associated with a poor prognosis . the ikaros family zinc finger protein 1 ( ikzf1 ) has been associated with high - risk all and poor outcomes . mutations of ikzf1 are common in bcr - abl1-positive all and in the lymphoid blast phase of chronic myeloid leukemia . the cytokine receptor - like factor 2 ( crlf2 ) has alterations in about 5% of adult all.7 abnormal expression of crlf2 can be detected by immunohistochemistry , and crlf2 rearranged all was associated with mutant jak2 in about 50% of cases . in pediatric all , elevated crlf2 expression a new finding of great interest is the identification of a gene expression profile in bcr - abl1-negative all that is similar to that seen in patients with the bcr - abl1 translocation . these cases also commonly harbor mutations of ikzf1 and have a poor prognosis.8,9 this phenotype is seen with increasing frequency in childhood all patients ( 10%14% , and up to 26% in young adults aged 2139 years).10 in vitro studies suggest that these cells may also be sensitive to tyrosine kinase inhibitors similar to their bcr - abl1-positive counterparts . intrachromosomal amplification of chromosome 21 ( iamp-21 ) is defined as a gain of at least five copies of the runx1 region of chromosome 21 . in pediatrics , this abnormality has been associated with significantly inferior survival.11 increasing identification of genetic abnormalities in all brings the hope that these abnormalities can translate into new therapeutic targets.12 all represents a remarkable odyssey of success in the era of cancer treatment . from the first report by sidney farber13 of temporary remissions induced by aminopterin in five children with all this was followed by reports in the 1960s of combination chemotherapy , including mercaptopurine and methotrexate , leading to 2-year survival rates of 20%.14 this led to the concept of using combination chemotherapy to treat malignancy . a recognition that all also could frequently involve the central nervous system ( cns ) led to the recognition of a need for cns - directed therapy , including cranial radiation and intrathecal methotrexate . , which was pioneered by donald pinkel and colleagues at st jude s research hospital in memphis , tennessee . their total therapy encompassed different phases of treatment , including remission induction , cns - directed therapy , intensification ( also referred to as consolidation ) therapy , and continuation ( or maintenance ) treatment.15 remarkably , these components of therapy remain the foundation of modern all therapy in both children and adults . as new chemotherapy agents were found to have activity in all , including anthracyclines , cytarabine , and asparaginase , they were incorporated into the treatment regimens . during this same time , it became apparent that radiation - induced complications could be severe and led to the use of triple intrathecal therapy with methotrexate , cytarabine , and hydrocortisone , along with higher doses of intravenous methotrexate to effectively prophylax the cns and replace prophylactic cranial irradiation . many of the advances in the treatment of childhood all during the 1980s and 1990s were related to optimizing the doses and schedules of existing agents , rather than simply the introduction of more new agents.14 these efforts led to remarkable improvement in survival for children with all , such that by the middle of the previous decade , 10-year survival estimates are at 91% ( figure 2).14 paralleling these advances in treatment were advances in the understanding of the biology of all , as outlined in the previous section . these advances led to the ability to risk - stratify patients and alter treatment intensity on the basis of prognosis . the identification of these cytogenetic and genetic markers has also begun to result in the development of new agents effective in the treatment of all , including the development of tyrosine kinase inhibitors such as imatinib , which in combination with chemotherapy , has significantly improved the outcome of children and adults with bcr - abl1 all ( philadelphia chromosome - positive).16,17 unfortunately , results of treatment in adults with all have not paralleled the success seen with children . these poorer results can be attributed to multiple factors including the inability of older adults to tolerate the intensive chemotherapy given to pediatric patients ; the relative rarity of all in adults compared with children , which makes it more challenging for adult oncologists to follow the complex treatment regimens developed for adult patients ; and most important , the different biology of the disease in adults compared with in children.18 in particular , adults with all more frequently harbor adverse cytogenetic abnormalities , including the t(9;22 ) ( philadelphia chromosome ) , t(4;11 ) , a complex karyotype ( five or more chromosomal abnormalities ) , or low hypodiploidy / near triploidy , and less frequently have favorable cytogenetic abnormalities such as the t(12;21 ) ( tel - aml1 ) or hyperdiploidy.5 thus , adults have increased rates of death from complications and more risk of relapse than children . survival rates in adults at 3 years are , therefore , only in the range of 30%40%.18 however , in the last 10 years , it has been increasingly recognized that adolescents and young adults ( aya ) fare differently if they are treated with a pediatric as opposed to an adult all chemotherapy regimen . this was first reported by stock et al19 in a retrospective comparison of outcome in 321 aya ( age , 1620 years ) who were treated on consecutive trials on either the children s cancer group or the cancer and leukemia group b from 19882001 . although complete remission rates were identical in the two cohorts of patients , the children s cancer group aya had a 67% overall survival at 7 years in contrast to the cancer and leukemia group b aya , for whom overall survival was 46% ( p0.001).19 multiple subsequent similar comparisons from other countries comparing aya treated on adult versus pediatric regimens showed similar results.20 the reasons for these differences in outcome are likely multiple , but an important factor is that pediatric regimens include much higher doses of nonmyelosuppressive chemotherapy drugs including corticosteroids , vincristine , and asparaginase . several studies have now prospectively assessed the use of pediatric - intensive regimens in adults . this was first reported by huguet et al,21 for the french group for research on adult acute lymphoblastic leukemia , who gave a pediatric intensive regimen to 225 adults with a median age of 31 years but a range of 1560 years . it was 95% in patients aged 1545 years and 87% in patients aged 4660 years . the overall complete response rate of 93.5% was superior to a previous trial ( leucmies aigus lymphoblastiques de ladulte-94 [ lala-94 ] ) , which used a traditional adult regimen and had a complete response rate of 88% ( p=0.02 ) . the overall survival rate was 66% in patients younger than 45 years , which compared favorably with the lala-94 trial , in which the overall survival rate was 44% at 42 months of follow - up . patients older than 45 years did not tolerate this pediatric - intensive regimen as well as patients younger than 45 years , as there was a higher cumulative incidence of chemotherapy - related deaths ( 23% versus 5% , respectively ; p0.001 ) and deaths in first complete remission ( 22% versus 5% , respectively ; p0.01).21 a recently published meta - analysis of trials with adult - intensive regimens has suggested that incorporation of allogeneic stem cell transplantation ( sct ) into the treatment of adults with all in first remission results in superior outcomes compared with chemotherapy or autologous sct.22 thus , the question has arisen as to whether a younger adult with all should be treated with a pediatric intensive regimen or directed to allogeneic transplant once they achieve first remission . many investigators feel that young adults without other high - risk features can be managed with a pediatric intensive chemotherapy regimen alone and only considered for transplant if they relapse.23 however , the therapy of older adults with all remains particularly challenging . they do not tolerate pediatric intensive regimens as well as younger adults but also do not tolerate intensive therapies such as allogeneic sct either . another option that is gaining increasing interest is the use of reduced - intensity conditioning allogenic sct . recent studies have suggested that the outcomes with reduced - intensity conditioning allogeneic sct may be comparable to myeloablative conditioning allogeneic sct , even though the patients who received reduced - intensity conditioning were older and likely had more comorbidities.24,25 the management of older adults with all has recently been summarized.26 the identification of the graft versus leukemia reaction after sct and the acknowledgment that it plays a key role in the cure of hematologic malignancies led to the recognition that the immune system could play a key role in the treatment of malignancy.27 this led to the increasing study of appropriate targets for immunotherapy , including tumor - specific and/or tumor - associated antigens . these can be attacked by different cellular components of the immune system , including t - cells , natural killer cells , and dendritic cells . the advent of the development of increasing sophisticated monoclonal antibodies has led to the approval of an ever - increasing number of such antibodies for the treatment of hematologic malignancies and solid tumors.28 conjugation of monoclonal antibodies with immunotoxins and chemotherapy drugs has also shown promise , as has the production of bispecific t - cell engaging antibodies . there is increasing interest in engineering cells that combine components of monoclonal antibodies with the t - cell to promote target - killing . examples include chimeric antigen receptors , modified t - cells that have shown recent promise in the treatment of all.29,30 during the course of b - cell ontogeny , multiple cell surface antigens are expressed , and several of them have become attractive targets for monoclonal antibody - directed therapy . these include cd20 and cd22 , although these two antigens are expressed somewhat later in b - cell development . cd19 has become one of the most attractive targets in the treatment of b - lineage all , as its surface expression on the cell begins around the time of ig gene rearrangement . the human cd19 antigen is a member of the ig superfamily and is a 95 kda transmembrane glycoprotein . the gene for cd19 is located on the short arm of chromosome 16 , contains 15 exons , and produces a protein composed of 556 amino acids . the cd19 protein contains an extracellular n - terminus , a single transmembrane domain , and a cytoplasmic c - terminus . it is categorized as a type 1 transmembrane protein without significant homology with any other known proteins ( figure 3).31 cd19 serves a vital function in establishing an effective immune response , as it takes part in antigen - independent development as well as in immunoglobulin - induced activation of b - cells . intracellular downstream targets of cd19 activation include protein kinase members of the src family , the ras family , abl , btk , and pi3k , among others . importantly , it functions as an adaptor protein by recruiting cytoplasmic signaling proteins to the cell membrane.31 mice that are deficient in cd19 have defects in the later stages of b - cell growth and maturation . this deficiency does not affect the number of b - cell precursors in the marrow , but cd19 mice have marked reductions in the frequency and number of splenic and peripheral blood b cells . humans have been described who have homozygous frame shift mutations of the cd19 gene , which produces truncation of three key cytoplasmic tyrosine residues . these individuals have normal numbers of precursor and total b cells but decreased numbers of cd5 b cells and cd27 memory b cells . the patients develop hypogammaglobulinemia and have poor antibody responses to rabies vaccination , and they are more prone to infection.32 as noted , cd19 is an attractive target for immunotherapy because of its restricted expression on b - lineage lymphomas and leukemias , as well as normal b cells , but not on other hematopoietic cells or normal tissues . it has a broader expression profile through b - cell development compared with other antigens , such as cd20 , and is more efficiently internalized.33 despite these attractive features , conventional antibodies alone targeted against cd19 had limited activity in preclinical models , although these models had high cd19 expression and were able to internalize antibodies.34 thus , multiple different antibody constructs and conjugations have been developed with cd19 as the target . these are summarized in table 1 and include cd19 antibodies conjugated to a maytansine derivative ; engineered anti - cd19 antibodies that enhance antibody - dependent cell - mediated cytotoxicity ; antibody conjugation to diphtheria toxin ; antibody conjugation to auristatin anti - cd19 ; the chimeric antigen receptor t - cells described earlier ; dual - affinity retargeting antibodies , which are encoded by two different polypeptide chains that contain a variable heavy chain domain fused to a variable light chain domain ; and a bispecific t - cell engager that comprises two single - chain antibodies ( scfvs ) that bind cd3 and cd19 , respectively . these various cd19 immunotherapy approaches were recently summarized in a review.33 blinatumomab s name is derived from the fact that it is a b - lineage - specific antitumor mouse monoclonal antibody . the basic characteristics of blinatumomab , including its structure , specificity , purification , and cytotoxicity , were first reported in 2000.35 blinatumomab is a construct of scfvs that forms a 55 kda fusion protein ( figure 4 ) . recombinant dna technology is used and takes the respective cdnas , which encode the four variable domains , and three linker sequences . a 5 amino acid linker sequence that is nonimmunogenic is used to recombinantly link the two scfvs in tandem . this is thought to give the two scfvs a significant degree of rotational ability to enhance binding of epitopes on separate cells.36 blinatumomab is produced in chinese hamster ovary cells in a both a monomeric and dimeric form . although both these forms are biologically active , only the monomeric form is purified for clinical use.36 in vitro , blinatumomab was found to be extremely potent , with a half - maximal concentration for redirected lysis of cd19-positive target cells by b cells in the range of 10100 pg / ml with t cells from healthy donors.37 all t - cell populations except naive t cells showed high - level redirected lysis . this effect was seen with resting t cells that were not previously activated and was associated with upregulation of t - cell antigen expression , including cd69 , cd25 , and cd2 , along with transient release of multiple inflammatory cytokines including interleukins 2 , 6 , and 10 ; tumor necrosis factor , and interferon .38 the first clinical trials in humans began in 2001 ( table 2 ) . phase i studies in patients with relapsed or refractory non - hodgkin lymphoma were carried out using doses ranging from 0.7513 g / m administered once , twice , or three times weekly as 2- or 4-hour intravenous infusions . significant neurologic events included aphasia , ataxia , disorientation , and seizures and led to discontinuation of therapy in 6 of 22 patients . cytokine release syndrome and infections were also observed.36 given the toxicity profile with shorter infusions , a continuous intravenous infusion over a period of 48 weeks was carried out in non - hodgkin lymphoma patients . after an initial observation period from 37 days , the blinatumomab infusion could be continued as an outpatient . all trials since this initial phase i study have administered blinatumomab as a continuous infusion for 4 weeks.36 the initial clinical experience with this infusion schedule was in patients with low - grade lymphoma and mantle cell lymphoma . dose escalation started as low as 0.5 g / m per day but was escalated up to 90 g / m per day . the trial was later amended to include patients with aggressive diffuse large b - cell lymphoma . the most recent update on 70 patients , of whom 93% had had prior rituximab - based regimens , showed that most patients had mild adverse events of a constitutional nature , generally of grade 1 or 2 . a transient release of inflammatory cytokines was noted in the first few days , along with a rapid decline in peripheral blood b cells . even with the continuous infusion , cns events were noted , again primarily during the first 3 days of treatment , and included encephalopathy , aphasia , tremor , disorientation , and convulsions . the etiology of the cns events is not entirely clear but may be related to activated t - cells that release neurotoxic cytokines into the cns . fortunately , these cns events have been reversible with discontinuation of treatment.39 the maximum tolerated dose in this study was 60 g / m per day . a step - wise approach to blinatumomab administration with lower doses given in the first week or 2 , followed by escalation to the full dose , has lessened the incidence of neurologic toxicity.39 complete remissions were noted with dose levels as low as 15 g / m per day , and it was noted that five of six patients at this dose had clearance of lymphomatous involvement of the marrow . the overall response rate at the 60 g / m per day dose was 76% ( 16/21 patients ) , with seven patients having a complete remission or unconfirmed complete remission.36,39 there is still some uncertainty related to the long - term effects of depletion of normal cd19-positive b - cells . patients do develop hypogammaglobulinemia as expected , but long - term infectious complications have not been prominent . in diffuse large b - cell non - hodgkin lymphoma , experience is more limited , but of a group of 13 patients treated , eleven were evaluable , with a total of six responses and two patients with stable disease . of the six responders , patients with extranodal disease and those with prior autologous transplant also responded ; these responses overall appeared to be durable . in three of six responders , allogeneic transplant was able to be performed.40 given the activity seen in non - hodgkin lymphoma in clearing lymphomatous cells from the marrow , it was felt that blinatumomab might be efficacious in b - lineage all . because patients who are in hematologic remission , but have persistent minimal residual disease ( mrd ) by molecular or immunophenotypic evidence , have a poor prognosis , it was decided that a pilot study in patients in hematologic remission who were mrd - positive or who had experienced an mrd relapse would be initially carried out . the dose chosen was 15 g / m per day by 4-week continuous intravenous infusion , as marrow clearing had been seen in lymphoma patients at this dose . of 21 patients treated , 20 were evaluable and 16 ( 80% ) became mrd - negative during the first 4-week cycle of therapy . no deaths were noted.41 longer - term follow - up from this study has been recently reported . after a median follow - up of 33 months , the relapse - free survival was 61% . nine patients were able to proceed to allogeneic sct and had a relapse - free survival of 65% . of six patients with bcr - abl1 all who responded and had no further therapy after blinatumomab , four remain in ongoing hematologic and molecular remission.42 these encouraging results have led to a larger trial in patients who are mrd - positive across several countries in europe , which was initiated in the fall of 2010 . these results have also led to a trial in patients with hematologic relapse or refractory b - lineage all . three dosing regimens were explored , including initiation of therapy at 15 g / m per day , and continued at that dose for the entire 4-week cycle versus an initial dose of 5 g / m per day for the first week and then escalation to 15 g / m per day for the subsequent 3 weeks . a third group initiated therapy at 5 g / m per day for the first week and then went to 15 g / m per day for the second week and to 30 g / m per day for the third and fourth weeks . the most recent update included 36 patients who had been treated.43 on the basis of the lowest incidence of adverse effects , the schedule selected for treatment of 18 of the 36 patients in a final cohort was the dose of 5 g / m per day for the first week , followed by 15 g / m per day for the subsequent 3 weeks . of the entire cohort of patients , 26 of 36 achieved a complete remission or complete remission without hematologic recovery ( 72% ) . of these 26 patients , 24 ( 92% ) achieved mrd negativity within the first two cycles . twenty of 21 ( 95% ) patients in first relapse responded , whereas only 6 ( 40% ) of 15 of the remaining patients achieved a hematologic complete remission or complete remission without hematologic recovery . two of these relapses were cd19-negative , three were cd19-positive , and three were unknown . the median overall survival for all 36 treated patients is 9 months , with a median follow - up time for overall survival of 10.7 months . for patients achieving a complete remission or complete remission without hematologic recovery , the median survival is 14.1 months , whereas for patients who failed blinatumomab , the median survival is 6.6 months . as noted previously , cytokine release syndrome and cns events were the most significant toxicities . three patients had seizures , and three patients had encephalopathy.43 an international phase ii trial of this regimen is currently ongoing . further studies have also confirmed that body surface area - based dosing is not essential , as pharmacokinetic studies have not shown a significant difference in levels with weight - based versus flat dosing . therefore , flat dosing with an initial dose of 9 g / day for the first week followed by 28 g / day is currently being used . the exciting results achieved with blinatumomab in both the hematologic relapsed / refractory setting and in the mrd setting have led to the development of a us national intergroup trial , which will randomize patients with b - lineage all between the ages of 35 and 70 years to induction chemotherapy to achieve remission followed by four cycles of blinatumomab versus chemotherapy alone . although more than 90% of children can now be cured of their all , these results have not been duplicated in adults . the use of pediatric intensive regimens has improved the outcome of young adults with all , but further progress is needed . in older adults , the limits of conventional chemotherapy have been reached , and new approaches are needed . the cd19 antigen is nearly universally expressed on b - lineage all and is an attractive target for therapy . multiple immunotherapeutic approaches using modified monoclonal antibodies have been developed as outlined in this review . one antibody construct , blinatumomab , a bispecific t - cell engager antibody , is in advanced stages of development and shows significant promise in improving outcomes of patients with b - lineage all . a large intergroup trial in the united states will test the efficacy of blinatumomab in newly diagnosed middle - aged and older adults with b - lineage all .

acute lymphoblastic leukemia ( all ) arises from immature b and t lymphoblasts . an increasing array of cytogenetic and molecular markers have been identified in all , which allows for increasingly sophisticated prognostication , as well as identification of potential new targets for therapy . the treatment of all in children has shown astounding success in the last 50 years , with more than 90% of children now able to be cured of their all . in adults , these success rates have not been duplicated . however , the use of pediatric - intensive regimens in young adults has shown increasing success . the use of monoclonal antibodies conjugated to drugs , immunotoxins , and cells also has shown early success and promises to enhance the outcome of newly diagnosed patients . blinatumomab , a bispecific t - cell engager antibody , brings a malignant b cell in proximity to a t cell with redirected lysis . this antibody construct has shown promising results in patients with relapsed and refractory disease and is entering randomized clinical trials in newly diagnosed patients . the addition of monoclonal antibody therapy to chemotherapy in adults promises to enhance outcomes while hopefully not increasing toxicity . after many years of stagnation , it appears that the therapy of adults with all is showing significant improvement .

Introduction Genetic abnormalities Current treatment options and patient outcomes Immunotherapy of cancer CD19 in immunology CD19 as an immunotherapeutic target Conclusion

acute lymphoblastic leukemia ( all ) arises from immature hematopoietic progenitors that are destined to develop into lymphocytes but acquire somatic gene mutations , which results in altered proliferation and arrest of differentiation . this includes rearrangement of their immunoglobulin ( ig ) and t - cell receptor ( tcr ) genes . similarly , cell surface antigens characteristic of normal b and t lymphocytes are expressed on the cell surface of the malignant lymphoid blast , and the pattern of this antigen expression can help delineate where in the maturation sequence the malignant transformation occurred . they progress through a series of stages of differentiation characterized by rearrangement of the tcr genes , lose expression of cd34 , and gain expression of cd1a.1 an early t - cell precursor phenotype has been identified that has a very high clinical risk and makes up 8%15% of t - all in children and a higher percentage in adults . these patients were previously felt to have a poorer prognosis , but this has not been borne out with the use of chemotherapy regimens in the modern era.4 genetic abnormalities play a key pathogenic role in the origin and development of all . recurring abnormalities have been identified , and the distribution of these abnormalities varies significantly between patients with pediatric all compared with those with adult all , with adult patients having a higher frequency of adverse cytogenetic abnormalities . in contrast , patients with a hyperdiploid karyotype or a t(12;21 ) ( tel - aml1 ) have a favorable prognosis and are much more frequently seen in pediatric all , where these latter two abnormalities make up more than 50% of cases.5 the molecular revolution has led to the ability to sequence the genome of patients with all and identified numerous recurring genetic mutations and other alterations in the genome of patients with all . some of the more common genetic alterations have included mutations in the paired box 5 ( pax5 ) gene.6 however , this has not been shown to have any prognostic significance . in adults , jak1 mutations are more prevalent in t - cell all and are associated with a poor prognosis . in pediatric all , elevated crlf2 expression a new finding of great interest is the identification of a gene expression profile in bcr - abl1-negative all that is similar to that seen in patients with the bcr - abl1 translocation . in pediatrics , this abnormality has been associated with significantly inferior survival.11 increasing identification of genetic abnormalities in all brings the hope that these abnormalities can translate into new therapeutic targets.12 all represents a remarkable odyssey of success in the era of cancer treatment . as new chemotherapy agents were found to have activity in all , including anthracyclines , cytarabine , and asparaginase , they were incorporated into the treatment regimens . during this same time , it became apparent that radiation - induced complications could be severe and led to the use of triple intrathecal therapy with methotrexate , cytarabine , and hydrocortisone , along with higher doses of intravenous methotrexate to effectively prophylax the cns and replace prophylactic cranial irradiation . many of the advances in the treatment of childhood all during the 1980s and 1990s were related to optimizing the doses and schedules of existing agents , rather than simply the introduction of more new agents.14 these efforts led to remarkable improvement in survival for children with all , such that by the middle of the previous decade , 10-year survival estimates are at 91% ( figure 2).14 paralleling these advances in treatment were advances in the understanding of the biology of all , as outlined in the previous section . the identification of these cytogenetic and genetic markers has also begun to result in the development of new agents effective in the treatment of all , including the development of tyrosine kinase inhibitors such as imatinib , which in combination with chemotherapy , has significantly improved the outcome of children and adults with bcr - abl1 all ( philadelphia chromosome - positive).16,17 unfortunately , results of treatment in adults with all have not paralleled the success seen with children . these poorer results can be attributed to multiple factors including the inability of older adults to tolerate the intensive chemotherapy given to pediatric patients ; the relative rarity of all in adults compared with children , which makes it more challenging for adult oncologists to follow the complex treatment regimens developed for adult patients ; and most important , the different biology of the disease in adults compared with in children.18 in particular , adults with all more frequently harbor adverse cytogenetic abnormalities , including the t(9;22 ) ( philadelphia chromosome ) , t(4;11 ) , a complex karyotype ( five or more chromosomal abnormalities ) , or low hypodiploidy / near triploidy , and less frequently have favorable cytogenetic abnormalities such as the t(12;21 ) ( tel - aml1 ) or hyperdiploidy.5 thus , adults have increased rates of death from complications and more risk of relapse than children . survival rates in adults at 3 years are , therefore , only in the range of 30%40%.18 however , in the last 10 years , it has been increasingly recognized that adolescents and young adults ( aya ) fare differently if they are treated with a pediatric as opposed to an adult all chemotherapy regimen . several studies have now prospectively assessed the use of pediatric - intensive regimens in adults . the overall survival rate was 66% in patients younger than 45 years , which compared favorably with the lala-94 trial , in which the overall survival rate was 44% at 42 months of follow - up . patients older than 45 years did not tolerate this pediatric - intensive regimen as well as patients younger than 45 years , as there was a higher cumulative incidence of chemotherapy - related deaths ( 23% versus 5% , respectively ; p0.001 ) and deaths in first complete remission ( 22% versus 5% , respectively ; p0.01).21 a recently published meta - analysis of trials with adult - intensive regimens has suggested that incorporation of allogeneic stem cell transplantation ( sct ) into the treatment of adults with all in first remission results in superior outcomes compared with chemotherapy or autologous sct.22 thus , the question has arisen as to whether a younger adult with all should be treated with a pediatric intensive regimen or directed to allogeneic transplant once they achieve first remission . many investigators feel that young adults without other high - risk features can be managed with a pediatric intensive chemotherapy regimen alone and only considered for transplant if they relapse.23 however , the therapy of older adults with all remains particularly challenging . recent studies have suggested that the outcomes with reduced - intensity conditioning allogeneic sct may be comparable to myeloablative conditioning allogeneic sct , even though the patients who received reduced - intensity conditioning were older and likely had more comorbidities.24,25 the management of older adults with all has recently been summarized.26 these were first identified by conventional cytogenetics and can be found in up to 75% of patients with all . recurring abnormalities have been identified , and the distribution of these abnormalities varies significantly between patients with pediatric all compared with those with adult all , with adult patients having a higher frequency of adverse cytogenetic abnormalities . in contrast , patients with a hyperdiploid karyotype or a t(12;21 ) ( tel - aml1 ) have a favorable prognosis and are much more frequently seen in pediatric all , where these latter two abnormalities make up more than 50% of cases.5 the molecular revolution has led to the ability to sequence the genome of patients with all and identified numerous recurring genetic mutations and other alterations in the genome of patients with all . in adults , jak1 mutations are more prevalent in t - cell all and are associated with a poor prognosis . in pediatric all , elevated crlf2 expression a new finding of great interest is the identification of a gene expression profile in bcr - abl1-negative all that is similar to that seen in patients with the bcr - abl1 translocation . these cases also commonly harbor mutations of ikzf1 and have a poor prognosis.8,9 this phenotype is seen with increasing frequency in childhood all patients ( 10%14% , and up to 26% in young adults aged 2139 years).10 in vitro studies suggest that these cells may also be sensitive to tyrosine kinase inhibitors similar to their bcr - abl1-positive counterparts . in pediatrics , this abnormality has been associated with significantly inferior survival.11 increasing identification of genetic abnormalities in all brings the hope that these abnormalities can translate into new therapeutic targets.12 all represents a remarkable odyssey of success in the era of cancer treatment . as new chemotherapy agents were found to have activity in all , including anthracyclines , cytarabine , and asparaginase , they were incorporated into the treatment regimens . during this same time , it became apparent that radiation - induced complications could be severe and led to the use of triple intrathecal therapy with methotrexate , cytarabine , and hydrocortisone , along with higher doses of intravenous methotrexate to effectively prophylax the cns and replace prophylactic cranial irradiation . many of the advances in the treatment of childhood all during the 1980s and 1990s were related to optimizing the doses and schedules of existing agents , rather than simply the introduction of more new agents.14 these efforts led to remarkable improvement in survival for children with all , such that by the middle of the previous decade , 10-year survival estimates are at 91% ( figure 2).14 paralleling these advances in treatment were advances in the understanding of the biology of all , as outlined in the previous section . the identification of these cytogenetic and genetic markers has also begun to result in the development of new agents effective in the treatment of all , including the development of tyrosine kinase inhibitors such as imatinib , which in combination with chemotherapy , has significantly improved the outcome of children and adults with bcr - abl1 all ( philadelphia chromosome - positive).16,17 unfortunately , results of treatment in adults with all have not paralleled the success seen with children . these poorer results can be attributed to multiple factors including the inability of older adults to tolerate the intensive chemotherapy given to pediatric patients ; the relative rarity of all in adults compared with children , which makes it more challenging for adult oncologists to follow the complex treatment regimens developed for adult patients ; and most important , the different biology of the disease in adults compared with in children.18 in particular , adults with all more frequently harbor adverse cytogenetic abnormalities , including the t(9;22 ) ( philadelphia chromosome ) , t(4;11 ) , a complex karyotype ( five or more chromosomal abnormalities ) , or low hypodiploidy / near triploidy , and less frequently have favorable cytogenetic abnormalities such as the t(12;21 ) ( tel - aml1 ) or hyperdiploidy.5 thus , adults have increased rates of death from complications and more risk of relapse than children . survival rates in adults at 3 years are , therefore , only in the range of 30%40%.18 however , in the last 10 years , it has been increasingly recognized that adolescents and young adults ( aya ) fare differently if they are treated with a pediatric as opposed to an adult all chemotherapy regimen . although complete remission rates were identical in the two cohorts of patients , the children s cancer group aya had a 67% overall survival at 7 years in contrast to the cancer and leukemia group b aya , for whom overall survival was 46% ( p0.001).19 multiple subsequent similar comparisons from other countries comparing aya treated on adult versus pediatric regimens showed similar results.20 the reasons for these differences in outcome are likely multiple , but an important factor is that pediatric regimens include much higher doses of nonmyelosuppressive chemotherapy drugs including corticosteroids , vincristine , and asparaginase . several studies have now prospectively assessed the use of pediatric - intensive regimens in adults . this was first reported by huguet et al,21 for the french group for research on adult acute lymphoblastic leukemia , who gave a pediatric intensive regimen to 225 adults with a median age of 31 years but a range of 1560 years . patients older than 45 years did not tolerate this pediatric - intensive regimen as well as patients younger than 45 years , as there was a higher cumulative incidence of chemotherapy - related deaths ( 23% versus 5% , respectively ; p0.001 ) and deaths in first complete remission ( 22% versus 5% , respectively ; p0.01).21 a recently published meta - analysis of trials with adult - intensive regimens has suggested that incorporation of allogeneic stem cell transplantation ( sct ) into the treatment of adults with all in first remission results in superior outcomes compared with chemotherapy or autologous sct.22 thus , the question has arisen as to whether a younger adult with all should be treated with a pediatric intensive regimen or directed to allogeneic transplant once they achieve first remission . many investigators feel that young adults without other high - risk features can be managed with a pediatric intensive chemotherapy regimen alone and only considered for transplant if they relapse.23 however , the therapy of older adults with all remains particularly challenging . they do not tolerate pediatric intensive regimens as well as younger adults but also do not tolerate intensive therapies such as allogeneic sct either . recent studies have suggested that the outcomes with reduced - intensity conditioning allogeneic sct may be comparable to myeloablative conditioning allogeneic sct , even though the patients who received reduced - intensity conditioning were older and likely had more comorbidities.24,25 the management of older adults with all has recently been summarized.26 the identification of the graft versus leukemia reaction after sct and the acknowledgment that it plays a key role in the cure of hematologic malignancies led to the recognition that the immune system could play a key role in the treatment of malignancy.27 this led to the increasing study of appropriate targets for immunotherapy , including tumor - specific and/or tumor - associated antigens . the advent of the development of increasing sophisticated monoclonal antibodies has led to the approval of an ever - increasing number of such antibodies for the treatment of hematologic malignancies and solid tumors.28 conjugation of monoclonal antibodies with immunotoxins and chemotherapy drugs has also shown promise , as has the production of bispecific t - cell engaging antibodies . there is increasing interest in engineering cells that combine components of monoclonal antibodies with the t - cell to promote target - killing . examples include chimeric antigen receptors , modified t - cells that have shown recent promise in the treatment of all.29,30 during the course of b - cell ontogeny , multiple cell surface antigens are expressed , and several of them have become attractive targets for monoclonal antibody - directed therapy . cd19 has become one of the most attractive targets in the treatment of b - lineage all , as its surface expression on the cell begins around the time of ig gene rearrangement . it is categorized as a type 1 transmembrane protein without significant homology with any other known proteins ( figure 3).31 cd19 serves a vital function in establishing an effective immune response , as it takes part in antigen - independent development as well as in immunoglobulin - induced activation of b - cells . the patients develop hypogammaglobulinemia and have poor antibody responses to rabies vaccination , and they are more prone to infection.32 as noted , cd19 is an attractive target for immunotherapy because of its restricted expression on b - lineage lymphomas and leukemias , as well as normal b cells , but not on other hematopoietic cells or normal tissues . it has a broader expression profile through b - cell development compared with other antigens , such as cd20 , and is more efficiently internalized.33 despite these attractive features , conventional antibodies alone targeted against cd19 had limited activity in preclinical models , although these models had high cd19 expression and were able to internalize antibodies.34 thus , multiple different antibody constructs and conjugations have been developed with cd19 as the target . these are summarized in table 1 and include cd19 antibodies conjugated to a maytansine derivative ; engineered anti - cd19 antibodies that enhance antibody - dependent cell - mediated cytotoxicity ; antibody conjugation to diphtheria toxin ; antibody conjugation to auristatin anti - cd19 ; the chimeric antigen receptor t - cells described earlier ; dual - affinity retargeting antibodies , which are encoded by two different polypeptide chains that contain a variable heavy chain domain fused to a variable light chain domain ; and a bispecific t - cell engager that comprises two single - chain antibodies ( scfvs ) that bind cd3 and cd19 , respectively . although both these forms are biologically active , only the monomeric form is purified for clinical use.36 in vitro , blinatumomab was found to be extremely potent , with a half - maximal concentration for redirected lysis of cd19-positive target cells by b cells in the range of 10100 pg / ml with t cells from healthy donors.37 all t - cell populations except naive t cells showed high - level redirected lysis . this effect was seen with resting t cells that were not previously activated and was associated with upregulation of t - cell antigen expression , including cd69 , cd25 , and cd2 , along with transient release of multiple inflammatory cytokines including interleukins 2 , 6 , and 10 ; tumor necrosis factor , and interferon .38 the first clinical trials in humans began in 2001 ( table 2 ) . phase i studies in patients with relapsed or refractory non - hodgkin lymphoma were carried out using doses ranging from 0.7513 g / m administered once , twice , or three times weekly as 2- or 4-hour intravenous infusions . of the six responders , patients with extranodal disease and those with prior autologous transplant also responded ; these responses overall appeared to be durable . of six patients with bcr - abl1 all who responded and had no further therapy after blinatumomab , four remain in ongoing hematologic and molecular remission.42 these encouraging results have led to a larger trial in patients who are mrd - positive across several countries in europe , which was initiated in the fall of 2010 . these results have also led to a trial in patients with hematologic relapse or refractory b - lineage all . the exciting results achieved with blinatumomab in both the hematologic relapsed / refractory setting and in the mrd setting have led to the development of a us national intergroup trial , which will randomize patients with b - lineage all between the ages of 35 and 70 years to induction chemotherapy to achieve remission followed by four cycles of blinatumomab versus chemotherapy alone . although more than 90% of children can now be cured of their all , these results have not been duplicated in adults . the use of pediatric intensive regimens has improved the outcome of young adults with all , but further progress is needed . in older adults , the limits of conventional chemotherapy have been reached , and new approaches are needed . one antibody construct , blinatumomab , a bispecific t - cell engager antibody , is in advanced stages of development and shows significant promise in improving outcomes of patients with b - lineage all . a large intergroup trial in the united states will test the efficacy of blinatumomab in newly diagnosed middle - aged and older adults with b - lineage all .

after cardiac abnormalities , neural tube defects ( ntds ) are the second most common group of serious congenital anomalies [ 13 ] . ntds include spina bifida ( sb ) and anencephaly , as well as cephalocele ( or encephalocoele ) , where the brain protrudes through a defect in the skull . each year , approximately 300,000 newborns worldwide are born with sb or anencephaly [ 5 , 6 ] . although prevalence rates of anencephaly and sb are similar , anencephaly results in more abortions because it is more easily detected in prenatal exams , and the condition is fatal to the child . cephaloceles are less common than anencephaly or sb , occurring in one to three per 10,000 live births . ntds typically occur when the neural tube fails to close properly , around day 28 following conception [ 4 , 810 ] . thus , closure of the neural tube often happens before a woman knows she is pregnant [ 4 , 9 , 11 ] . folate is a water - soluble b vitamin that is found naturally in foods , such as fruits , dark green vegetables , potatoes , beans , and yeast extract . folic acid is the synthetic form of folate found in dietary supplements and added to enriched flour and grain products , such as breads , pasta , rice , and cereals [ 1315 ] . when taken before conception , adequate use of folic acid reduces the incidence of ntds . in light of this , healthy people objectives for 2010 related to ntds , to reduce the number of ntds and to ensure that women have appropriate folate levels prior to conception . also , public health bodies worldwide , such as the centers for disease control and prevention ( cdc ) and the world health organization ( who ) , recommend a daily folic acid intake of 0.4 mg [ 1820 ] taken at least 1 month before pregnancy and in the first trimester of pregnancy to reduce the risk of ntds ( since folic acid only appears in plasma in subjects receiving doses above 0.2 mg ) . there are three public health strategies for reaching the recommended daily dose : ( 1 ) folic acid supplements combined with a healthy diet ; ( 2 ) voluntary fortification of food with synthetic folic acid ; and ( 3 ) mandatory fortification of a staple food . in addition , various public campaigns educating both healthcare professionals and the general public have increased international awareness and helped prevent ntds . however , whilst studies generally demonstrate increased awareness , knowledge , and consumption of folic acid post campaigns , the long - term effects of these campaigns are unknown and campaigns are limited to a particular time for a cross - section of their target audience ( from as little as 2 days up to 6 years [ 22 , 24 ] ) . therefore , the key audience , women of child - bearing age , may not be reached . in the us , however , an innovative approach to increasing folate levels can now be achieved through the simple use of combined oral contraceptives ( cocs ) . in 2010 , two new cocs known as beyaz and safyral ( bayer healthcare pharmaceuticals , leverkusen , germany ) approved by the us food and drug administration ( fda ) were launched . beside a label for contraception , these two cocs are indicated to raise folate levels for the purpose of reducing the risk of a neural tube defect in a pregnancy conceived while taking the product or shortly after discontinuing the product in women who choose an oral contraceptive as their method of contraception . the combination of health education programs , recommendations , and food fortification programs has contributed to declines in ntd rates ( declines of 2670% over a period of 15 years ) . in addition , the reduction might in part be attributable to secondary prevention , as the number of terminations due to improved early diagnosis is rising . however , the rate of decline has not been as dramatic as expected [ 2529 ] and despite measures to reduce ntds , approximately 4,500 pregnancies every year in europe result in a live birth , stillbirth , or termination where a baby or fetus has been affected by an ntd , and in the us there are 2,500 live births of children with ntds each year . parents ( often the sole caregivers ) face great distress at the diagnosis of an ntd . they are confronted with either the grief of a termination or stillbirth , or the lifelong emotional and financial challenges of caring for a child with an ntd . individuals with ntds that survive , such as those with sb , are often at risk of psychosocial maladjustment and have acute , life - long disabilities [ 32 , 33 ] . the diverse symptoms can be associated with ntds adversely impact quality of life ( qol ) , which can manifest in extensive physical and psychosocial burden [ 3442 ] . there is also associated economic burden incurred , including substantial direct medical treatment costs , direct nonmedical costs ( such as special education and developmental costs ) , as well as indirect costs related to increased morbidity and mortality of patients with ntds . health policy makers worldwide increasingly require insight from caregivers and patients perspectives , in addition to information about direct and indirect costs , to demonstrate the overall impact of a condition [ 44 , 45 ] . although this review was intended to demonstrate the impact of all types of ntds , since anencephaly is inevitably fatal , the authors main focus was on the impact of sb on patients and caregivers . using guidelines defined by the university of york national health service ( nhs ) centre for reviews and dissemination , a comprehensive search strategy was developed . the search strategy was implemented using three electronic databases ( pubmed , psycinfo , and embase ) to identify relevant studies from january 1976 to november 2010 . terms included health - related quality of life , hrqol , quality of life , qol symptoms , satisfaction , body image , self - image , emotional , physical , psychological , psychosocial , self - esteem , impact , relationships , caregiver burden , family impact , work , productivity , absenteeism , presenteeism , qualitative , interviews , grounded theory , and interpretive phenomenological analysis . cost , economic , burden / impact of illness , resource use , hospitalization , and economic evaluation . family planning , unplanned pregnancy , prenatal care , abortion , and termination were other keywords used . in addition to the electronic database searches , abstracts from the international federation for sb and hydrocephalus 17th international conference were hand - searched to capture recent information that may have been presented but not yet published in journals . internet searches of family caregiver associations and societies were also conducted to access information from grey literature ; these included the association for sb and hydrocephalus ( uk ) , the sb association ( us ) , the scottish sb association ( uk ) , and sb family support ( us ) . following completion of the search , all titles and abstracts were screened for possible inclusion in the study by two independent researchers ( dr and lm ) . to satisfy the inclusion criteria , selected abstracts included an appropriate clinical term and at least one of the pro terms or cost terms . the review pool was restricted to english language studies , human subjects , and articles published from january 1976 to november 2010 . studies were excluded only if the reviewers could be sure that they did not fulfill the criteria . due to the high number of seemingly relevant articles , following the inclusion / exclusion criteria , abstracts were ranked 1 , 2 , or 3 , according to the following three criteria : ( 1 ) the journal article included terms of interest in the title and abstract and the terms of interest were the main focus ; ( 2 ) the journal article included the terms of interest as secondary or exploratory analyses ; or ( 3 ) the abstract contained supportive information , but there was no real data ( e.g. , there was a background comment in the introduction or conclusions ) . following the ranking process , articles ranked 1 were included and all others were excluded from this review . to facilitate comparison of economic studies , costs were inflated to 2010 us dollar prices using the consumer price index inflation calculator ( available at www.bls.gov/data/inflation_calculator.htm ) ( original costs are reported in brackets ) . although this review was intended to demonstrate the impact of all types of ntds , since anencephaly is inevitably fatal , the authors main focus was on the impact of sb on patients and caregivers . using guidelines defined by the university of york national health service ( nhs ) centre for reviews and dissemination , a comprehensive search strategy was developed . the search strategy was implemented using three electronic databases ( pubmed , psycinfo , and embase ) to identify relevant studies from january 1976 to november 2010 . terms included health - related quality of life , hrqol , quality of life , qol symptoms , satisfaction , body image , self - image , emotional , physical , psychological , psychosocial , self - esteem , impact , relationships , caregiver burden , family impact , work , productivity , absenteeism , presenteeism , qualitative , interviews , grounded theory , and interpretive phenomenological analysis . cost , economic , burden / impact of illness , resource use , hospitalization , and economic evaluation . family planning , unplanned pregnancy , prenatal care , abortion , and termination were other keywords used . in addition to the electronic database searches , abstracts from the international federation for sb and hydrocephalus 17th international conference were hand - searched to capture recent information that may have been presented but not yet published in journals . internet searches of family caregiver associations and societies were also conducted to access information from grey literature ; these included the association for sb and hydrocephalus ( uk ) , the sb association ( us ) , the scottish sb association ( uk ) , and sb family support ( us ) . following completion of the search , all titles and abstracts were screened for possible inclusion in the study by two independent researchers ( dr and lm ) . to satisfy the inclusion criteria , selected abstracts included an appropriate clinical term and at least one of the pro terms or cost terms . the review pool was restricted to english language studies , human subjects , and articles published from january 1976 to november 2010 . studies were excluded only if the reviewers could be sure that they did not fulfill the criteria . due to the high number of seemingly relevant articles , following the inclusion / exclusion criteria , abstracts were ranked 1 , 2 , or 3 , according to the following three criteria : ( 1 ) the journal article included terms of interest in the title and abstract and the terms of interest were the main focus ; ( 2 ) the journal article included the terms of interest as secondary or exploratory analyses ; or ( 3 ) the abstract contained supportive information , but there was no real data ( e.g. , there was a background comment in the introduction or conclusions ) . following the ranking process , articles ranked 1 were included and all others were excluded from this review . to facilitate comparison of economic studies , costs were inflated to 2010 us dollar prices using the consumer price index inflation calculator ( available at www.bls.gov/data/inflation_calculator.htm ) ( original costs are reported in brackets ) . the titles and abstracts were then examined in further detail and a total of 4,288 were excluded for not containing all of the search terms in the title or abstract following ranking , or due to duplication between the databases . the impact of sb from the patient s perspective has been extensively documented [ 34 , 3739 , 4870 ] . using information from the articles included in this review , a conceptual model was developed to demonstrate the relationship between various factors associated with sb in individuals ( fig . 1 ) . a conceptual model compartmentalizes potential causes , consequences , and signs and symptoms of the disease while showing how they are linked with one another.fig . hrqol health - related quality of life , ntds neural tube defects impact of spina bifida on patients . hrqol health - related quality of life , ntds neural tube defects areas of patients lives affected include physical functioning , activities of daily living , role functioning , bodily pain , vitality , emotional functioning , mental health , self - esteem , self - image , social functioning , relationships , and sexual functioning ( see fig . 1 ) . long - term health problems of sb include urinary tract infections , calculi ( kidney stones ) , and skin infections . grimby looked at the differences between two groups of subjects : one with cerebral palsy , the other with sb , in their dependence and their perceived difficulty in performing daily activities . subjects in both groups needed help in basic activities of daily living ; however , sb subjects were more impacted by toileting problems and lack of bladder and bowel control than the cerebral palsy subjects . the impact of sb on caregivers has also been well documented [ 2 , 38 , 40 , 43 , 7292 ] . they are confronted with either the grief of a termination or stillbirth , or extensive emotional and financial challenges of caring for a child with an ntd . caring for patients with sb who may have comorbidities can also exert a substantial burden on caregivers , including the impact on carer workload , decreased qol , less time for work , and additional responsibilities . areas of caregivers lives affected include activities of daily living , work impact , time consumption ( including the need to always be on hand to provide the level of care required for individuals with sb ) [ 74 , 76 , 80 ] , parental responsibilities ( including responsibilities to other children ) , confidence , feelings and emotions , mental health , stress , social impact , psychological adjustment , relationships ( with sb child , siblings , and other members of the family ) , social support , coping strategies , and termination decisions . in one study on caregivers of children with cerebral palsy or sb , caring for an affected child took up to 29% of their waking time . this equated to more time than spent cooking , cleaning , and doing the laundry ( 26% ) . leisure activities and work took up the least amount of their time [ 81 , 94 ] . the average lifetime direct medical cost per person with sb ranges from $ 285,959 ( $ 235,839 in 2002 dollars ) to $ 378,000 ( $ 319,000 in 2003 dollars ) in 2010 dollars . this does not include lifetime direct nonmedical costs ( such as special education and development services ) of $ 52,570 per person ( $ 43,371 in 2002 dollars ) . the average lifetime indirect cost per person with sb in the us was estimated to be $ 432,176 ( $ 356,553 in 2002 dollars ) in 2010 dollars , or 57% of the average total lifetime cost per person with sb . the cost burden on individuals and caregivers includes out - of - pocket costs , lost wages / household production due to increased morbidity and mortality , transportation , and other nonmedical costs . very few studies examine the costs of ntds from the perspective of patients and/or caregivers . ouyang et al . reported the out - of - pocket cost burden to privately insured patients in the us . according to this study , individuals with sb in a private health insurance plan shared on average 11% of their total health expenditure ( 8% of costs for their inpatient care , 11% of costs for outpatient visits , and 17% of costs for prescription drugs ) , which in 2006 was $ 40,928 ( $ 34,536 in 2003 dollars ) per person ( taken as an average across all age groups of people with sb ) . despite the potential important contribution of caregiver time costs to the total cost estimate of birth defects , only three studies estimated caregiver time costs related specifically to birth defects [ 9799 ] . average reductions of 14 h per week in paid work time for mothers and 5 h per week for fathers of children with sb were reported . differences in work hours by caregivers of children with sb translated into lifetime costs of $ 162,124 in 2010 dollars ( $ 133,755 in 2002 dollars ) using a 3% discount rate , and an age- and sex - adjusted earnings profile . the titles and abstracts were then examined in further detail and a total of 4,288 were excluded for not containing all of the search terms in the title or abstract following ranking , or due to duplication between the databases . the impact of sb from the patient s perspective has been extensively documented [ 34 , 3739 , 4870 ] . using information from the articles included in this review , a conceptual model was developed to demonstrate the relationship between various factors associated with sb in individuals ( fig . 1 ) . a conceptual model compartmentalizes potential causes , consequences , and signs and symptoms of the disease while showing how they are linked with one another.fig . hrqol health - related quality of life , ntds neural tube defects impact of spina bifida on patients . hrqol health - related quality of life , ntds neural tube defects areas of patients lives affected include physical functioning , activities of daily living , role functioning , bodily pain , vitality , emotional functioning , mental health , self - esteem , self - image , social functioning , relationships , and sexual functioning ( see fig . 1 ) . long - term health problems of sb include urinary tract infections , calculi ( kidney stones ) , and skin infections . grimby looked at the differences between two groups of subjects : one with cerebral palsy , the other with sb , in their dependence and their perceived difficulty in performing daily activities . subjects in both groups needed help in basic activities of daily living ; however , sb subjects were more impacted by toileting problems and lack of bladder and bowel control than the cerebral palsy subjects . the impact of sb on caregivers has also been well documented [ 2 , 38 , 40 , 43 , 7292 ] . they are confronted with either the grief of a termination or stillbirth , or extensive emotional and financial challenges of caring for a child with an ntd . caring for patients with sb who may have comorbidities can also exert a substantial burden on caregivers , including the impact on carer workload , decreased qol , less time for work , and additional responsibilities . areas of caregivers lives affected include activities of daily living , work impact , time consumption ( including the need to always be on hand to provide the level of care required for individuals with sb ) [ 74 , 76 , 80 ] , parental responsibilities ( including responsibilities to other children ) , confidence , feelings and emotions , mental health , stress , social impact , psychological adjustment , relationships ( with sb child , siblings , and other members of the family ) , social support , coping strategies , and termination decisions . in one study on caregivers of children with cerebral palsy or sb , caring for an affected child took up to 29% of their waking time . this equated to more time than spent cooking , cleaning , and doing the laundry ( 26% ) . leisure activities and work took up the least amount of their time [ 81 , 94 ] . the average lifetime direct medical cost per person with sb ranges from $ 285,959 ( $ 235,839 in 2002 dollars ) to $ 378,000 ( $ 319,000 in 2003 dollars ) in 2010 dollars . this does not include lifetime direct nonmedical costs ( such as special education and development services ) of $ 52,570 per person ( $ 43,371 in 2002 dollars ) . the average lifetime indirect cost per person with sb in the us was estimated to be $ 432,176 ( $ 356,553 in 2002 dollars ) in 2010 dollars , or 57% of the average total lifetime cost per person with sb . the cost burden on individuals and caregivers includes out - of - pocket costs , lost wages / household production due to increased morbidity and mortality , transportation , and other nonmedical costs . very few studies examine the costs of ntds from the perspective of patients and/or caregivers . ouyang et al . reported the out - of - pocket cost burden to privately insured patients in the us . according to this study , individuals with sb in a private health insurance plan shared on average 11% of their total health expenditure ( 8% of costs for their inpatient care , 11% of costs for outpatient visits , and 17% of costs for prescription drugs ) , which in 2006 was $ 40,928 ( $ 34,536 in 2003 dollars ) per person ( taken as an average across all age groups of people with sb ) . despite the potential important contribution of caregiver time costs to the total cost estimate of birth defects , only three studies estimated caregiver time costs related specifically to birth defects [ 9799 ] . average reductions of 14 h per week in paid work time for mothers and 5 h per week for fathers of children with sb were reported . differences in work hours by caregivers of children with sb translated into lifetime costs of $ 162,124 in 2010 dollars ( $ 133,755 in 2002 dollars ) using a 3% discount rate , and an age- and sex - adjusted earnings profile . the results from this review demonstrated the profound impact of sb on individuals and caregivers . for patients , this lifetime impact is apparent in physical , emotional , mental , educational , sexual , social , and financial aspects of everyday life . the conceptual model developed based on the literature highlights the notable range and variety of the impact on patients with sb . for caregivers , the emotional and financial burden along with the toll on their social lives and work were the greatest impacts . caregivers also experience additional financial burden , including reduced income due to the necessity of working a reduced number of hours in paid employment . the diverse humanistic impact and economic burden of sb for individuals and caregivers emphasizes the importance of providing substantial care and support to both . in addition , as outlined earlier , sustained and persistent education about the benefits of preconceptional folates is important to help prevent ntds , especially since the burden is so often avoidable with adequate folate consumption at the right time . however , whilst long - term , effective health campaigns educating the public about the benefits of preconceptional folate may help reduce ntd risk , given the limitations of such campaigns , additional strategies , such as different types of targeted fortification , may be warranted to reduce this risk even further it is important to acknowledge that this literature review and its findings are based on published english literature studies that emerged from searching electronic databases . further research could use statistical methods to explore the net effect of ntds and treatment . furthermore , the majority of the papers in this review were us focused , especially economic - related papers . little is known about the cost that sb has on individuals health - related qol in europe and other regions outside of the us . therefore , there is a need for additional studies in europe and asia , and future studies could be designed to address this . in addition , while sb is a major type of ntds , other rarer forms of ntds also exist . although there is a significant body of evidence to demonstrate the impact of sb on individuals and caregivers , this comprehensive literature review may under - represent the impact of anencephaly on prospective parents and rarer types of ntds , which are also likely to have a profound effect on individuals and caregivers . given the substantial range and variety of humanistic impact and economic burden of sb , this review highlights the need to provide care and support to individuals with sb and their caregivers . results also emphasize the importance of effective long - term public health campaigns and/or newer strategies to prevent ntds , such as sb . this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use , distribution , and reproduction in any medium , provided the original author(s ) and the source are credited .

introductiondespite measures to reduce the incidence of neural tube defects ( ntds ) , the rate of decline has not been as dramatic as expected . at least 300,000 newborns worldwide are known to be affected by ntds each year . this comprehensive literature review summarizes the human and economic burden of ntds to patients and caregivers , with particular focus on spina bifida ( sb).methodspubmed , psycinfo , and embase were searched for studies from january 1976 to november 2010 that included clinical terms , such as ntd , and at least one patient - reported outcome or cost term . a conceptual model was also developed.resultsareas of peoples lives affected by sb included physical and role functioning , activities of daily living , bodily pain , vitality , emotional functioning , mental health , self - esteem , self - image , social functioning , relationships , and sexual functioning . areas of caregivers lives affected included activities of daily living , work impact , time consumption , parental responsibilities ( including responsibilities to other children ) , confidence , feelings and emotions , mental health , stress , social impact , psychological adjustment , relationships ( with sb child , siblings , other family members ) , social support , coping strategies , and termination decisions . cost burdens on patients and caregivers also include out - of - pocket costs , lost wages , or household production due to increased morbidity and mortality , transportation and other nonmedical costs.conclusionsthis review highlights the need to provide care and support to individuals with sb and their caregivers . results also emphasize the importance of effective long - term public health campaigns and/or newer strategies to prevent ntds , such as sb .

Introduction Materials and Methods Search Strategy for Literature Search Conference Proceeding Abstract Searches and Internet Searches Inclusion and Exclusion Criteria Ranking Process Currency Conversion Results Study Selection Impact of SB on Individuals Impact on Caregivers Economic Impact of NTDs on Individuals and Caregivers Discussion Conclusion Conflict of interest Open Access

after cardiac abnormalities , neural tube defects ( ntds ) are the second most common group of serious congenital anomalies [ 13 ] . ntds include spina bifida ( sb ) and anencephaly , as well as cephalocele ( or encephalocoele ) , where the brain protrudes through a defect in the skull . each year , approximately 300,000 newborns worldwide are born with sb or anencephaly [ 5 , 6 ] . folate is a water - soluble b vitamin that is found naturally in foods , such as fruits , dark green vegetables , potatoes , beans , and yeast extract . folic acid is the synthetic form of folate found in dietary supplements and added to enriched flour and grain products , such as breads , pasta , rice , and cereals [ 1315 ] . when taken before conception , adequate use of folic acid reduces the incidence of ntds . in light of this , healthy people objectives for 2010 related to ntds , to reduce the number of ntds and to ensure that women have appropriate folate levels prior to conception . also , public health bodies worldwide , such as the centers for disease control and prevention ( cdc ) and the world health organization ( who ) , recommend a daily folic acid intake of 0.4 mg [ 1820 ] taken at least 1 month before pregnancy and in the first trimester of pregnancy to reduce the risk of ntds ( since folic acid only appears in plasma in subjects receiving doses above 0.2 mg ) . there are three public health strategies for reaching the recommended daily dose : ( 1 ) folic acid supplements combined with a healthy diet ; ( 2 ) voluntary fortification of food with synthetic folic acid ; and ( 3 ) mandatory fortification of a staple food . in addition , various public campaigns educating both healthcare professionals and the general public have increased international awareness and helped prevent ntds . however , whilst studies generally demonstrate increased awareness , knowledge , and consumption of folic acid post campaigns , the long - term effects of these campaigns are unknown and campaigns are limited to a particular time for a cross - section of their target audience ( from as little as 2 days up to 6 years [ 22 , 24 ] ) . therefore , the key audience , women of child - bearing age , may not be reached . beside a label for contraception , these two cocs are indicated to raise folate levels for the purpose of reducing the risk of a neural tube defect in a pregnancy conceived while taking the product or shortly after discontinuing the product in women who choose an oral contraceptive as their method of contraception . the combination of health education programs , recommendations , and food fortification programs has contributed to declines in ntd rates ( declines of 2670% over a period of 15 years ) . in addition , the reduction might in part be attributable to secondary prevention , as the number of terminations due to improved early diagnosis is rising . however , the rate of decline has not been as dramatic as expected [ 2529 ] and despite measures to reduce ntds , approximately 4,500 pregnancies every year in europe result in a live birth , stillbirth , or termination where a baby or fetus has been affected by an ntd , and in the us there are 2,500 live births of children with ntds each year . individuals with ntds that survive , such as those with sb , are often at risk of psychosocial maladjustment and have acute , life - long disabilities [ 32 , 33 ] . the diverse symptoms can be associated with ntds adversely impact quality of life ( qol ) , which can manifest in extensive physical and psychosocial burden [ 3442 ] . there is also associated economic burden incurred , including substantial direct medical treatment costs , direct nonmedical costs ( such as special education and developmental costs ) , as well as indirect costs related to increased morbidity and mortality of patients with ntds . health policy makers worldwide increasingly require insight from caregivers and patients perspectives , in addition to information about direct and indirect costs , to demonstrate the overall impact of a condition [ 44 , 45 ] . although this review was intended to demonstrate the impact of all types of ntds , since anencephaly is inevitably fatal , the authors main focus was on the impact of sb on patients and caregivers . the search strategy was implemented using three electronic databases ( pubmed , psycinfo , and embase ) to identify relevant studies from january 1976 to november 2010 . terms included health - related quality of life , hrqol , quality of life , qol symptoms , satisfaction , body image , self - image , emotional , physical , psychological , psychosocial , self - esteem , impact , relationships , caregiver burden , family impact , work , productivity , absenteeism , presenteeism , qualitative , interviews , grounded theory , and interpretive phenomenological analysis . cost , economic , burden / impact of illness , resource use , hospitalization , and economic evaluation . family planning , unplanned pregnancy , prenatal care , abortion , and termination were other keywords used . in addition to the electronic database searches , abstracts from the international federation for sb and hydrocephalus 17th international conference were hand - searched to capture recent information that may have been presented but not yet published in journals . internet searches of family caregiver associations and societies were also conducted to access information from grey literature ; these included the association for sb and hydrocephalus ( uk ) , the sb association ( us ) , the scottish sb association ( uk ) , and sb family support ( us ) . to satisfy the inclusion criteria , selected abstracts included an appropriate clinical term and at least one of the pro terms or cost terms . the review pool was restricted to english language studies , human subjects , and articles published from january 1976 to november 2010 . due to the high number of seemingly relevant articles , following the inclusion / exclusion criteria , abstracts were ranked 1 , 2 , or 3 , according to the following three criteria : ( 1 ) the journal article included terms of interest in the title and abstract and the terms of interest were the main focus ; ( 2 ) the journal article included the terms of interest as secondary or exploratory analyses ; or ( 3 ) the abstract contained supportive information , but there was no real data ( e.g. although this review was intended to demonstrate the impact of all types of ntds , since anencephaly is inevitably fatal , the authors main focus was on the impact of sb on patients and caregivers . the search strategy was implemented using three electronic databases ( pubmed , psycinfo , and embase ) to identify relevant studies from january 1976 to november 2010 . terms included health - related quality of life , hrqol , quality of life , qol symptoms , satisfaction , body image , self - image , emotional , physical , psychological , psychosocial , self - esteem , impact , relationships , caregiver burden , family impact , work , productivity , absenteeism , presenteeism , qualitative , interviews , grounded theory , and interpretive phenomenological analysis . cost , economic , burden / impact of illness , resource use , hospitalization , and economic evaluation . family planning , unplanned pregnancy , prenatal care , abortion , and termination were other keywords used . in addition to the electronic database searches , abstracts from the international federation for sb and hydrocephalus 17th international conference were hand - searched to capture recent information that may have been presented but not yet published in journals . internet searches of family caregiver associations and societies were also conducted to access information from grey literature ; these included the association for sb and hydrocephalus ( uk ) , the sb association ( us ) , the scottish sb association ( uk ) , and sb family support ( us ) . to satisfy the inclusion criteria , selected abstracts included an appropriate clinical term and at least one of the pro terms or cost terms . the review pool was restricted to english language studies , human subjects , and articles published from january 1976 to november 2010 . due to the high number of seemingly relevant articles , following the inclusion / exclusion criteria , abstracts were ranked 1 , 2 , or 3 , according to the following three criteria : ( 1 ) the journal article included terms of interest in the title and abstract and the terms of interest were the main focus ; ( 2 ) the journal article included the terms of interest as secondary or exploratory analyses ; or ( 3 ) the abstract contained supportive information , but there was no real data ( e.g. the titles and abstracts were then examined in further detail and a total of 4,288 were excluded for not containing all of the search terms in the title or abstract following ranking , or due to duplication between the databases . using information from the articles included in this review , a conceptual model was developed to demonstrate the relationship between various factors associated with sb in individuals ( fig . a conceptual model compartmentalizes potential causes , consequences , and signs and symptoms of the disease while showing how they are linked with one another.fig . hrqol health - related quality of life , ntds neural tube defects impact of spina bifida on patients . hrqol health - related quality of life , ntds neural tube defects areas of patients lives affected include physical functioning , activities of daily living , role functioning , bodily pain , vitality , emotional functioning , mental health , self - esteem , self - image , social functioning , relationships , and sexual functioning ( see fig . long - term health problems of sb include urinary tract infections , calculi ( kidney stones ) , and skin infections . grimby looked at the differences between two groups of subjects : one with cerebral palsy , the other with sb , in their dependence and their perceived difficulty in performing daily activities . subjects in both groups needed help in basic activities of daily living ; however , sb subjects were more impacted by toileting problems and lack of bladder and bowel control than the cerebral palsy subjects . they are confronted with either the grief of a termination or stillbirth , or extensive emotional and financial challenges of caring for a child with an ntd . caring for patients with sb who may have comorbidities can also exert a substantial burden on caregivers , including the impact on carer workload , decreased qol , less time for work , and additional responsibilities . areas of caregivers lives affected include activities of daily living , work impact , time consumption ( including the need to always be on hand to provide the level of care required for individuals with sb ) [ 74 , 76 , 80 ] , parental responsibilities ( including responsibilities to other children ) , confidence , feelings and emotions , mental health , stress , social impact , psychological adjustment , relationships ( with sb child , siblings , and other members of the family ) , social support , coping strategies , and termination decisions . this equated to more time than spent cooking , cleaning , and doing the laundry ( 26% ) . this does not include lifetime direct nonmedical costs ( such as special education and development services ) of $ 52,570 per person ( $ 43,371 in 2002 dollars ) . the average lifetime indirect cost per person with sb in the us was estimated to be $ 432,176 ( $ 356,553 in 2002 dollars ) in 2010 dollars , or 57% of the average total lifetime cost per person with sb . the cost burden on individuals and caregivers includes out - of - pocket costs , lost wages / household production due to increased morbidity and mortality , transportation , and other nonmedical costs . very few studies examine the costs of ntds from the perspective of patients and/or caregivers . reported the out - of - pocket cost burden to privately insured patients in the us . according to this study , individuals with sb in a private health insurance plan shared on average 11% of their total health expenditure ( 8% of costs for their inpatient care , 11% of costs for outpatient visits , and 17% of costs for prescription drugs ) , which in 2006 was $ 40,928 ( $ 34,536 in 2003 dollars ) per person ( taken as an average across all age groups of people with sb ) . average reductions of 14 h per week in paid work time for mothers and 5 h per week for fathers of children with sb were reported . differences in work hours by caregivers of children with sb translated into lifetime costs of $ 162,124 in 2010 dollars ( $ 133,755 in 2002 dollars ) using a 3% discount rate , and an age- and sex - adjusted earnings profile . the titles and abstracts were then examined in further detail and a total of 4,288 were excluded for not containing all of the search terms in the title or abstract following ranking , or due to duplication between the databases . using information from the articles included in this review , a conceptual model was developed to demonstrate the relationship between various factors associated with sb in individuals ( fig . a conceptual model compartmentalizes potential causes , consequences , and signs and symptoms of the disease while showing how they are linked with one another.fig . hrqol health - related quality of life , ntds neural tube defects impact of spina bifida on patients . hrqol health - related quality of life , ntds neural tube defects areas of patients lives affected include physical functioning , activities of daily living , role functioning , bodily pain , vitality , emotional functioning , mental health , self - esteem , self - image , social functioning , relationships , and sexual functioning ( see fig . long - term health problems of sb include urinary tract infections , calculi ( kidney stones ) , and skin infections . grimby looked at the differences between two groups of subjects : one with cerebral palsy , the other with sb , in their dependence and their perceived difficulty in performing daily activities . subjects in both groups needed help in basic activities of daily living ; however , sb subjects were more impacted by toileting problems and lack of bladder and bowel control than the cerebral palsy subjects . they are confronted with either the grief of a termination or stillbirth , or extensive emotional and financial challenges of caring for a child with an ntd . caring for patients with sb who may have comorbidities can also exert a substantial burden on caregivers , including the impact on carer workload , decreased qol , less time for work , and additional responsibilities . areas of caregivers lives affected include activities of daily living , work impact , time consumption ( including the need to always be on hand to provide the level of care required for individuals with sb ) [ 74 , 76 , 80 ] , parental responsibilities ( including responsibilities to other children ) , confidence , feelings and emotions , mental health , stress , social impact , psychological adjustment , relationships ( with sb child , siblings , and other members of the family ) , social support , coping strategies , and termination decisions . this equated to more time than spent cooking , cleaning , and doing the laundry ( 26% ) . the average lifetime direct medical cost per person with sb ranges from $ 285,959 ( $ 235,839 in 2002 dollars ) to $ 378,000 ( $ 319,000 in 2003 dollars ) in 2010 dollars . this does not include lifetime direct nonmedical costs ( such as special education and development services ) of $ 52,570 per person ( $ 43,371 in 2002 dollars ) . the average lifetime indirect cost per person with sb in the us was estimated to be $ 432,176 ( $ 356,553 in 2002 dollars ) in 2010 dollars , or 57% of the average total lifetime cost per person with sb . the cost burden on individuals and caregivers includes out - of - pocket costs , lost wages / household production due to increased morbidity and mortality , transportation , and other nonmedical costs . very few studies examine the costs of ntds from the perspective of patients and/or caregivers . reported the out - of - pocket cost burden to privately insured patients in the us . according to this study , individuals with sb in a private health insurance plan shared on average 11% of their total health expenditure ( 8% of costs for their inpatient care , 11% of costs for outpatient visits , and 17% of costs for prescription drugs ) , which in 2006 was $ 40,928 ( $ 34,536 in 2003 dollars ) per person ( taken as an average across all age groups of people with sb ) . average reductions of 14 h per week in paid work time for mothers and 5 h per week for fathers of children with sb were reported . differences in work hours by caregivers of children with sb translated into lifetime costs of $ 162,124 in 2010 dollars ( $ 133,755 in 2002 dollars ) using a 3% discount rate , and an age- and sex - adjusted earnings profile . for patients , this lifetime impact is apparent in physical , emotional , mental , educational , sexual , social , and financial aspects of everyday life . the conceptual model developed based on the literature highlights the notable range and variety of the impact on patients with sb . for caregivers , the emotional and financial burden along with the toll on their social lives and work were the greatest impacts . caregivers also experience additional financial burden , including reduced income due to the necessity of working a reduced number of hours in paid employment . the diverse humanistic impact and economic burden of sb for individuals and caregivers emphasizes the importance of providing substantial care and support to both . in addition , as outlined earlier , sustained and persistent education about the benefits of preconceptional folates is important to help prevent ntds , especially since the burden is so often avoidable with adequate folate consumption at the right time . however , whilst long - term , effective health campaigns educating the public about the benefits of preconceptional folate may help reduce ntd risk , given the limitations of such campaigns , additional strategies , such as different types of targeted fortification , may be warranted to reduce this risk even further it is important to acknowledge that this literature review and its findings are based on published english literature studies that emerged from searching electronic databases . further research could use statistical methods to explore the net effect of ntds and treatment . furthermore , the majority of the papers in this review were us focused , especially economic - related papers . little is known about the cost that sb has on individuals health - related qol in europe and other regions outside of the us . therefore , there is a need for additional studies in europe and asia , and future studies could be designed to address this . in addition , while sb is a major type of ntds , other rarer forms of ntds also exist . although there is a significant body of evidence to demonstrate the impact of sb on individuals and caregivers , this comprehensive literature review may under - represent the impact of anencephaly on prospective parents and rarer types of ntds , which are also likely to have a profound effect on individuals and caregivers . given the substantial range and variety of humanistic impact and economic burden of sb , this review highlights the need to provide care and support to individuals with sb and their caregivers . results also emphasize the importance of effective long - term public health campaigns and/or newer strategies to prevent ntds , such as sb .

although it closed its doors in 1950 , the pioneer health centre ( phc ) in peckham , south london ( also known as the peckham health centre or the peckham experiment ) remains a project whose significance and legacy are vigorously contested . on the one hand , there are those who have criticised what they see as its over - ambitious and ill - defined aims ; its lack of scientific and statistical rigour ; its sinister extension of the medical gaze and its essentially conservative conception of the family . on the other , there are those who regard the national health service ( nhs ) as more accurately a sickness service , and seek to establish initiatives actively promoting health rather than attending only to people already unwell . in particular , the pioneer health foundation ( phf ) keeps the peckham flame burning , with some success . the phc s founders , dr george scott williamson ( 18841953 ) and dr innes h. pearse ( 18891978 ) , were hostile to the nhs , but there are doctors active in the phf who work in the nhs and believe that the service would benefit from a re - consideration of the peckham approach . this article s chief purpose is to examine the circumstances surrounding the phc s closure , and the reasons for it , and to assess which organisations and individuals might be held responsible for that event . however , a brief summary of the centre s history , principles and practice is necessary . its first incarnation , from 1926 to 1929 , was a response to widespread concern about the fitness of the british people . in effect an exercise in positive eugenics , it aimed to encourage the health of the fit rather than working to reverse the law of nature by procuring the survival of the unfit. in 1925 scott williamson was director of pathological studies at the london school of medicine for women , specialising , with his colleague innes pearse , in thyroid research . despite their professional commitment to pathology , scott williamson and pearse believed that medical scientists were wrong to ignore the uncomplaining or so - called healthy members of the population . if the nation s health was to be improved , focusing on disease was unlikely to provide the clue to how this might be achieved ; similarly , scott williamson and pearse were sceptical about the scientific validity of the methods which geneticists advocated . they envisaged instead an experiment in social biology , in order to explore the environmental factor in inheritance , and with a small group of non - medical associates opened in april 1926 a family club at 142 queens road , peckham . they chose this area because its population was chiefly of good artisan type , but including as wide an admixture of cultural types as it is possible to find within any small area in the metropolis , unaffected by long - term unemployment or serious social problems . the centre s staff consisted of a resident medical officer , a social secretary and a housekeeper , while the facilities included a changing room , a consulting room , a bathroom and a club - room . membership was for families only , and in return for a small weekly sum they received periodic medical and dental examinations , a parents clinic , an orthopaedic clinic , and ante- and post - natal clinics . the centre s service was purely advisory , its aim being to identify the early stages of disease and to evoke a desire for health. during the centre s three years in queens road , 112 families joined , and the doctors concluded that there were many families who , in the right circumstances , would welcome a service which encouraged health rather than dealing with sickness . scott williamson and pearse therefore closed the first stage of the centre and spent the next six years planning and raising funds for the second , which required an environment providing families with an outlet for all their members . sufficient money was obtained , mainly from private individuals , to begin the project , and the pioneer health centre building , designed by the noted architectural engineer sir owen williams , opened in 1935 in st mary s road , peckham . it contained a swimming - pool , separated only by glass from the rooms surrounding it ; a gymnasium , dance hall and cafeteria , all similarly visible ; a billiard room ; a small theatre ; a pool for babies ; nurseries and a children s playground . the centre s staff did not run the leisure activities ; it was the members who discovered what they wanted to do and then organised it . after an initial period of chaos , a spontaneous order emerged which combined a feeling of heightened individuality with strong communal integration , giving many members a lifelong spirit of confidence . the doctors had two main roles : observing members activities and providing an annual medical overhaul for each family member . acceptance of this overhaul was a necessary condition of membership , which was for families only and required payment of a token fee for use of the centre and for its various activities . the doctors shared with the parents information gleaned via the overhaul , and offered advice about conception , pregnancy and post - natal matters . no treatment was provided : parents were left to decide what action they should take . scott williamson and pearse saw themselves more as biologists than as doctors , and from their biologists perspective the family was the basic social unit . by creating the best conditions possible for the development of body , mind and spirit they hoped to discover how health could be encouraged , and to see if any laws of healthy development revealed themselves . pathology which studied disease . it is important to emphasise here that scott williamson and pearse did not regard their work as a form of preventive medicine . to think in such terms was , they believed , to prioritise the concept of disease over the concept of health . it is true that the regular physical overhauls required of the phc s members revealed medical problems ; but , as alison stallibrass has pointed out , the doctors were only enabling the members to alleviate or remove the pathological conditions preventing them from engaging in the process of health. the peckham doctors saw health as a process of realising one s potential for maturity [ and ] a mutual subjective synthesis of organism and environment and of person and group [ which was ] not something that members of the medical profession give us. the word health in common usage is intimately bound up with its opposite , being associated with remedial and therapeutic institutions and the care of sickness . the peckham experiment s primary aim was to foster an environment in which health ( physical vitality , social confidence and development of skills and talents ) could be cultivated : reduction of illness was a by - product of this . although many assessments of the pioneer health centre s work have been broadly , and at times strongly , sympathetic to its aims and achievements , it has not been without critics . david armstrong goes so far as to describe its implications for observation of its members as terrifying , seeing it as part of a mid - twentieth - century extension of the medical gaze : something akin to bentham s panopticon now widening its scope to include not merely the unwell but the behaviour of the community as a whole . pyrs gruffudd offers a more focused critique of the phc by comparing it unfavourably with another modernist project of the 1930s , the finsbury health centre in north london ( 1938 ) . gruffudd notes the phc s political ambiguity as a retreat from state provision of health care , essentially a form of private medicine which regarded its members as distinctly gendered and autonomous individuals , in contrast with the self - consciously progressive message of finsbury s labour council , which saw the centre s members as citizens for condensing through the social and ideological mechanisms of the health centre. whereas the peckham doctors refused to compromise with the demands of state - led medicine , the finsbury health centre was an initiative by the local state of the type that formed the bedrock on which would be built the welfare state in the 1940s. however , gruffudd concludes by commenting that the pioneer health centre ltd charity has provided continued inspiration for practitioners of community - based medicine and speculating that its ecological brand of conservative modernity [ might ] yet win out over the welfare state that embraced finsbury. it must be emphasised here that the term health centre as used by the peckham doctors had a different meaning from the concept referred to in the 1946 national health service act . as michael ryan points out , health centres were intended as a key feature of the nhs , since it was widely considered that bringing together curative and preventive work under one roof would do much to improve the quality of first - line medical care. under section 21(1 ) of the act , local authorities were required to provide , equip and maintain premises to be called health centres , where facilities would be available for general medical , dental and pharmaceutical services , any services local health authorities were required or empowered to provide , and facilities both for specialist and for outpatient treatment . for scott williamson and pearse , these proposed centres were merely a variety of services under one roof , dealing chiefly with sickness and remedial treatment . only the phc , they believed , seeking as it did to encourage health and to observe and understand its laws , could genuinely claim to be a health centre . there has been a persistent sense that the pioneer health centre offered an approach to health preferable to that which the nhs adopted , and that many of the problems with which the nhs has had to struggle not least its cost could have been avoided had the peckham philosophy been put more widely into practice : a hope which seemed , in the mid-1940s , to have some justification . in the nation target for tomorrow ( whose editorial board included sir william beveridge , john boyd orr and julian huxley ) , the phc was praised for its belief that a doctor s task should be to make people as healthy as possible , not simply to attend to them once they are ill , and for its recognition of the part played by social activities and relationships in total health. the book ended with praise for the centre , recommending that similar centres should in time be set up elsewhere , both for the collection of data about people in supposedly normal health , and for the re - creation of family and community life. so why did this not happen , and why , when during the late 1940s the phc had been receiving a constant stream of visitors , many of them highly distinguished , and was the subject of a film by the noted documentary - maker paul rotha , did it have to close early in 1950 ? the nhs has been a chief suspect or scapegoat for the death of the pioneer health centre , apparently on the basis of the philosophical fallacy post hoc ergo propter hoc , since the centre s demise occurred little more than eighteen months after the nhs was established . an editorial in the medical press and circular saw the phc as not fitting into the pattern of the new service , which it deemed too rigid and inflexible to contain it. mary langman ( 19082004 ) , who was secretary to scott williamson and later ran the centre s farm at bromley common in kent , believed that the centre closed because of post - war inflation and lack of political will : it was an embarrassment to the architects of the national health service , which took no thought for the cultivation of health. in 1974 , the architect peter ray , who designed a field centre for the practical study of human ethology , wrote dramatically in the introduction to his project that the peckham experiment had been swept away on the high tide of socialism which rose after the second world war. former members of the centre , interviewed by john nye in 1984 , said with some bitterness that the government would nt help and that the national health smashed it. david goodway , in a more recent assessment , quotes an article in the anarchist journal freedom which blamed the post - war labour government for rejecting the peckham example in favour of a top - down , state - controlled bureaucratic national sickness system , and urged the transformation of the nhs into a decentralized , community - led organization. this article will argue that the reasons for the phc s closure were more varied and complex than such comments imply , and that the nhs was by no means the chief villain of the piece . drawing on hitherto unseen material from mary langman s papers , it will show that the personalities of scott williamson and pearse contributed to the phc s problems ; it will consider the part played by the medical research council ; it will examine the nature of the debates which took place between the centre and london county council ; and it will suggest some broader medical and cultural reasons why the centre s approach came to be seen as marginal or irrelevant in the post - war period . the simplest possible answer to the question why did the pioneer health centre have to close? is lack of money ; but this merely raises the further question of why it could not find the necessary funds . the important fact to note is that the centre had always struggled for funds ; its history had been one of begging all the way , according to the first edition of its glossy journal peckham . certainly it had been struggling ten years before the nhs was established . the phc s report for 1938/39 revealed that by august 1938 its position had been desperate , with its overdraft almost at the 10,000 limit . a 2000 loan kept things afloat until the situation was saved in july 1939 by a 5000 gift from lord nuffield , a gift of 1000 from an anonymous donor , and a further 1000 the following month from the pilgrim trust . the executive committee had anticipated that the centre could be run , within three years , at a cost coverable by subscriptions and members expenditure , but this proved not to be so . aiming to enrol 2 000 families in order to achieve financial self - sufficiency , the centre had by december 1938 attracted only 673 , despite having that autumn extended its catchment area fivefold , thereby creating a potential membership of 25 000 families . but there was a very disappointing response to this change : only 5% of those circulated went to visit the centre , and only 2% actually joined . reasons suggested in the 1938/39 report included inconvenience ( because of a greater distance to walk or push a pram ) ; the cost of membership , and worry about unaccompanied children having to cross main roads . nevertheless , the report was optimistic that over a six- to ten - year period , the health service which the centre provided could become self - financing . it was not just members that the centre sought , however ; it wanted to gain official recognition and public support for its work and so qualify for grants for various aspects of its research : medical , biological , social and educational . to this end it had been in touch with the medical research council ( mrc ) , who were considering the nature of [ the centre s ] material with a view to grants in aid. before the war , the mrc appears to have been well - disposed , although not without some doubts . in a letter to the statistician austin bradford hill , dated 4 may 1939 , mrc secretary sir edward mellanby said that he wanted to help improve the phc s status and find money for it . however , he added the caveat that the work may be of such a nature that it can lead nowhere from a scientific point of view. bradford hill visited the centre and , in a letter to mellanby of 25 may 1939 , said he thought that it was doing very useful work in preventive medicine but that the records were scribbled down so that only the compilers could interpret the entries . he felt that scott williamson needed to develop a clearer research scheme and a well - defined form of record - keeping . on 30 may 1939 , lord de la warr , at the board of education , wrote to mellanby saying that herbert morrison of the london county council ( lcc ) thought the lcc could make a grant to the phc under section 86 of the education act relating to community centres . at this point , there starts to emerge what would become a familiar pattern in the history of the pioneer health centre , with one body expressing its willingness to help the phc if another was also willing to do so . de la warr said that the mrc would be willing to pay the phc to undertake work on its behalf , but only if it could be assured that the centre would continue to exist . a certain obrien at the rockefeller foundation would be willing to recommend help for the centre , but mellanby would need to show that he was officially interested . move at the mrc to help as soon as possible , but that the council could only take action on advice from people like hill and others. in a letter to lord nuffield of 8 june 1939 , de la warr identified another problem which would hamper the phc s quest for funding : the centre s work did not fall within the limits of any recognised public health or education service , and , although this was in itself a valuable feature of that work , it made it hard for the government to act under its statutory powers . it appears that it was largely thanks to de la warr that lord nuffield made his substantial donation to the phc , for in this same letter he described the centre s work as of quite outstanding value and importance. eight days later , scott williamson was able to write to mellanby to say that he had received nuffield s 5000 . de la warr also encouraged thomas jones of the pilgrim trust to put pressure on mellanby , who , in a letter to jones ( 4 july 1939 ) said that the mrc would be only too happy to promote scientific investigation at the phc , but that the effort to get the place going has up to the present been incompatible with the close control necessary for scientific observation. if the pilgrim trust put some money up for this interesting social experiment , the medical research council would probably interest themselves in it and endeavour to make it a more effective place. the pilgrim trust did so , but the outbreak of war intervened and the centre had to close . in any case , the word probably has all the appearance of a let - out . the war over , scott williamson was rapidly back in contact with mellanby , who remained cagey , saying in a typed note dated 7 september 1945 that the mrc would be glad to use for investigation the facilities offered by the centre if a suitable scheme were put forward. presumably none was , for two years later the centre having re - opened in the spring of 1946 the situation was still at stalemate . by this time , j. a. charles of the ministry of health was involved . in reply to a communication from charles , mellanby wrote on 13 august 1947 as follows : we were anxious to help them just before the war but found that all their records were untidy , and we were unable to make use of them for statistical analysis . we then had the idea of asking bradford hill to look into their methods of recording , in order to see whether we could improve them , and this was all agreed upon when war broke out charles thought that the ministry of health might be able to help under the wide powers of section 16 of the national health service act , but first he wanted to know if the mrc was still interested . from this point on mellanby s mrc colleague arthur landsborough thomson was downright dismissive in an undated memorandum referring to a memorandum about the phc which had been forwarded to him with a letter from j. a. charles dated 15 september 1948 . illustrate what the writers mean by research apparently investigation of cases with a view to their treatment. among the material underlined is with the purpose of cultivating health , and to provide for efficient medical attention for these cases what is needed is a small research team to deal with the 80%. here indeed is some evidence for the view of the epidemiologist archibald cochrane that the mrc had a well known bias towards pure research and a limited record of initiating research into problems of immediate importance ; also for lord moynihan s view that the mrc was more concerned with mice than with humans and was aloof from day - to - day medical practice . the mrc thereafter played an important part in stifling the hopes of the pioneer health centre . a confidential document of april 1950 [ no day specified ] about the centre which by then had closed was sent to the lord president of the council by the labour mps freda corbet ( peckham ) , john cooper ( deptford ) and wilfred vernon ( dulwich ) . they argued that the centre s work fell naturally within the mrc s province , quoting , from its 194548 report , its stated aim of promoting scientific investigation for the acquisition of knowledge likely to be of value for the prevention , diagnosis and treatment of disease , and for the maintenance of normal health and full human efficiency. the mrc , they believed , concentrated too much on disease . we believe that the present governors would agree to transfer the whole enterprise to the nation if the medical research council would assume responsibility for its future , and continue the work on the lines already laid down. major vernon invited harold himsworth of the mrc to lunch at the house of commons ( 13 may 1950 ) , and scott williamson wrote to himsworth ten days later enclosing the phc s research programme . however , this included statements unlikely to have strengthened the centre s case . he wrote , for instance , that the experiment had no relation to laboratory experiments in the exact sciences ; that it could not use exact control as in physiology , and that it was not a medical experiment in the sense of studying specific diseases . on the day that scott williamson sent this letter , vernon wrote to lord addison , saying that the mrc was the only body competent to direct the phc s researches . there followed a rapid exchange of letters between himsworth , on the one hand , and scott williamson and vernon on the other . himsworth proved a master of delaying tactics , writing to scott williamson on 30 may 1950 : i think it would be wiser to postpone any discussion as to the possible role of the medical research council in the future scheme , until an approach has been made through the official channels. scott williamson replied the next day that as both the ministry of health and the london county council had found it impossible to use the phc for research into health , the centre would either go up for sale or be given to anyone willing to carry on the work . i would prefer to give the assets of the centre for research purposes into health not for medical research . sorry to have troubled you. scott williamson seems to have been offering the centre to the medical research council in the hope that it would not be used for purposes of medical research . an undated draft by himsworth of a suggested letter that addison might write contained the following comments on the phc s apparently dilatory approach : no precise proposals for research have yet been submitted to the council , which had been unable to discover that investigations which they could justifiably support were in progress. on 15 june 1950 he wrote to addison about the draft and admitted to having made a it used himsworth s draft , but , where himsworth had suggested saying that any proposals from the phc would be considered sympathetically , addison struck out that adverb . according to scott williamson , writing to j.g.s . administrators ( not the government) by which , given the context , he appears to have meant the mrc found the phc a thorn in their flesh ; privately they have told us so . [ ] i am angry that they would not even risk making the p.h.c . a government research station in a letter to olive of 6 february 1952 , we could have got all the support we needed and more from all the research foundations if we had had the goodwill of the l.c.c . and the m.o.h . [ ministry of health]. we shall look later at the role of these other bodies . on the face of it , the point made by the three local mps , quoted above , that the pioneer health centre s work fell within the remit of the mrc , was quite valid . why , then , might the mrc have found the centre a thorn its flesh ? we have seen that the council concentrated its efforts on investigating disease rather than looking at how health might be encouraged and maintained . according to mary langman , everything at the centre that could be measured , was ; but , she asked : [ h]ow do you measure serenity ? [ ] [ t]his was the sort of problem in which the medical research council showed no interest at all. it was in fact one aspect of a broader problem : quantity could be measured , but how was quality to be scientifically assessed ? for we should be clear that scott williamson insisted that he was only interested in pure science , but believed that science in britain was affected by the fell disease the mrc would have been , to scott williamson s eyes , a prime exemplar of this tendency . we saw above that scott williamson held the london county council and the ministry of health accountable for the pioneer health centre s inability to attract funding from research establishments , and material in the london metropolitan archives illustrates how these two bodies responded when the centre approached them for support . the centre did not help its cause by stating explicitly that it was not a medical organisation , but cultural and educative , and that its breadth meant that it was no - one s specific responsibility . this was a point with which sir allen daley , medical officer of health for the lcc , and sir graham savage , the lcc s education officer , wrestled as they tried to solve the problem of the phc s future . writing to a. reginald stamp , chairman of the lcc s health committee ( 10 may 1949 ) , daley said truistically that the phc was doing pioneer work , but was outside the provisions of the 1946 national health service act . perhaps , he suggested , it could be given a grant under section 28 , relating to care and after - care ; or perhaps it could get one under section 53 of the 1944 education act , relating to voluntary organisations maintaining community centres . daley had discussed the matter with savage , who agreed that the phc was doing very good work , but thought it difficult to bring [ the centre ] under any new scheme which the local authority could subsidise. two days later , stamp told j.w . bowen , chairman of the lcc , that the phc insisted on family membership , including payment , and that this was not permissible under the 1946 act . bowen had been approached by lord lindsay , a long - term supporter of the phc , and now responded ( 24 may 1949 ) by saying that the centre was a praiseworthy many - sided social experiment , but that many difficulties [ stood ] in the way of granting assistance to the centre and were likely to prove insuperable. the following month , stamp and daley held an informal conference with c. d. wilson and major short of the phc , at which short said that his chief interest was in the phc s health work . stamp emphasised that voluntary organisations had to conform to the education and national health service acts ; if the centre did not , it was practically impossible for local authorities to make contributions to its work . under the national health service act it would be impossible to provide help if non - members could not be admitted . he suggested that the centre might alter its constitution in the light of the acts , but wilson rejected this option . by february 1950 the centre was again in the position where no - one would jump unless someone else did . in a letter to daley ( 23 february ) , wilson said that no trusts would make a grant unless the lcc and the government would officially guarantee the centre s continuation and development . the lcc claimed that it had made an offer of help to the centre , but scott williamson disputed this in a letter of 7 march . all he had received , he said , was a verbal assurance that the council would help if the phc were prepared to abandon the policies of family membership , a defined locality for the member families and a periodic health overhaul . to have done so would have changed the whole purpose of the centre . on 16 march , the lcc s clerk wrote to scott williamson , emphasising that any financial assistance it might provide would be dependent on the phc s facilities being generally available to the public. scott williamson was adamant that the centre would not alter its constitution , and his reply ( 21 march ) read , in toto : thank you for your letter of 16th march , which clarifies the situation in a most valuable way. it is evident that by then the lcc had its own purposes in mind . reginald stamp ( 9 march 1950 ) sent a memo to the leader of the council expressing impatience with the whole situation and suggesting that the only way to deal with this centre is for us to get into a position to purchase the whole premises for educational and health purposes. however , it looked for a while as though the halley stewart trust might find a continuing scientific purpose for the phc . this charitable body had helped fund the centre from its re - opening in 1946 , and its withdrawal of funding it undertook only limited periods of support had helped precipitate the centre s terminal crisis . in the spring of 1950 it was looking at ways in which the phc might undertake more effective scientific work on a different basis , and it commissioned an interim report by a scientific advisory committee whose members included professor d.v . morris of the mrc s social medicine research unit and dr max rosenheim of university college hospital . the trust wanted to know the centre s scientific value , and the committee s report formed the appendix to a joint report , dated 9 march 1950 , by the lcc s medical officer of health and education officer . the committee felt that the phc did not provide proper facilities for scientific research , on account of its inadequate research staff and its community being probably highly selected. the best plan of development would be to turn the centre into a health centre in terms of the 1946 act , with a research institute of human biology attached and a family club continuing . the research institute would qualify for a grant under section 16 of the act , with research carried out in , and from , the centre , by medical practitioners providing general services , others engaged in those services and others specially appointed. nothing came of this proposal , and the lcc benefited from the phc s governors abandonment of any hope that the centre could continue on its original lines . a public health department memo of 27 april 1950 discussed possible uses of the pioneer health centre building for health services ; it said that the lcc could not give support by subsidising present activities for 600800 families , and it demonstrated a lack of understanding of the centre s purpose by suggesting that if the family club idea is essential , it could be achieved by hiring accommodation for evenings to a voluntary body. this lack of understanding was further demonstrated at a conference at london s county hall on 1 may . major short , mrs iris montagu and jack donaldson represented the phc ; others present included the mp freda corbet , drs godber and fenton from the ministry of health , allen daley , sir graham savage and reginald stamp , who chaired the meeting . stamp felt that the conception of health centres under the 1946 act went a long way to meeting the existing aims of the phc . dr godber said that he could not commit the ministry of health to any decision , but he had no doubt that a good case could be made for some form of medico - social research. allen daley wanted to set up health and community services first , then local gp and dental group practices ; only when these were established could a research institute be considered , but this was unlikely to happen for some time . on 26 wilson wrote to sir howard roberts , clerk of the council at the lcc , to emphasise that the phc s primary objects included positive health , regular health overhauls , family membership and a social field for family use . the institution which the lcc envisaged would not be a continuation of the original experiment and should bear some other name . a letter ( 28 july 1950 ) from herbert morrison to reginald stamp advised him on how to make the phc s representatives bear responsibility for any breaking off of negotiations : they had the choice of either accepting the lcc s offer or of selling off the centre and losing everything that had already been done . the centre s representatives opted for the latter course , and from then on a major concern was the haggling over the price at which the lcc would buy the centre and its assets . one final point worth making concerns the noted health centre on the woodberry down estate in green lanes , north london . in a ministry of health circular ( 3/48 ) , issued on 14 january 1948 , aneurin bevan wrote that he wanted intense expert scrutiny by a special committee or working party to be set up of the likely lines of first experiments in the building and establishment of health centres . at a meeting of the lcc s health committee on 2 march 1948 , it was agreed that the lcc should get an experimental health centre started [ at woodberry down ] as soon as possible. this centre s opening in 1952 provides a somewhat symbolic contrast with the recent closure of the pioneer health centre , demonstrating the lcc s firm sense of obligation to the provisions of the 1946 national health service act . a memorandum from sir allen daley makes it clear that the proposal for a health centre at woodberry down was for gps and public health staff the curative and as we have seen , such an institution was not a health centre in the peckham sense of the term . in their booklet the passing of peckham , published in the summer of 1951 , scott williamson and pearse gave an account of the pioneer health centre s history , purpose and re - opening , and offered various reasons for the problems it faced . the doctors hostility to the direction of post - war politics emerged at the point where they quoted an unnamed director of a foundation as saying that it could not support the phc since to do so this appears to be the general attitude adopted by all foundations at the present time. one would want to know , though , which foundation was being referred to , and whether this was what it really thought or whether it was making excuses . the doctors attributed the centre s passing to its administrative irregularities : it was concerned with studying and cultivating health , not treating disease ; it was based on the family , not the individual ; it was based on a locality , having no open door policy ; it was not free , and it was based on autonomous administration , not conforming to the lines laid down by the ministry . above all , it arose out of an original conception which extends into a region beyond the ambit of present day medicine and hygiene [ italics in original ] , and therefore is not eligible to receive the enabling stamp of authority. they concluded that : a welfare state must be the sole arbiter of its nation s destiny . to maintain its integrity it can brook no influence that comes from outside its own programme of compelling care . it is not yet ready to consider the possibility that the cultivation of order , ease and virtue in society , might prove an even greater power for the welfare of the people than the abiding care of the administrator. a welfare state must be the sole arbiter of its nation s destiny . to maintain its integrity it can brook no influence that comes from outside its own programme of compelling care . it is not yet ready to consider the possibility that the cultivation of order , ease and virtue in society , might prove an even greater power for the welfare of the people than the abiding care of the administrator. scott williamson was prepared to accept that the centre had re - opened too soon after the war ; it was dilapidated and lacked adequate research equipment . but there had been strong pressure to do so , not just from former members , but from influential supporters such as lord horder , sir malcolm stewart and the publisher stanley unwin . with poignantly ironic optimism , unwin opined that when the centre re - opened the universities will fall over themselves . [ ] we know that it will grow and that nothing can stop it. there is evidence , though , that scott williamson did not help his own cause , and that he and innes pearse antagonised other senior figures at the centre by the way they reacted to its problems . in january 1984 , mary langman wrote a long letter to douglas trotter about the phc s history and its demise . as a young man training for ordination trotter had been sent by george macleod , founder of the iona community , to see what was happening at peckham ; macleod was particularly interested in other experiments in community . trotter , who later became an academic theologian at st andrews university , met his future wife at the phc ( they married in 1949 ) and remained committed to the centre for the rest of his life . letter is very frank about the inadequacies of scott williamson and pearse ( whom she nevertheless much admired ) , implying that not all the blame for the phc s problems could be projected on to the welfare state . he was , she said , unable to convince anyone ( ? also himself ) that he was able to cope with the research problem he had opened up even where , which was not often , he was able to make them see what it was . that was , to devise means of assessment and measurement for quality. langman s letter confirms that harold himsworth s suspicion of a split within the pioneer health centre was correct . tactical unwisdom and of the dialogue of the deaf that they conducted with those who tried to help them . the tag that went through my mind was those whom the gods wish to destroy they first make mad . they suffered , in langman s view , from the character flaw of an inability to recognise that any of their colleagues had any essential contribution to make to the experiment. major short , who , as we have noted , at times negotiated on their behalf , considered them megalomaniacs , and r. p. winfrey , who had through the halley stewart trust given the doctors much support over many years , found that at the end he wilson found himself in the position of trying to save the centre in spite of the doctors . minutes of a meeting held on 23 march 1950 provide refer to internal dissensions and misunderstandings which could not be tolerated any longer . langman was open in her private letter to douglas trotter , but prepared to be economical with the truth , as we would now say , where the outside world was concerned . she expressed her relief that jane lewis , who with barbara brookes had published two major articles on the centre in 1983 , got no wind of all this. we should note , though , that lewis and brookes do attribute scott williamson s problems in part to his personality . a letter from him to aneurin bevan ( undated , but perhaps from 1946 , as it refers to bevan s bill ) exhibits a distinctly abrasive and impatient tone . it now appears, he wrote , that my research work which created peckham as a unique and original approach to the social problems of health is not likely to get funds under your bill because it does not fit into orthodox medical research the phc was a research proposition having nothing to do with medicine or the care of the sick , he emphasised ; but how would that have helped persuade bevan that the centre deserved assistance ? scott williamson asked forgiveness for his indignation but we are wasting precious time begging for money to keep the door open in peckham and i would prefer to give time to research original not orthodox ; for we do not fit into any existing line of research. scott williamson particularly resented the criticism that the pioneer health centre was not self - supporting . had it achieved this aim , its independence would have been guaranteed ; but it did not do so , either before or after the war . for scott williamson , living sociological entity with its own inherent rate of growth , that could not be forced . the centre s post - war social environment , though , meant that the rate of growth was likely to be slow . war had dislocated the life of the district and dissipated many of the family - members of the centre. this in itself , though , does not explain why the centre , which was internationally renowned , could not attract sufficient member families to enable it to break even . it may be that the introduction of the nhs , ironically , encouraged people to be less concerned about health because they could now have free treatment for illness ; but that theory would apply only from the summer of 1948 , about eighteen months before the phc closed , by which time it had been open again for more than two years . it may be that adolescents were reluctant to spend their leisure time with their families , and that there was a desire to enjoy the privacy of domestic life after the upheavals of war - time and the collectivism which they necessitated . no definitive explanation , however , can be arrived at . only those peckham families the overwhelming majority who did not join the centre it should be clear by now that the nhs can hardly be regarded as the chief cause of the pioneer health centre s demise . we can therefore widen the focus and mention some of the more general features of medicine and science which provided an unsympathetic context for the centre s struggles . in 1935 , the year that the pioneer health centre s modernist building opened , the british postgraduate medical school was established at hammersmith hospital , committed to promoting specialist research in order to advance medical knowledge . its work , according to james le fanu , ensured the style of medicine epitomised by the school clinical science would revolutionise the practice and philosophy of medicine. le fanu contrasts the attitudes of two prominent clinicians , lord horder and sir thomas lewis : a particularly interesting choice for our purposes here , since horder was closely involved with the work of the phc . horder was a traditional doctor in the osler mode , excellent at providing a diagnosis based on a patient s history and physical symptoms . the essence of his approach was a personal relationship between doctor and patient , with little or no use of technology . lewis , on the other hand , used electrocardiogram technology to produce a magisterial study of the heartbeat , and paved the way for applying the methods of physiological investigation to human patients . one of his most important followers , john mcmichael , experimented with inserting catheters into the heart to measure the fall in pressure following blood loss . le fanu describes the significance of this as follows : here , then , is the decisive moment when the focus of medicine shifts from the horder view of a professional contract to one where the welfare of the patient is subordinated to the progress of science. in this new world , patients became interesting clinical material on whom the ambitious young doctor performs his experiments with a view to publication in a prestigious medical journal. one consequence of this approach was that specialist research gained enormously in status . another factor posing problems for the phc s work was the growing influence of statistical analysis in medical science . as we saw earlier , interpretation of the centre s records had been a matter for discussion before the second world war , with the medical research council involving austin bradford hill in examining the data . in 1945 , bradford hill became professor of medical statistics at the london school of hygiene and tropical medicine , and honorary director of the mrc s statistical research unit , at a time when the importance of statistics was growing rapidly . his classic textbook quotes helmholtz : all science is measurement. hill believed that records were without value unless kept as part of a planned experiment , and that random sampling was essential . winner had made a similar point in the journal scientific worker at the time of the centre s closure . winner argued that the peckham experiment ( 1943 ) by innes pearse and lucy crocker , was full of woolly statements about a variety of sociological topics which would not stand scientific examination. there had been no statistical sampling , whereas nearly all academic centres of social medicine were chiefly concerned with statistical studies . more was exactly what scott williamson was hoping could be achieved , when he approached the medical research council . ) sir henry dale praised the calm and ruthless logic of the statistical method for giving a higher level of scientific authority to the planning of investigations and the appraisement of their results ; this branch of science had quietly but irresistibly come to exercise a corrective influence , substituting a numerical measure for the vague and speculative methods of appraisement which formerly prevailed. the phc s work was inadequate according to these criteria , as a particular definition and understanding of science began to overshadow alternatives . the mrc s ethos had been shaped by the dominant influence of walter morley fletcher , who believed that research work required concentration on some particular disease or some special aspect of disease . his approach fostered small specialised committees of a scientific elite able to exert control through the selective support of certain individuals and areas of research at the expense of others , believing that medical practitioners were incapable of co - ordinating scientific activities . landsborough thomson , at the beginning of his two - volume history of the mrc , quotes approvingly what rockefeller s adviser f.t . gates wrote in 1897 : medicine can hardly hope to become a science until it can be endowed , and qualified men enabled to give themselves to uninterrupted study and investigation , on ample salary [ my italics ] , entirely independent of practice. early in the second volume , landsborough thomson is scathing about certain proposals for treatment of tb , but his comments might equally apply to a project such as the pioneer health centre : the promoters tended to stimulate pressure groups of patients and busybodies , anxious to champion the supposed pioneer against the alleged tyranny of the professional closed shop . the disdain is palpable . it is interesting to discover in appendix c of thomson s history , which lists the mrc s research establishments , that more than 30 of these establishments were disbanded when their directors either retired or died . to approach the same issue from a more personal angle , sir peter medawar s memoirs offer an insight into how things worked in the world of the mrc . some time in the summer of 1960 the then administrative head of the medical research council , sir harold himsworth , invited me to lunch at his club , abetting his advocacy by that well known to be exercised by chateau cheval blanc . he put it to me that when the then incumbent sir charles harington retired in 1962 , i should take over the directorship of the national institute for medical research at mill hill. despite landsborough thomson s lofty pretensions to purity of research and the advancement of natural knowledge that might , eventually , and perhaps only indirectly , help towards the solution of practical problems of medicine , it is hard to avoid the suspicion that this was a world of one notable feature of the cultural context in the post - war period was what might be termed a the achievements of science and technology during the first half of the twentieth century created at least among scientists and industrialists a sense that humanity s problems could be solved by the application of ever more exciting and effective discoveries . dr willard dow of dow chemicals provided an example of such optimism on the occasion of his receipt of the chemical industry medal in november 1946 , referring to science s successful interpretation of nature s laws and to industry s successful employment of scientific discoveries for the useful purposes of mankind , which led in turn to the progress of civilisation . nature s immense riches were ready to be tapped by industry , or , as dow expressed it : the industries of the world are standing ready to offer their every assistance. the people who maligned these efforts were , said dow , traitors to civilization and economic parasites who were destined to destroy themselves ; for in an advancing civilization there is less and less room for recalcitrants. a similar mind - set could be found in britain . professor c. n. hinshelwood , president of the chemical society , spoke at the society s centenary dinner in 1947 on how chemistry offered rich gifts for conquering disease , providing food and enabling us to obtain all [ sic ] that made life worth living. the agricultural scientist sir john russell was similarly optimistic . from 1912 until 1943 russell had been the director of rothamsted experimental station in hertfordshire , the chief centre in britain for research into chemical fertilisers . as president of the british association for 1949 , he waxed lyrical about the limitless possibilities of science and how the efforts of chemists could not fail to bear fruit so long as scientists had a sustaining faith , a high purpose in life and unflinching courage to pursue it. russell s views are directly relevant to the history of the pioneer health centre , as he was a leading opponent of the organic school of farming , which during the second world war had started to organise its opposition to industrial - chemical agriculture . jane lewis and barbara brookes refer disparagingly to the fact that some peckham staff members were known to be involved in a related , dubiously scientific venture , called the living soil society , which was founded by lady evelyn balfour. this was in fact the soil association , founded in 1946 , which for seventy years has been the leading body in the promotion of organic food and farming . scott williamson , along with eve balfour ( 18981990 ) and the farmer and racehorse breeder friend sykes ( 18881965 ) of chute , near andover , was one of its three begetters . innes pearse , mary langman and dr kenneth barlow ( 19062000 ) , a coventry gp who worked at peckham for a time in the late 1940s , were founder members of the association ; langman , the daughter of a somerset dairy farmer , ran the farm at bromley common in kent which supplied the phc with fresh humus - grown foodstuffs . in 1943 balfour s book the living soil had appeared , subtitled evidence of the importance to human health of soil vitality , with special reference to post - war planning. the soil association had as its first aim to bring together all those working for a fuller understanding of the vital relationships between soil , plant , animal and man ; in other words , its prime concern was with health , and it believed that it was crucial to investigate whether agricultural methods affected the health of plants , animals and humans . there was some evidence to suggest that the enrichment of soil through the creation of humus did indeed have beneficial effects , and balfour and her colleagues argued that if this were so ( further experimentation being essential ) , then organic farming and the production of humus - grown crops would become a form of health service . a humus - rich soil would produce healthy plants and animals , which would feed an increasingly healthy population . any need for the paraphernalia of sprays , weed - killers and other features of the chemical weaponry would similarly be reduced . chemical fertilisers , with their potential danger to soil bacteria , earthworms , and human health , would become increasingly unnecessary . the centre provided a social soil in which healthy human plants could develop ; the reparative work and expense of treating disease would be reduced . the philosophy of both the phc and the soil association was biological and ecological , looking at processes of growth and at the living systems of which the organisms whether botanical , animal or human were part . it is easy to see why such an approach would have been unpopular with those scientific and commercial interests which stood to gain from applying chemical methods to medicine and food production . in medicine , a prime reason for the mood of chemical triumphalism was the development of the sulphonamide drugs , whose identification in 1933 by the german scientist gerhard domagk , a researcher for the chemical company bayer , was followed by a succession of breakthroughs during the next twenty years . whereas doctors in the 1920s had relatively few remedies to offer , by the 1950s they had at their disposal in the region of two thousand as one new compound followed another . what need , then , for a scientifically doubtful attempt to seek the laws of health , when dangerous infectious illnesses could be killed off by the silver bullets which the pharmaceutical industry was making available ? a speech by the biochemist professor e. c. dodds at the seventieth agm of the society of chemical industry , in july 1951 , exemplified the pride with which the industry regarded itself at this time . dodds outlined the decisive influence of chemical industry on medicine , claiming that it was entirely helpful and beneficial ; he summarised the advances made possible in therapeutics through aspirin , salvarsan , sulphonamides , antibiotics and , particularly , cortisone ; and he rejoiced that certain illnesses , such as pneumonia , had now been relegated to the past . private enterprise and competition provided the medical practitioner with that wonderful array of alternatives which today he is privileged to use. the chemical industry could , thanks to those advances , claim the moral high ground . it was saving lives , curing serious illnesses and helping to grow increased amounts of crops in a world desperately short of food . to start criticising it , or warning of possible but unproven malign side - effects , ran the risk of appearing inhumane and reactionary . the pioneer health centre was swimming against a cultural tide , and it would be more than two decades before a critique of the newly dominant medical model emerged through the writings of , among others , ivan illich , thomas mckeown and ian kennedy . kennedy s the unmasking of medicine condemned the dominant philosophy of medicine for its emphasis on disease , its military metaphors , its neglect of the idea of the patient as a whole person and its separation of the malady from the patient . kennedy wrote of the cachet which doctors hoped to gain from being considered scientists , and attacked the baleful influence of teaching hospitals , with their emphasis on technology . one finds gps expressing similar views in jonathan gathorne - hardy s book doctors , with their criticism of the exclusively scientific nature of medical training , which occurs in hospitals and gives no idea of the work of gps or of their patients lives . it was with the patients lives beyond the hospital or the gp s consulting room that scott williamson , pearse and their colleagues were particularly concerned . the view that the national health service was chiefly to blame for the closure of the pioneer health centre ignores the complexity of the circumstances in which the closure occurred . if any one body is responsible , the medical research council appears to have a strong claim ; but various factors contributed to the centre s demise . scott williamson sought official recognition for the centre in order to find the money that he could not raise from the families of peckham , but his antagonism towards government policy and orthodox science were guaranteed to alienate those whom he approached . nor could he offer a clear philosophical basis for his radical approach to the study of human biology ; his ideas , painstakingly pieced together by innes pearse from innumerable notes , were not published until twelve years after he died , as science , synthesis and sanity ( 1965 ) . this book is regarded as exceptionally difficult to understand even by those favourable to what scott williamson had tried to achieve . scott williamson and pearse wanted to develop a science of ethology ( the study of human health ) as opposed to the centrality in medicine of pathology ( the study of sickness ) ; orthodox medical science , increasingly dominated by pharmaceuticals , technology and statistics , and looking to cure existing illness and disability , was not interested . experiment , did not meet the criteria being established by the medical research council . today , though , its work might well be considered to fall within the parameters of the science of epidemiology , which , according to rodolfo saracci , explores health and disease in connection with a range of factors , from the level of the molecule to the level of society this broad perspective makes epidemiology at the same time a biomedical and a social science. saracci distinguishes between observational and experimental studies or but in the former , epidemiologists observe what happens in a group of people , record health - related events , ask questions , take measurements of the body or on blood specimens , but do not intervene actively in the lives or the environments of the subjects under study. the phc s bias in favour of self - selection would still be problematic , though . as the epidemiologist dr julian tudor hart wrote to dr peter mansfield : if we really are human biologists , we need the discipline of facing up to the needs and wishes of genuinely random samples of the population , rather than self - selected volunteers i realise that much preventive work must depend on volunteer groups , but the population at risk from which they are drawn must remain very important. regrettably , we can never know how the peckham doctors would have responded to this criticism of their methodology .

the pioneer health centre , based in south london before and after the second world war , remains a source of interest for advocates of a positive approach to health promotion in contrast with the treatment of those already ill . its closure in 1950 for lack of funds has been blamed on the then recently established national health service , but this article argues that such an explanation is over - simplified and ignores a number of other factors . the centre had struggled financially during the 1930s and tried to gain support from the medical research council . the council appeared interested in the centre before the war , but was less sympathetic in the 1940s . around the time of its closure and afterwards , the centre was also involved in negotiations with london county council ; these failed because the centre s directors would not accept the changes which the council would have needed to make . unpublished documents reveal that the centre s directors were uncompromising and that their approach to the situation antagonised their colleagues . changes in medical science also worked against the centre . the success of sulphonamide drugs appeared to render preventive medicine less significant , while the development of statistical techniques cast doubt on the centre s experimental methods . the centre was at the heart of the nascent organic farming movement , which opposed the rapid growth of chemical cultivation . but what might be termed chemical triumphalism was on the march in both medicine and agriculture , and the centre was out of tune with the mood of the times .

Introduction Relations with the Medical Research Council Relations with London County Council Tensions within the Pioneer Health Centre The Wider Context: Developments in Medical Science The Wider Context: Chemical Triumphalism Concluding Comments

although it closed its doors in 1950 , the pioneer health centre ( phc ) in peckham , south london ( also known as the peckham health centre or the peckham experiment ) remains a project whose significance and legacy are vigorously contested . on the one hand , there are those who have criticised what they see as its over - ambitious and ill - defined aims ; its lack of scientific and statistical rigour ; its sinister extension of the medical gaze and its essentially conservative conception of the family . on the other , there are those who regard the national health service ( nhs ) as more accurately a sickness service , and seek to establish initiatives actively promoting health rather than attending only to people already unwell . the phc s founders , dr george scott williamson ( 18841953 ) and dr innes h. pearse ( 18891978 ) , were hostile to the nhs , but there are doctors active in the phf who work in the nhs and believe that the service would benefit from a re - consideration of the peckham approach . the centre s staff consisted of a resident medical officer , a social secretary and a housekeeper , while the facilities included a changing room , a consulting room , a bathroom and a club - room . during the centre s three years in queens road , 112 families joined , and the doctors concluded that there were many families who , in the right circumstances , would welcome a service which encouraged health rather than dealing with sickness . scott williamson and pearse therefore closed the first stage of the centre and spent the next six years planning and raising funds for the second , which required an environment providing families with an outlet for all their members . sufficient money was obtained , mainly from private individuals , to begin the project , and the pioneer health centre building , designed by the noted architectural engineer sir owen williams , opened in 1935 in st mary s road , peckham . although many assessments of the pioneer health centre s work have been broadly , and at times strongly , sympathetic to its aims and achievements , it has not been without critics . gruffudd notes the phc s political ambiguity as a retreat from state provision of health care , essentially a form of private medicine which regarded its members as distinctly gendered and autonomous individuals , in contrast with the self - consciously progressive message of finsbury s labour council , which saw the centre s members as citizens for condensing through the social and ideological mechanisms of the health centre. whereas the peckham doctors refused to compromise with the demands of state - led medicine , the finsbury health centre was an initiative by the local state of the type that formed the bedrock on which would be built the welfare state in the 1940s. however , gruffudd concludes by commenting that the pioneer health centre ltd charity has provided continued inspiration for practitioners of community - based medicine and speculating that its ecological brand of conservative modernity [ might ] yet win out over the welfare state that embraced finsbury. it must be emphasised here that the term health centre as used by the peckham doctors had a different meaning from the concept referred to in the 1946 national health service act . there has been a persistent sense that the pioneer health centre offered an approach to health preferable to that which the nhs adopted , and that many of the problems with which the nhs has had to struggle not least its cost could have been avoided had the peckham philosophy been put more widely into practice : a hope which seemed , in the mid-1940s , to have some justification . in the nation target for tomorrow ( whose editorial board included sir william beveridge , john boyd orr and julian huxley ) , the phc was praised for its belief that a doctor s task should be to make people as healthy as possible , not simply to attend to them once they are ill , and for its recognition of the part played by social activities and relationships in total health. the nhs has been a chief suspect or scapegoat for the death of the pioneer health centre , apparently on the basis of the philosophical fallacy post hoc ergo propter hoc , since the centre s demise occurred little more than eighteen months after the nhs was established . an editorial in the medical press and circular saw the phc as not fitting into the pattern of the new service , which it deemed too rigid and inflexible to contain it. mary langman ( 19082004 ) , who was secretary to scott williamson and later ran the centre s farm at bromley common in kent , believed that the centre closed because of post - war inflation and lack of political will : it was an embarrassment to the architects of the national health service , which took no thought for the cultivation of health. in 1974 , the architect peter ray , who designed a field centre for the practical study of human ethology , wrote dramatically in the introduction to his project that the peckham experiment had been swept away on the high tide of socialism which rose after the second world war. former members of the centre , interviewed by john nye in 1984 , said with some bitterness that the government would nt help and that the national health smashed it. this article will argue that the reasons for the phc s closure were more varied and complex than such comments imply , and that the nhs was by no means the chief villain of the piece . drawing on hitherto unseen material from mary langman s papers , it will show that the personalities of scott williamson and pearse contributed to the phc s problems ; it will consider the part played by the medical research council ; it will examine the nature of the debates which took place between the centre and london county council ; and it will suggest some broader medical and cultural reasons why the centre s approach came to be seen as marginal or irrelevant in the post - war period . the simplest possible answer to the question why did the pioneer health centre have to close? the important fact to note is that the centre had always struggled for funds ; its history had been one of begging all the way , according to the first edition of its glossy journal peckham . the executive committee had anticipated that the centre could be run , within three years , at a cost coverable by subscriptions and members expenditure , but this proved not to be so . but there was a very disappointing response to this change : only 5% of those circulated went to visit the centre , and only 2% actually joined . nevertheless , the report was optimistic that over a six- to ten - year period , the health service which the centre provided could become self - financing . it was not just members that the centre sought , however ; it wanted to gain official recognition and public support for its work and so qualify for grants for various aspects of its research : medical , biological , social and educational . to this end it had been in touch with the medical research council ( mrc ) , who were considering the nature of [ the centre s ] material with a view to grants in aid. on 30 may 1939 , lord de la warr , at the board of education , wrote to mellanby saying that herbert morrison of the london county council ( lcc ) thought the lcc could make a grant to the phc under section 86 of the education act relating to community centres . at this point , there starts to emerge what would become a familiar pattern in the history of the pioneer health centre , with one body expressing its willingness to help the phc if another was also willing to do so . a certain obrien at the rockefeller foundation would be willing to recommend help for the centre , but mellanby would need to show that he was officially interested . de la warr also encouraged thomas jones of the pilgrim trust to put pressure on mellanby , who , in a letter to jones ( 4 july 1939 ) said that the mrc would be only too happy to promote scientific investigation at the phc , but that the effort to get the place going has up to the present been incompatible with the close control necessary for scientific observation. if the pilgrim trust put some money up for this interesting social experiment , the medical research council would probably interest themselves in it and endeavour to make it a more effective place. the pilgrim trust did so , but the outbreak of war intervened and the centre had to close . we then had the idea of asking bradford hill to look into their methods of recording , in order to see whether we could improve them , and this was all agreed upon when war broke out charles thought that the ministry of health might be able to help under the wide powers of section 16 of the national health service act , but first he wanted to know if the mrc was still interested . the mrc thereafter played an important part in stifling the hopes of the pioneer health centre . they argued that the centre s work fell naturally within the mrc s province , quoting , from its 194548 report , its stated aim of promoting scientific investigation for the acquisition of knowledge likely to be of value for the prevention , diagnosis and treatment of disease , and for the maintenance of normal health and full human efficiency. we believe that the present governors would agree to transfer the whole enterprise to the nation if the medical research council would assume responsibility for its future , and continue the work on the lines already laid down. himsworth proved a master of delaying tactics , writing to scott williamson on 30 may 1950 : i think it would be wiser to postpone any discussion as to the possible role of the medical research council in the future scheme , until an approach has been made through the official channels. scott williamson replied the next day that as both the ministry of health and the london county council had found it impossible to use the phc for research into health , the centre would either go up for sale or be given to anyone willing to carry on the work . scott williamson seems to have been offering the centre to the medical research council in the hope that it would not be used for purposes of medical research . an undated draft by himsworth of a suggested letter that addison might write contained the following comments on the phc s apparently dilatory approach : no precise proposals for research have yet been submitted to the council , which had been unable to discover that investigations which they could justifiably support were in progress. on the face of it , the point made by the three local mps , quoted above , that the pioneer health centre s work fell within the remit of the mrc , was quite valid . [ ] [ t]his was the sort of problem in which the medical research council showed no interest at all. we saw above that scott williamson held the london county council and the ministry of health accountable for the pioneer health centre s inability to attract funding from research establishments , and material in the london metropolitan archives illustrates how these two bodies responded when the centre approached them for support . writing to a. reginald stamp , chairman of the lcc s health committee ( 10 may 1949 ) , daley said truistically that the phc was doing pioneer work , but was outside the provisions of the 1946 national health service act . bowen , chairman of the lcc , that the phc insisted on family membership , including payment , and that this was not permissible under the 1946 act . bowen had been approached by lord lindsay , a long - term supporter of the phc , and now responded ( 24 may 1949 ) by saying that the centre was a praiseworthy many - sided social experiment , but that many difficulties [ stood ] in the way of granting assistance to the centre and were likely to prove insuperable. stamp emphasised that voluntary organisations had to conform to the education and national health service acts ; if the centre did not , it was practically impossible for local authorities to make contributions to its work . he suggested that the centre might alter its constitution in the light of the acts , but wilson rejected this option . scott williamson was adamant that the centre would not alter its constitution , and his reply ( 21 march ) read , in toto : thank you for your letter of 16th march , which clarifies the situation in a most valuable way. reginald stamp ( 9 march 1950 ) sent a memo to the leader of the council expressing impatience with the whole situation and suggesting that the only way to deal with this centre is for us to get into a position to purchase the whole premises for educational and health purposes. the research institute would qualify for a grant under section 16 of the act , with research carried out in , and from , the centre , by medical practitioners providing general services , others engaged in those services and others specially appointed. nothing came of this proposal , and the lcc benefited from the phc s governors abandonment of any hope that the centre could continue on its original lines . a public health department memo of 27 april 1950 discussed possible uses of the pioneer health centre building for health services ; it said that the lcc could not give support by subsidising present activities for 600800 families , and it demonstrated a lack of understanding of the centre s purpose by suggesting that if the family club idea is essential , it could be achieved by hiring accommodation for evenings to a voluntary body. this centre s opening in 1952 provides a somewhat symbolic contrast with the recent closure of the pioneer health centre , demonstrating the lcc s firm sense of obligation to the provisions of the 1946 national health service act . a memorandum from sir allen daley makes it clear that the proposal for a health centre at woodberry down was for gps and public health staff the curative and as we have seen , such an institution was not a health centre in the peckham sense of the term . in their booklet the passing of peckham , published in the summer of 1951 , scott williamson and pearse gave an account of the pioneer health centre s history , purpose and re - opening , and offered various reasons for the problems it faced . the doctors attributed the centre s passing to its administrative irregularities : it was concerned with studying and cultivating health , not treating disease ; it was based on the family , not the individual ; it was based on a locality , having no open door policy ; it was not free , and it was based on autonomous administration , not conforming to the lines laid down by the ministry . scott williamson was prepared to accept that the centre had re - opened too soon after the war ; it was dilapidated and lacked adequate research equipment . langman s letter confirms that harold himsworth s suspicion of a split within the pioneer health centre was correct . major short , who , as we have noted , at times negotiated on their behalf , considered them megalomaniacs , and r. p. winfrey , who had through the halley stewart trust given the doctors much support over many years , found that at the end he wilson found himself in the position of trying to save the centre in spite of the doctors . it now appears, he wrote , that my research work which created peckham as a unique and original approach to the social problems of health is not likely to get funds under your bill because it does not fit into orthodox medical research the phc was a research proposition having nothing to do with medicine or the care of the sick , he emphasised ; but how would that have helped persuade bevan that the centre deserved assistance ? scott williamson particularly resented the criticism that the pioneer health centre was not self - supporting . it may be that adolescents were reluctant to spend their leisure time with their families , and that there was a desire to enjoy the privacy of domestic life after the upheavals of war - time and the collectivism which they necessitated . only those peckham families the overwhelming majority who did not join the centre it should be clear by now that the nhs can hardly be regarded as the chief cause of the pioneer health centre s demise . we can therefore widen the focus and mention some of the more general features of medicine and science which provided an unsympathetic context for the centre s struggles . in 1935 , the year that the pioneer health centre s modernist building opened , the british postgraduate medical school was established at hammersmith hospital , committed to promoting specialist research in order to advance medical knowledge . le fanu describes the significance of this as follows : here , then , is the decisive moment when the focus of medicine shifts from the horder view of a professional contract to one where the welfare of the patient is subordinated to the progress of science. as we saw earlier , interpretation of the centre s records had been a matter for discussion before the second world war , with the medical research council involving austin bradford hill in examining the data . winner had made a similar point in the journal scientific worker at the time of the centre s closure . early in the second volume , landsborough thomson is scathing about certain proposals for treatment of tb , but his comments might equally apply to a project such as the pioneer health centre : the promoters tended to stimulate pressure groups of patients and busybodies , anxious to champion the supposed pioneer against the alleged tyranny of the professional closed shop . some time in the summer of 1960 the then administrative head of the medical research council , sir harold himsworth , invited me to lunch at his club , abetting his advocacy by that well known to be exercised by chateau cheval blanc . despite landsborough thomson s lofty pretensions to purity of research and the advancement of natural knowledge that might , eventually , and perhaps only indirectly , help towards the solution of practical problems of medicine , it is hard to avoid the suspicion that this was a world of one notable feature of the cultural context in the post - war period was what might be termed a the achievements of science and technology during the first half of the twentieth century created at least among scientists and industrialists a sense that humanity s problems could be solved by the application of ever more exciting and effective discoveries . russell s views are directly relevant to the history of the pioneer health centre , as he was a leading opponent of the organic school of farming , which during the second world war had started to organise its opposition to industrial - chemical agriculture . there was some evidence to suggest that the enrichment of soil through the creation of humus did indeed have beneficial effects , and balfour and her colleagues argued that if this were so ( further experimentation being essential ) , then organic farming and the production of humus - grown crops would become a form of health service . in medicine , a prime reason for the mood of chemical triumphalism was the development of the sulphonamide drugs , whose identification in 1933 by the german scientist gerhard domagk , a researcher for the chemical company bayer , was followed by a succession of breakthroughs during the next twenty years . the pioneer health centre was swimming against a cultural tide , and it would be more than two decades before a critique of the newly dominant medical model emerged through the writings of , among others , ivan illich , thomas mckeown and ian kennedy . the view that the national health service was chiefly to blame for the closure of the pioneer health centre ignores the complexity of the circumstances in which the closure occurred . if any one body is responsible , the medical research council appears to have a strong claim ; but various factors contributed to the centre s demise . today , though , its work might well be considered to fall within the parameters of the science of epidemiology , which , according to rodolfo saracci , explores health and disease in connection with a range of factors , from the level of the molecule to the level of society this broad perspective makes epidemiology at the same time a biomedical and a social science.

traumatic brain injury is a major cause of death and disability in children and adolescents , more so among boys than girls ( kraus et al 1986 ; rivara 1994 ; arnarson and halldorsson 1995 ; emanuelson and wendt 1997 ; jennett 1998 ) . young children are at relatively high risk of minimal and mild traumatic head injuries ( rivara 1994 ; jennett 1998 ; lovasik et al 2001 ) . an increase in the more severe and fatal traumatic brain injuries has been found in late adolescence ( kraus et al 1986 ; rivara 1994 ; kraus and mcarthur 1996 ; jennett 1998 ; lovasik et al 2001 ) . many slight injuries may never reach the attention of healthcare personnel . fortunately , most recorded head injuries are minimal or mild with fast recovery and no apparent complications ( kraus and mcarthur 1996 ) . nonetheless , every head injury may have the potential of leading to serious damage ( jennett 1998 ) . however , it can be problematic , especially in infants and young children , due to less marked clinical signs and different responses to trauma compared with older individuals ( bernardi et al 1993 ; dietrich et al 1993 ; quayle et al 1997 ; greenes and schutzman 1998 ; savitsky and votey 2000 ; schutzman et al 2001 ) . pediatric head injuries , even those considered mild , irrespective of cause , may in some cases have debilitating long - term consequences ( jennett 1998 ) . sometimes the consequences of early brain injury do not fully manifest until adolescence or early adulthood ( brooke 1988 ; eslinger et al 1992 ) . the previously held assumption that young children recover better from brain injury than older children due to developmental plasticity has not been substantiated . to the contrary , early brain damage disrupts normal maturation and development , and neuronal plasticity may not always lead to optimal outcome ( chapman and mckinnon 2000 ) . due to unpredictable , hidden , and sometimes serious consequences , prevention of traumatic pediatric head injuries is imperative . bearing in mind methodological considerations when comparing results ( rivara 1994 ; kraus and mcarthur 1996 ; emanuelson and wendt 1997 ; jennett 1998 ) , previous studies on the epidemiology of pediatric traumatic head injuries have indicated that each geographical area may have its special characteristics with regard to incidence , age and gender distribution , and severity of injury , important from a prophylactic point of view ( kraus et al 1986 ; rivara 1994 ; arnarson and halldorsson 1995 ; kraus and mcarthur 1996 ; emanuelson 1997 ; jennett 1998 ; lovasik et al 2001 ) . at the time of the present study , the icelandic population numbered 262,202 . the environment was diverse , with one major urban area , small towns , villages , and farmland . because of a social security system , icelanders have had good access to comprehensive medical services with health - care insurance for all , including the underprivileged . standard of living is overall similar to the neighboring scandinavian countries . however , working days are longer , for both men and women ( olafsson 1990 ) . compared with the scandinavian countries , iceland has the highest incidence of childhood injuries and pediatric accident mortality rate ( stefansdottir and mogensen 1997 ) . the mortality rate is higher for rural than urban areas ( stefansdottir and mogensen 1997 ) . they may be related to less parental supervision , due to long working hours , certain values and views characteristic of the icelandic population , emphasizing the need for independence and personal freedom , even at a young age , and underestimating environmental hazards ( stefansdottir and mogensen 1997 ) . in spite of the high incidence of childhood injuries in iceland , the annual incidence of hospitalizations due to pediatric head trauma in the reykjavik area has been comparable with other countries ( arnarson and halldorsson 1995 ) . falls have been the most common cause of traumatic head injuries among the youngest children , with an increase in traffic - related injuries with age ( arnarson and halldorsson 1995 ) . in the present study , we had the opportunity to collect information for one year on all recorded cases of pediatric traumatic head injuries nationwide . the study was prospective , which enhanced the control of data collection , classifications , and recordings . a search of the literature did not reveal any other prospective nationwide studies on the incidence of traumatic pediatric head injuries , with the same inclusion criteria , definitions , and methodology for comparison . the aims of the study were to estimate the incidence of recorded head injuries by gender , age , severity , and geographical area . the uniqueness of the study is related to its prospective , nationwide scope , including all recorded traumatic head injuries of different severity in both urban and rural areas . this study comprised all 550 children and adolescents 019 years old , consecutively recorded for head injury , icd-9 850854 ( world health organization [ who ] 1977 ) , at all hospitals , emergency units , and healthcare centers in iceland during the period april 15 1992 to april 14 1993 . table 1 shows the population at risk by gender , age , and geographical area . although by law , icelandic adolescents receive most adult responsibilities at 18 years of age , we decided to have the upper age limit at 19 years instead of 17 . the majority of icelandic adolescents do not complete grammar school or trade school until age 20 and are living with their parents and are still reliant on their support during that time , not in the least when traumatic events occur . at the time of the study , the only neurosurgical unit and the only computed tomography ( ct ) scanners in iceland practically all patients in iceland diagnosed with or suspected of moderate or severe brain injury ( icd-9 851854 ) were brought there by ambulance , helicopter , airplane , or by sea . when the diagnosis and degree of severity was uncertain , expert advice was available by telephone and transport to reykjavik encouraged . by the end of the one - year period , all healthcare institutions outside the city of reykjavik supplied available information from their computerized patient registry regarding age , gender , diagnosis , and residence of head injury patients . in the city of reykjavik , the primary author , through information provided by neurosurgeons , other hospital personnel , and written and computerized hospital records ascertained the information daily . care was taken not to count twice patients who were transferred from one healthcare institution to another . private practitioners or health clinics were not contacted , as they did not provide emergency medical services for patients with traumatic head injuries . data were collected from the icelandic death register ( statistics iceland 2001 ) on patients who received fatal traumatic brain injuries during the same period . included were patients who died after being admitted to hospital and those who died at the scene or during transport to hospital . all patients were classified according to international classification of diseases 9 ( icd-9 ) ( who 1977 ) diagnostic codes 850854 : icd-9 850 denotes concussion ; 851 cerebral laceration and contusion ; 852 subarachnoid , subdural , and extradural hemorrhage following injury ; 853 other and unspecified intracranial hemorrhage following injury ; and 854 intracranial injury of unspecified nature . patients with more than one icd-9 diagnosis were included if at least one of the diagnoses was in the 850854 range . patients with more than one 850854 diagnosis were recorded according to the most serious one . physicians diagnosed concussion following traumatic head injury , based on clinical symptoms , such as loss of or reduced consciousness , confusion , dizziness , somnolence , nausea , or amnesia . 851853 diagnoses were made by neurosurgeons , based on cerebral ct findings . according to icelandic guidelines , concussed patients with glasgow coma scale ( gcs ) ( teasdale and jennett 1974 ) score 15 , no loss of consciousness ( loc ) and no signs of skull fracture or other complications , were not admitted to hospital , but discharged from emergency units with head injury instructions . concussed patients with gcs scores lower than 15 , loc , amnesia , signs of skull fracture , or other complications were hospitalized . referral to cerebral ct was based on neurosurgical consultation and expert opinion on injury severity and complications . concussions ( icd-9 850 ) treated at emergency units and subsequently discharged were classified as minimal head injuries . concussions leading to hospitalization were classified as mild head injuries , and nonfatal icd-9 851854 diagnoses as moderate / severe injuries . a distinction was made between patients living in the reykjavik area , the only major urban area in iceland , including the city of reykjavik and the surrounding towns and suburbs ( total population 019 years : 47,327 ) , and patients living in the rural areas and small towns and villages in other parts of iceland ( total population 019 years : 38,419 ) ( see table 1 ) . the largest town outside the reykjavik area was akureyri with total population of 019 years : 4,903 . a log - linear model with two - way interactions was used for statistical analysis of the data . inspection of residuals supported the validity of the log - linear assumption and showed no alarming outliers . confidence intervals ( ci ) were calculated for all incidences in tables 24 . while total population numbers were high , incidence rates were extremely low . therefore , confidence intervals were computed with the wilson score procedure ( agresti and coull 1998 ) . the icelandic data protection commission , the icelandic medical ethics committee , and the medical directors concerned approved the protocol . permission was obtained from statistics iceland regarding use of data from the icelandic death register . this study comprised all 550 children and adolescents 019 years old , consecutively recorded for head injury , icd-9 850854 ( world health organization [ who ] 1977 ) , at all hospitals , emergency units , and healthcare centers in iceland during the period april 15 1992 to april 14 1993 . table 1 shows the population at risk by gender , age , and geographical area . although by law , icelandic adolescents receive most adult responsibilities at 18 years of age , we decided to have the upper age limit at 19 years instead of 17 . the majority of icelandic adolescents do not complete grammar school or trade school until age 20 and are living with their parents and are still reliant on their support during that time , not in the least when traumatic events occur . at the time of the study , the only neurosurgical unit and the only computed tomography ( ct ) scanners in iceland were located in reykjavik . practically all patients in iceland diagnosed with or suspected of moderate or severe brain injury ( icd-9 851854 ) were brought there by ambulance , helicopter , airplane , or by sea . when the diagnosis and degree of severity was uncertain , expert advice was available by telephone and transport to reykjavik encouraged . by the end of the one - year period , all healthcare institutions outside the city of reykjavik supplied available information from their computerized patient registry regarding age , gender , diagnosis , and residence of head injury patients . in the city of reykjavik , the primary author , through information provided by neurosurgeons , other hospital personnel , and written and computerized hospital records ascertained the information daily . care was taken not to count twice patients who were transferred from one healthcare institution to another . private practitioners or health clinics were not contacted , as they did not provide emergency medical services for patients with traumatic head injuries . data were collected from the icelandic death register ( statistics iceland 2001 ) on patients who received fatal traumatic brain injuries during the same period . included were patients who died after being admitted to hospital and those who died at the scene or during transport to hospital . all patients were classified according to international classification of diseases 9 ( icd-9 ) ( who 1977 ) diagnostic codes 850854 : icd-9 850 denotes concussion ; 851 cerebral laceration and contusion ; 852 subarachnoid , subdural , and extradural hemorrhage following injury ; 853 other and unspecified intracranial hemorrhage following injury ; and 854 intracranial injury of unspecified nature . patients with more than one icd-9 diagnosis were included if at least one of the diagnoses was in the 850854 range . patients with more than one 850854 diagnosis were recorded according to the most serious one . physicians diagnosed concussion following traumatic head injury , based on clinical symptoms , such as loss of or reduced consciousness , confusion , dizziness , somnolence , nausea , or amnesia . 851853 diagnoses were made by neurosurgeons , based on cerebral ct findings . according to icelandic guidelines , concussed patients with glasgow coma scale ( gcs ) ( teasdale and jennett 1974 ) score 15 , no loss of consciousness ( loc ) and no signs of skull fracture or other complications , were not admitted to hospital , but discharged from emergency units with head injury instructions . concussed patients with gcs scores lower than 15 , loc , amnesia , signs of skull fracture , or other complications were hospitalized . referral to cerebral ct was based on neurosurgical consultation and expert opinion on injury severity and complications . concussions ( icd-9 850 ) treated at emergency units and subsequently discharged were classified as minimal head injuries . concussions leading to hospitalization were classified as mild head injuries , and nonfatal icd-9 851854 diagnoses as moderate / severe injuries . a distinction was made between patients living in the reykjavik area , the only major urban area in iceland , including the city of reykjavik and the surrounding towns and suburbs ( total population 019 years : 47,327 ) , and patients living in the rural areas and small towns and villages in other parts of iceland ( total population 019 years : 38,419 ) ( see table 1 ) . the largest town outside the reykjavik area was akureyri with total population of 019 years : 4,903 . a log - linear model with two - way interactions was used for statistical analysis of the data . inspection of residuals supported the validity of the log - linear assumption and showed no alarming outliers . confidence intervals ( ci ) were calculated for all incidences in tables 24 . while total population numbers were high , incidence rates were extremely low . therefore , confidence intervals were computed with the wilson score procedure ( agresti and coull 1998 ) . the icelandic data protection commission , the icelandic medical ethics committee , and the medical directors concerned approved the protocol . permission was obtained from statistics iceland regarding use of data from the icelandic death register . table 2 shows the annual incidence with calculated confidence intervals of minimal , mild , moderate / severe , and fatal traumatic head injury by gender and age . the total annual incidence of head injuries was 6.4 ( ci 5.9 , 7.0 ) per 1000 population . boys ( 7.1 per 1000 ) were more likely to sustain head injury than girls ( 5.7 per 1000 ) ( = 9.987 , df = 1 , p = 0.002 ) . there was an interaction between age and severity ( = 24.920 , df = 9 , p = 0.003 ) , mainly due to a relatively high incidence of minimal head injuries among the youngest children . table 3 provides information on the incidence of traumatic head injuries in the city of reykjavik and the surrounding urban area compared with the more rural areas of iceland . the results show an interaction between place of residence and severity ( = 37.799 , df = 3 , p = 0.000 ) . considering the confidence intervals , there was clear evidence of a significant difference between minimal head injuries in the reykjavik area and in rural areas . this was not so for mild , moderate / severe , or fatal injuries ( table 3 ) . in rural areas , age - related differences were less marked than in the reykjavik area , although not statistically significant . clinically this rural urban difference was most striking in the youngest age group and related to minimal head injuries ( tables 3 and 4 ) . no three - way interactions of age , gender , severity , and residence were significant . in the reykjavik area , 49% of the head injured patients were admitted during the six winter months , october to march . in this one - year nationwide sample in the 019 years age range , the total incidence of traumatic head injuries was 6.4 ( ci 5.9 , 7.0 ) per 1000 population . the national incidence of mild , moderate / severe , and fatal head injuries was 1.8 ( ci 1.5 , 2.1 ) per 1000 population . this compared well with the average annual incidence in the reykjavik area 19871991 , and in neighboring countries , while the incidence of minimal head injury , 4.7 ( ci 4.2 , 5.1 ) per 1000 was considerably lower ( arnarson and halldorsson 1995 ) . the incidence of traumatic head injuries was lower in rural ( 3.7 , ci 3.1 , 4.3 per 1000 ) than urban ( 8.6 , ci 7.9 , 9.5 per 1000 ) areas , predominantly due to relatively few recorded minimal head injuries . the incidence of minimal head injuries was 1.9 ( ci 1.5 , 2.4 ) per 1000 in rural areas , but 6.9 ( ci 6.2 , 7.7 ) in the reykjavik area . as age differences were less marked outside the reykjavik area , young head injured children may have been less likely to be brought to medical attention than were older children . studies have shown higher rates of the more severe head trauma and fatal injuries in rural compared with urban areas ( vane and shackford 1995 ; triebel et al 1998 ; zietlow and swanson 1999 ; reid et al 2001 ; eberhardt and pamuk 2004 ) . as the incidence of mild , moderate / severe , and fatal brain injuries was comparable to the reykjavik area , it may be inferred that children and adolescents in rural areas with minimal head injuries were less likely to be brought to the attention of medical personnel and receive diagnosis and treatment . the reasons for this may be related to cultural , parental , socio - economic status , and seasonal factors , as well as accessibility to healthcare services . head trauma that seems to be minimal or mild in the acute phase can lead to intracranial injury and long - term consequences ( tulipan 1998 ; schutzman and greenes 2001 ) . these injuries can be difficult to detect in the acute phase , especially in young children and infants , calling for careful observation of clinical signs , and in selected cases radiographic imaging ( greenes and schutzman 1998 ; schutzman et al 2001 ) . delayed identification of intracranial injuries can lead to secondary injuries due to intracranial hemorrhage , cerebral swelling and elevated intracranial pressure , causing progressive irreversible brain damage , permanent disability , and death ( dietrich et al 1993 ; savitsky and votey 2000 ) . due to the increased likelihood of delayed emergency services in rural areas ( olafsson and sigurdsson 2000 ) , , we have no evidence suggesting that a low incidence of recorded minimal head injuries in rural areas led to increased morbidity or mortality . nonetheless , the findings presented have implications for public health - care services . in rural areas , there may be increased need for providing information on dangers related to primary and secondary brain injuries and to emphasize preventive strategies . people should be made aware of clinical symptoms , signs of deterioration , and the effects of repeated minimal or mild head injuries . caregivers should be encouraged to seek medical evaluation regarding acute pediatric head injuries and ensure easy and fast access to services . because of the possibly long - term consequences of head injuries , healthcare personnel should keep records of medical advice provided via telephone regarding such instances . there was an increased risk for minimal traumatic head injury in the 04 year age range . the reasons for this may be cultural , lack of parental supervision ( stefansdottir and mogensen 1997 ) , and related to physical characteristics of young children ( brudvik 2000 ) . for mild , moderate / severe , and fatal head injuries , young age was not a specific risk factor , and there was not a marked increase in incidence in the oldest age group , which has been found in the usa ( kraus et al 1986 ; rivara 1994 ; kraus and mcarthur 1996 ; lovasik et al 2001 ) . increased parental supervision , safety awareness and safer environment for infants and young children should be encouraged . this is a common finding in similar studies , in both iceland and elsewhere ( kraus et al 1986 ; rivara 1994 ; arnarson and halldorsson 1995 ; emanuelson and wendt 1997 ; jennett 1998 ) , and may reflect a behavioral pattern and increased exposure to environmental hazards related to the male gender ( rivara 1994 ) . the data presented show the value of using a nationwide estimate of the incidence of pediatric head injury , to highlight important differences between urban and rural areas as well as between different age groups . these differences , important for public health planning , may be missed in studies relying solely on local samples . in the present study , information on the causes of traumatic head injuries was not available for the total group of patients . the study took place prior to a formal introduction and implementation of the head injury severity scale ( hiss ) ( stein and spettell 1995 ) and the scandinavian guidelines for the initial management of head injuries ( ingebrigtsen et al 2000 ) . in the present study , the criteria for hospital admissions following concussion are identical to those suggested by hiss and the scandinavian guidelines , but due to lack of detailed information related to injury severity for the total group corresponding criteria for mild and moderate head injuries could not be adopted . in recent years , there has been increased awareness in iceland regarding the importance of injury prevention and safety measures . in 1999 , legislation was passed requiring children under 15 years of age to wear helmets when riding bicycles . in spite of this , there are indications that the annual incidence of moderate , severe , and fatal traumatic brain injuries has been stable in the reykjavik area from 1990 until 2006 . the design of the present study , including a nationwide sample of all recorded instances of pediatric traumatic head injuries of different severity during a one - year period , provides the opportunity to study the long - term consequences of such injuries . in the present study , information on the causes of traumatic head injuries was not available for the total group of patients . the study took place prior to a formal introduction and implementation of the head injury severity scale ( hiss ) ( stein and spettell 1995 ) and the scandinavian guidelines for the initial management of head injuries ( ingebrigtsen et al 2000 ) . in the present study , the criteria for hospital admissions following concussion are identical to those suggested by hiss and the scandinavian guidelines , but due to lack of detailed information related to injury severity for the total group corresponding criteria for mild and moderate head injuries could not be adopted . in recent years , there has been increased awareness in iceland regarding the importance of injury prevention and safety measures . in 1999 , legislation was passed requiring children under 15 years of age to wear helmets when riding bicycles . in spite of this , there are indications that the annual incidence of moderate , severe , and fatal traumatic brain injuries has been stable in the reykjavik area from 1990 until 2006 . the design of the present study , including a nationwide sample of all recorded instances of pediatric traumatic head injuries of different severity during a one - year period , provides the opportunity to study the long - term consequences of such injuries . urban / rural differences are to be expected . there may be urban / rural differences regarding the discrepancy between actual and recorded traumatic head injury incidences . the findings of the present study have implications for public health policy and practice in rural areas , where increased awareness regarding minimal pediatric traumatic head injuries should be encouraged . preventive effort should consider geographical location , age , gender , and cultural and socio - economic factors .

aimsto estimate differences in the incidence of recorded traumatic head injuries by gender , age , severity , and geographical area.methodsthe study was prospective and nationwide . data were collected from all hospitals , emergency units and healthcare centers in iceland regarding all icelandic children and adolescents 019 years old consecutively diagnosed with traumatic head injuries ( n = 550 ) during a one - year period.resultsannual incidence of minimal , mild , moderate / severe , and fatal head injuries ( icd-9 850854 ) was 6.41 per 1000 , with 95% confidence interval ( ci ) 5.9 , 7.0 . annual incidence of minimal head injuries ( icd-9 850 ) treated at emergency units was 4.65 ( ci 4.2 , 5.1 ) per 1000 , mild head injuries admitted to hospital ( icd-9 850 ) was 1.50 ( ci 1.3 , 1.8 ) per 1000 , and moderate / severe nonfatal injuries ( icd-9 851854 ) was 0.21 ( ci 0.1 , 0.3 ) per 1000 . death rate was 0.05 ( ci 0.0 , 0.1 ) per 1000 . young children were at greater risk of sustaining minimal head injuries than older ones . boys were at greater risk than girls were . in rural areas , incidence of recorded minimal head injuries was low.conclusionsuse of nationwide estimate of the incidence of pediatric head injury shows important differences between urban and rural areas as well as between different age groups .

Introduction Material and methods Patients Procedures Classifications Statistical analysis Ethics Results Discussion Limitations and future directions Conclusion

traumatic brain injury is a major cause of death and disability in children and adolescents , more so among boys than girls ( kraus et al 1986 ; rivara 1994 ; arnarson and halldorsson 1995 ; emanuelson and wendt 1997 ; jennett 1998 ) . young children are at relatively high risk of minimal and mild traumatic head injuries ( rivara 1994 ; jennett 1998 ; lovasik et al 2001 ) . an increase in the more severe and fatal traumatic brain injuries has been found in late adolescence ( kraus et al 1986 ; rivara 1994 ; kraus and mcarthur 1996 ; jennett 1998 ; lovasik et al 2001 ) . nonetheless , every head injury may have the potential of leading to serious damage ( jennett 1998 ) . pediatric head injuries , even those considered mild , irrespective of cause , may in some cases have debilitating long - term consequences ( jennett 1998 ) . the previously held assumption that young children recover better from brain injury than older children due to developmental plasticity has not been substantiated . due to unpredictable , hidden , and sometimes serious consequences , prevention of traumatic pediatric head injuries is imperative . bearing in mind methodological considerations when comparing results ( rivara 1994 ; kraus and mcarthur 1996 ; emanuelson and wendt 1997 ; jennett 1998 ) , previous studies on the epidemiology of pediatric traumatic head injuries have indicated that each geographical area may have its special characteristics with regard to incidence , age and gender distribution , and severity of injury , important from a prophylactic point of view ( kraus et al 1986 ; rivara 1994 ; arnarson and halldorsson 1995 ; kraus and mcarthur 1996 ; emanuelson 1997 ; jennett 1998 ; lovasik et al 2001 ) . the environment was diverse , with one major urban area , small towns , villages , and farmland . compared with the scandinavian countries , iceland has the highest incidence of childhood injuries and pediatric accident mortality rate ( stefansdottir and mogensen 1997 ) . they may be related to less parental supervision , due to long working hours , certain values and views characteristic of the icelandic population , emphasizing the need for independence and personal freedom , even at a young age , and underestimating environmental hazards ( stefansdottir and mogensen 1997 ) . in spite of the high incidence of childhood injuries in iceland , the annual incidence of hospitalizations due to pediatric head trauma in the reykjavik area has been comparable with other countries ( arnarson and halldorsson 1995 ) . falls have been the most common cause of traumatic head injuries among the youngest children , with an increase in traffic - related injuries with age ( arnarson and halldorsson 1995 ) . in the present study , we had the opportunity to collect information for one year on all recorded cases of pediatric traumatic head injuries nationwide . the study was prospective , which enhanced the control of data collection , classifications , and recordings . a search of the literature did not reveal any other prospective nationwide studies on the incidence of traumatic pediatric head injuries , with the same inclusion criteria , definitions , and methodology for comparison . the aims of the study were to estimate the incidence of recorded head injuries by gender , age , severity , and geographical area . the uniqueness of the study is related to its prospective , nationwide scope , including all recorded traumatic head injuries of different severity in both urban and rural areas . this study comprised all 550 children and adolescents 019 years old , consecutively recorded for head injury , icd-9 850854 ( world health organization [ who ] 1977 ) , at all hospitals , emergency units , and healthcare centers in iceland during the period april 15 1992 to april 14 1993 . table 1 shows the population at risk by gender , age , and geographical area . although by law , icelandic adolescents receive most adult responsibilities at 18 years of age , we decided to have the upper age limit at 19 years instead of 17 . at the time of the study , the only neurosurgical unit and the only computed tomography ( ct ) scanners in iceland practically all patients in iceland diagnosed with or suspected of moderate or severe brain injury ( icd-9 851854 ) were brought there by ambulance , helicopter , airplane , or by sea . by the end of the one - year period , all healthcare institutions outside the city of reykjavik supplied available information from their computerized patient registry regarding age , gender , diagnosis , and residence of head injury patients . in the city of reykjavik , the primary author , through information provided by neurosurgeons , other hospital personnel , and written and computerized hospital records ascertained the information daily . private practitioners or health clinics were not contacted , as they did not provide emergency medical services for patients with traumatic head injuries . data were collected from the icelandic death register ( statistics iceland 2001 ) on patients who received fatal traumatic brain injuries during the same period . included were patients who died after being admitted to hospital and those who died at the scene or during transport to hospital . all patients were classified according to international classification of diseases 9 ( icd-9 ) ( who 1977 ) diagnostic codes 850854 : icd-9 850 denotes concussion ; 851 cerebral laceration and contusion ; 852 subarachnoid , subdural , and extradural hemorrhage following injury ; 853 other and unspecified intracranial hemorrhage following injury ; and 854 intracranial injury of unspecified nature . patients with more than one icd-9 diagnosis were included if at least one of the diagnoses was in the 850854 range . physicians diagnosed concussion following traumatic head injury , based on clinical symptoms , such as loss of or reduced consciousness , confusion , dizziness , somnolence , nausea , or amnesia . according to icelandic guidelines , concussed patients with glasgow coma scale ( gcs ) ( teasdale and jennett 1974 ) score 15 , no loss of consciousness ( loc ) and no signs of skull fracture or other complications , were not admitted to hospital , but discharged from emergency units with head injury instructions . concussions ( icd-9 850 ) treated at emergency units and subsequently discharged were classified as minimal head injuries . concussions leading to hospitalization were classified as mild head injuries , and nonfatal icd-9 851854 diagnoses as moderate / severe injuries . a distinction was made between patients living in the reykjavik area , the only major urban area in iceland , including the city of reykjavik and the surrounding towns and suburbs ( total population 019 years : 47,327 ) , and patients living in the rural areas and small towns and villages in other parts of iceland ( total population 019 years : 38,419 ) ( see table 1 ) . the largest town outside the reykjavik area was akureyri with total population of 019 years : 4,903 . inspection of residuals supported the validity of the log - linear assumption and showed no alarming outliers . confidence intervals ( ci ) were calculated for all incidences in tables 24 . while total population numbers were high , incidence rates were extremely low . permission was obtained from statistics iceland regarding use of data from the icelandic death register . this study comprised all 550 children and adolescents 019 years old , consecutively recorded for head injury , icd-9 850854 ( world health organization [ who ] 1977 ) , at all hospitals , emergency units , and healthcare centers in iceland during the period april 15 1992 to april 14 1993 . table 1 shows the population at risk by gender , age , and geographical area . the majority of icelandic adolescents do not complete grammar school or trade school until age 20 and are living with their parents and are still reliant on their support during that time , not in the least when traumatic events occur . at the time of the study , the only neurosurgical unit and the only computed tomography ( ct ) scanners in iceland were located in reykjavik . practically all patients in iceland diagnosed with or suspected of moderate or severe brain injury ( icd-9 851854 ) were brought there by ambulance , helicopter , airplane , or by sea . by the end of the one - year period , all healthcare institutions outside the city of reykjavik supplied available information from their computerized patient registry regarding age , gender , diagnosis , and residence of head injury patients . in the city of reykjavik , the primary author , through information provided by neurosurgeons , other hospital personnel , and written and computerized hospital records ascertained the information daily . private practitioners or health clinics were not contacted , as they did not provide emergency medical services for patients with traumatic head injuries . data were collected from the icelandic death register ( statistics iceland 2001 ) on patients who received fatal traumatic brain injuries during the same period . included were patients who died after being admitted to hospital and those who died at the scene or during transport to hospital . all patients were classified according to international classification of diseases 9 ( icd-9 ) ( who 1977 ) diagnostic codes 850854 : icd-9 850 denotes concussion ; 851 cerebral laceration and contusion ; 852 subarachnoid , subdural , and extradural hemorrhage following injury ; 853 other and unspecified intracranial hemorrhage following injury ; and 854 intracranial injury of unspecified nature . patients with more than one icd-9 diagnosis were included if at least one of the diagnoses was in the 850854 range . physicians diagnosed concussion following traumatic head injury , based on clinical symptoms , such as loss of or reduced consciousness , confusion , dizziness , somnolence , nausea , or amnesia . according to icelandic guidelines , concussed patients with glasgow coma scale ( gcs ) ( teasdale and jennett 1974 ) score 15 , no loss of consciousness ( loc ) and no signs of skull fracture or other complications , were not admitted to hospital , but discharged from emergency units with head injury instructions . concussions ( icd-9 850 ) treated at emergency units and subsequently discharged were classified as minimal head injuries . concussions leading to hospitalization were classified as mild head injuries , and nonfatal icd-9 851854 diagnoses as moderate / severe injuries . a distinction was made between patients living in the reykjavik area , the only major urban area in iceland , including the city of reykjavik and the surrounding towns and suburbs ( total population 019 years : 47,327 ) , and patients living in the rural areas and small towns and villages in other parts of iceland ( total population 019 years : 38,419 ) ( see table 1 ) . the largest town outside the reykjavik area was akureyri with total population of 019 years : 4,903 . a log - linear model with two - way interactions was used for statistical analysis of the data . confidence intervals ( ci ) were calculated for all incidences in tables 24 . while total population numbers were high , incidence rates were extremely low . permission was obtained from statistics iceland regarding use of data from the icelandic death register . table 2 shows the annual incidence with calculated confidence intervals of minimal , mild , moderate / severe , and fatal traumatic head injury by gender and age . the total annual incidence of head injuries was 6.4 ( ci 5.9 , 7.0 ) per 1000 population . boys ( 7.1 per 1000 ) were more likely to sustain head injury than girls ( 5.7 per 1000 ) ( = 9.987 , df = 1 , p = 0.002 ) . there was an interaction between age and severity ( = 24.920 , df = 9 , p = 0.003 ) , mainly due to a relatively high incidence of minimal head injuries among the youngest children . table 3 provides information on the incidence of traumatic head injuries in the city of reykjavik and the surrounding urban area compared with the more rural areas of iceland . considering the confidence intervals , there was clear evidence of a significant difference between minimal head injuries in the reykjavik area and in rural areas . this was not so for mild , moderate / severe , or fatal injuries ( table 3 ) . in rural areas , age - related differences were less marked than in the reykjavik area , although not statistically significant . clinically this rural urban difference was most striking in the youngest age group and related to minimal head injuries ( tables 3 and 4 ) . no three - way interactions of age , gender , severity , and residence were significant . in the reykjavik area , 49% of the head injured patients were admitted during the six winter months , october to march . in this one - year nationwide sample in the 019 years age range , the total incidence of traumatic head injuries was 6.4 ( ci 5.9 , 7.0 ) per 1000 population . the national incidence of mild , moderate / severe , and fatal head injuries was 1.8 ( ci 1.5 , 2.1 ) per 1000 population . this compared well with the average annual incidence in the reykjavik area 19871991 , and in neighboring countries , while the incidence of minimal head injury , 4.7 ( ci 4.2 , 5.1 ) per 1000 was considerably lower ( arnarson and halldorsson 1995 ) . the incidence of traumatic head injuries was lower in rural ( 3.7 , ci 3.1 , 4.3 per 1000 ) than urban ( 8.6 , ci 7.9 , 9.5 per 1000 ) areas , predominantly due to relatively few recorded minimal head injuries . the incidence of minimal head injuries was 1.9 ( ci 1.5 , 2.4 ) per 1000 in rural areas , but 6.9 ( ci 6.2 , 7.7 ) in the reykjavik area . studies have shown higher rates of the more severe head trauma and fatal injuries in rural compared with urban areas ( vane and shackford 1995 ; triebel et al 1998 ; zietlow and swanson 1999 ; reid et al 2001 ; eberhardt and pamuk 2004 ) . as the incidence of mild , moderate / severe , and fatal brain injuries was comparable to the reykjavik area , it may be inferred that children and adolescents in rural areas with minimal head injuries were less likely to be brought to the attention of medical personnel and receive diagnosis and treatment . the reasons for this may be related to cultural , parental , socio - economic status , and seasonal factors , as well as accessibility to healthcare services . head trauma that seems to be minimal or mild in the acute phase can lead to intracranial injury and long - term consequences ( tulipan 1998 ; schutzman and greenes 2001 ) . these injuries can be difficult to detect in the acute phase , especially in young children and infants , calling for careful observation of clinical signs , and in selected cases radiographic imaging ( greenes and schutzman 1998 ; schutzman et al 2001 ) . delayed identification of intracranial injuries can lead to secondary injuries due to intracranial hemorrhage , cerebral swelling and elevated intracranial pressure , causing progressive irreversible brain damage , permanent disability , and death ( dietrich et al 1993 ; savitsky and votey 2000 ) . due to the increased likelihood of delayed emergency services in rural areas ( olafsson and sigurdsson 2000 ) , , we have no evidence suggesting that a low incidence of recorded minimal head injuries in rural areas led to increased morbidity or mortality . in rural areas , there may be increased need for providing information on dangers related to primary and secondary brain injuries and to emphasize preventive strategies . people should be made aware of clinical symptoms , signs of deterioration , and the effects of repeated minimal or mild head injuries . caregivers should be encouraged to seek medical evaluation regarding acute pediatric head injuries and ensure easy and fast access to services . because of the possibly long - term consequences of head injuries , healthcare personnel should keep records of medical advice provided via telephone regarding such instances . there was an increased risk for minimal traumatic head injury in the 04 year age range . the reasons for this may be cultural , lack of parental supervision ( stefansdottir and mogensen 1997 ) , and related to physical characteristics of young children ( brudvik 2000 ) . for mild , moderate / severe , and fatal head injuries , young age was not a specific risk factor , and there was not a marked increase in incidence in the oldest age group , which has been found in the usa ( kraus et al 1986 ; rivara 1994 ; kraus and mcarthur 1996 ; lovasik et al 2001 ) . increased parental supervision , safety awareness and safer environment for infants and young children should be encouraged . the data presented show the value of using a nationwide estimate of the incidence of pediatric head injury , to highlight important differences between urban and rural areas as well as between different age groups . in the present study , information on the causes of traumatic head injuries was not available for the total group of patients . the study took place prior to a formal introduction and implementation of the head injury severity scale ( hiss ) ( stein and spettell 1995 ) and the scandinavian guidelines for the initial management of head injuries ( ingebrigtsen et al 2000 ) . in the present study , the criteria for hospital admissions following concussion are identical to those suggested by hiss and the scandinavian guidelines , but due to lack of detailed information related to injury severity for the total group corresponding criteria for mild and moderate head injuries could not be adopted . in recent years , there has been increased awareness in iceland regarding the importance of injury prevention and safety measures . in spite of this , there are indications that the annual incidence of moderate , severe , and fatal traumatic brain injuries has been stable in the reykjavik area from 1990 until 2006 . the design of the present study , including a nationwide sample of all recorded instances of pediatric traumatic head injuries of different severity during a one - year period , provides the opportunity to study the long - term consequences of such injuries . in the present study , information on the causes of traumatic head injuries was not available for the total group of patients . the study took place prior to a formal introduction and implementation of the head injury severity scale ( hiss ) ( stein and spettell 1995 ) and the scandinavian guidelines for the initial management of head injuries ( ingebrigtsen et al 2000 ) . in the present study , the criteria for hospital admissions following concussion are identical to those suggested by hiss and the scandinavian guidelines , but due to lack of detailed information related to injury severity for the total group corresponding criteria for mild and moderate head injuries could not be adopted . in recent years , there has been increased awareness in iceland regarding the importance of injury prevention and safety measures . in spite of this , there are indications that the annual incidence of moderate , severe , and fatal traumatic brain injuries has been stable in the reykjavik area from 1990 until 2006 . the design of the present study , including a nationwide sample of all recorded instances of pediatric traumatic head injuries of different severity during a one - year period , provides the opportunity to study the long - term consequences of such injuries . there may be urban / rural differences regarding the discrepancy between actual and recorded traumatic head injury incidences . the findings of the present study have implications for public health policy and practice in rural areas , where increased awareness regarding minimal pediatric traumatic head injuries should be encouraged . preventive effort should consider geographical location , age , gender , and cultural and socio - economic factors .

cancer patients are unable to continue their lives as it used to be and the appearance of problems in all aspects of personal , familial , and social life eventually leads the quality of life to decrease ( 2 ) . therefore , to prevent such events , palliative care is followed for this group of patients . in fact , palliative care is a comprehensive approach that focuses on improving the quality of life in its final stages and usually it is provided for life - threatening diseases with unpleasant prognosis ( 3 ) . palliative care for cancer patients includes physical , psychological , social , and spiritual dimensions ( 4 ) . the main objective of this type of care is to reduce the suffering through early identification of problems in these patients ( 5 ) . on the other hand , human relationships in this type of care for cancer patients are mainly formed in the framework of nurse - patient relationship . in this respect , mok and chiu ( 2004 ) in their qualitative study in china described that one of the key elements of good palliative care is the nurse - patient relationship . these relationships provided by palliative care not only improve the cancer patients physical and emotional conditions but also facilitate their acceptance of disease , lower their pain , and eventually lead them towards a good experience over the last stages of life ( 6 ) . a phenomenological study carried out in cultural texture of iran also showed that the nurse - patient relationships are associated with altruism and support and in general the concept of care is regarded as sacred ( 7 ) . iran is an ancient country located in the middle east with more than 5000 years of civilization and a population of about 70 millions , mostly muslim ( 95% ) and with a life expectancy of 71 years(8 ) . cancer is the third cause of death in iran , after the heart disease and road accidents as the two leading causes of fatality . cancer occurrence in iran is estimated to be around 48 - 112 and 51 - 144 cases per million for women and men , respectively ( 9 ) . as mentioned , the rate of cancer is high in iran however , despite widespread medical centers for treating cancer patients , palliative care centers in this country are very limited and at present there is only one palliative care center located in the capital of iran ( tehran ) which is affiliated to tehran university of medical sciences . unfortunately , the wrong cultural beliefs about cancer are still present among people indicating that the cancer taboo has not yet broken and many iranians believe that cancer is equivalent to death and end of life ( 10 ) . according to our literature review , we found that the number of researches in particular those associated with qualitative studies on palliative care of cancer patients in iran is so limited , therefore it seemed rational to the authors of the present study to perform a research in this field . the purpose of our study was to explore the experiences of nurses providing palliative care to cancer patients within the context of iranian culture . a qualitative approach was chosen because it describes lived experiences , gives meaning to them and increases the understanding of human experiences ( 11 ) . hermeneutic phenomenological approach in our study , would allow participants to focus on their life experiences through description of their personal experiences of giving palliative care to cancer patients . van manen(2001 ) presented a phenomenological method based on elements of husserl and heidegger s philosophies ( 12 ) . he proposed 6 research activities for conducting hermeneutic phenomenology research , which are used in this study : activity 1 : returning to the nature of the lived experience . activity 6 : balancing the research context by considering the parts and the whole . during the first activity , the researcher should make sense of a specific experience by posing profound questions and having abiding concern about the subject . in our study , the research question was raised when the second author was really impressed by the experience of giving care to cancer patients . in the second activity , we searched the literature about the lived experiences of individuals from around the world to be able to guide our participants . in the third activity , we used thematic analysis to identify what signified the experience of giving palliative care to cancer patients . in the fourth activity , we provided an exhaustive description of the findings to present the experience of giving palliative care as it emerged . in the fifth activity , we tried to understand the experience of giving palliative care in a more humanitarian sense and not to settle for superficialities and falsities . in the final activity , we simultaneously considered the whole and the contextual data to comprehend the contribution of each part to the formation of the phenomenon . we recruited a purposive sample of 10 baccalaureate nurses ( 7 women and 3 men ) . the nurses ranged from 27 to 44 years of age , and had worked in the cancer wards for 6 - 10 years . the main criterion for inclusion was the experience of giving palliative care to cancer patients , the phenomena under the study . we employed a face - to - face , semi - structured interview method , lasting 40 - 65 minutes , for data collection . second author performed all the interviews in persian and translated them into english after transcription . since 2 participants were inter - viewed twice ( due to their long stories or fatigue ) , a total number of 12 interviews were conducted . at the end of each interview session , each interview was transcribed verbatim by the jet audio lyric maker and converted to rich text format to be compatible with the maxqda software which was used for data management . our interviews were conducted using what is the meaning of giving palliative care to cancer patients ? as the main research question . after the nurses answered the above question , further questions were asked to gain richer data , such as : would you explain more about this ? , what is the meaning of that notion ? , and could you please give me an example in order to help us properly understand your point ? analysis of data was followed by activities 36 of the methodology suggested by van manen . in the activity 3 , each transcription was read several times and a short description was written using a holistic approach ( macro thematic reflection ) . in this interpretive act after understanding the whole , a selective approach was employed to isolate the thematic statements ( micro thematic reflection ) . the transcriptions were hence read repeatedly to identify the statement(s ) that seemed particularly essential to or revealing about the palliative care experience of the nurses . the process of analysis included moving back and forth between the whole and the parts of the transcripts to increase the understanding and compare the text and critical reflection on it . collaborative analysis was then performed by presenting the preliminary findings to the co - researchers . free imaginative variation was employed to differentiate and verify essential and incidental themes . in the activity 4 , we used the art of writing and rewriting to thoughtfully bring the assessed phenomenon into written words . for achieving depth , the results were written exhaustively . in the activity 5 , the researchers tried to deeply understand the phenomenon studied and to avoid superficialities and falsities in the whole process of data analysis . in the activity 6 , using the hermeneutic circle , the researchers repeatedly referred to the whole and the parts of the text to analyze how they were connected . in addition , the interviews transcribed and coded in an iterative fashion because coding in this fashion allows the interviewer to incorporate information learned from previous interviews into subsequent interviews . in our study , all the transcripts were read repeatedly and compared with the audio - taped interviews to confirm the accuracy of the data . the credibility of the results and interpretations were assured through prolonged engagement with the data during all stages of the study . this tactic helped to reduce the effect of any particular researcher s preconceived ideas on the final outcome . further , a member check for validation of the interpretation and classification of the interviews was conducted . the data collection was carried out after obtaining verbal consent and a signed informed consent form from the participants . they had the right to withdraw from the study at any time during or after the interviews and could ask the researchers to return their audio - taped interviews . the interview setting was a quiet location in the palliative care center , on the basis of participant convenience and preference . we recruited a purposive sample of 10 baccalaureate nurses ( 7 women and 3 men ) . the nurses ranged from 27 to 44 years of age , and had worked in the cancer wards for 6 - 10 years . the main criterion for inclusion was the experience of giving palliative care to cancer patients , the phenomena under the study . we employed a face - to - face , semi - structured interview method , lasting 40 - 65 minutes , for data collection . second author performed all the interviews in persian and translated them into english after transcription . since 2 participants were inter - viewed twice ( due to their long stories or fatigue ) , a total number of 12 interviews were conducted . at the end of each interview session , each interview was transcribed verbatim by the jet audio lyric maker and converted to rich text format to be compatible with the maxqda software which was used for data management . our interviews were conducted using what is the meaning of giving palliative care to cancer patients ? as the main research question . after the nurses answered the above question , further questions were asked to gain richer data , such as : would you explain more about this ? , what is the meaning of that notion ? , and could you please give me an example in order to help us properly understand your point ? analysis of data was followed by activities 36 of the methodology suggested by van manen . in the activity 3 , each transcription was read several times and a short description was written using a holistic approach ( macro thematic reflection ) . in this interpretive act , we expressed the overall meaning of the text . after understanding the whole , a selective approach was employed to isolate the thematic statements ( micro thematic reflection ) . the transcriptions were hence read repeatedly to identify the statement(s ) that seemed particularly essential to or revealing about the palliative care experience of the nurses . the process of analysis included moving back and forth between the whole and the parts of the transcripts to increase the understanding and compare the text and critical reflection on it . collaborative analysis was then performed by presenting the preliminary findings to the co - researchers . free imaginative variation was employed to differentiate and verify essential and incidental themes . in the activity 4 , we used the art of writing and rewriting to thoughtfully bring the assessed phenomenon into written words . for achieving depth , the researchers tried to deeply understand the phenomenon studied and to avoid superficialities and falsities in the whole process of data analysis . in the activity 6 , using the hermeneutic circle , the researchers repeatedly referred to the whole and the parts of the text to analyze how they were connected . in addition , the interviews transcribed and coded in an iterative fashion because coding in this fashion allows the interviewer to incorporate information learned from previous interviews into subsequent interviews . in our study , all the transcripts were read repeatedly and compared with the audio - taped interviews to confirm the accuracy of the data . the credibility of the results and interpretations were assured through prolonged engagement with the data during all stages of the study . this tactic helped to reduce the effect of any particular researcher s preconceived ideas on the final outcome . further , a member check for validation of the interpretation and classification of the interviews was conducted . the data collection was carried out after obtaining verbal consent and a signed informed consent form from the participants . they had the right to withdraw from the study at any time during or after the interviews and could ask the researchers to return their audio - taped interviews . the interview setting was a quiet location in the palliative care center , on the basis of participant convenience and preference . in this study , the main theme of human relationships was consisted of two sub - themes including the comprehensive acceptance and psychological support . in other words , the experiences of participants indicated that the human relationships in palliative care given to cancer patients bear two entities of comprehensive acceptance and psychological support together . here are some examples of the interviews presented to give the readers the opportunity to make a better judgment on the results provided with more details . one important aspect of human relationships in the palliative care of cancer patients was the issue of comprehensive acceptance . this is evident in the narratives of the participants : i have accepted the particular conditions of a cancer patient and agreed to deal with this group of patients ; i have admitted that the patients in this place spend the end - stage of their lives ; i have learned that a patient must be accepted as a human . if as a nurse i reject accepting a cancer patient , i should not expect the other members of the public to accept such patient . a cancer patient , even in the last days of life , has feelings indigenous to man , understands the reactions shown by a nurse , and needs the nurse s acceptance . i think this characteristic is specific to cancer patients ward , where a nurse with all her heart and soul understands a human who is facing the final days of life . i used to work in icu and giving care to patients who were also spending the last days of their lives but this place is different because the patients are fully conscious . these expressions were made by a female nurse with 7 years work experience . by working in cancer patients ward i have accepted that it does nt matter if the cancer patient has just one day left to live in the world but what is important is to extend the useful load of this day into one month and even longer for the patient . another aspect of human relationships in the palliative care of cancer patients was related to psychological support . are some examples of psychological support expressed by our participants in their narratives : sometimes cancer patient are looking for someone to trust in order to decrease the load of their excitement through self - expression . for example , one patient came to me seeking for consultation over a problem and asking what decision is better to make to solve the problem i can tell you that i and my colleagues in this place are sponsors for cancer patients and they do not come to us only as a nurse but they refer to us as a sister or a friend and seek counseling . i personally think that i have to support cancer patients and give them necessary guidance . these expressions were made by a female nurse with 8 years of work experience while talking with great enthusiasm . when i listen to a patient and sit next to him , he realizes that there is someone acting as his patron , a person who is the listener of his problems . although many cancer patients of this place know that they will die soon but i think i can help them . sometimes i come to the conclusion that the patient is just one step away from the cemetery but it does not cause me to leave the patient alone , instead , i try to be with patient under any circumstances to increase the patient s tolerance against cancer . one important aspect of human relationships in the palliative care of cancer patients was the issue of comprehensive acceptance . this is evident in the narratives of the participants : i have accepted the particular conditions of a cancer patient and agreed to deal with this group of patients ; i have admitted that the patients in this place spend the end - stage of their lives ; i have learned that a patient must be accepted as a human . if as a nurse i reject accepting a cancer patient , i should not expect the other members of the public to accept such patient . a cancer patient , even in the last days of life , has feelings indigenous to man , understands the reactions shown by a nurse , and needs the nurse s acceptance . i think this characteristic is specific to cancer patients ward , where a nurse with all her heart and soul understands a human who is facing the final days of life . i used to work in icu and giving care to patients who were also spending the last days of their lives but this place is different because the patients are fully conscious . these expressions were made by a female nurse with 7 years work experience . by working in cancer patients ward i have accepted that it does nt matter if the cancer patient has just one day left to live in the world but what is important is to extend the useful load of this day into one month and even longer for the patient . another aspect of human relationships in the palliative care of cancer patients was related to psychological support . are some examples of psychological support expressed by our participants in their narratives : sometimes cancer patient are looking for someone to trust in order to decrease the load of their excitement through self - expression . for example , one patient came to me seeking for consultation over a problem and asking what decision is better to make to solve the problem i can tell you that i and my colleagues in this place are sponsors for cancer patients and they do not come to us only as a nurse but they refer to us as a sister or a friend and seek counseling . i personally think that i have to support cancer patients and give them necessary guidance . these expressions were made by a female nurse with 8 years of work experience while talking with great enthusiasm . when i listen to a patient and sit next to him , he realizes that there is someone acting as his patron , a person who is the listener of his problems . although many cancer patients of this place know that they will die soon but i think i can help them . sometimes i come to the conclusion that the patient is just one step away from the cemetery but it does not cause me to leave the patient alone , instead , i try to be with patient under any circumstances to increase the patient s tolerance against cancer . in present study , the human relationships in palliative care of cancer patients were revealed as the nurses described their experiences . on the other hand , this study introduces the human relationships as a key aspect of experiences found by the iranian nurses while providing palliative care to cancer patients . the experiences of nurses participating in this study suggest that the human relationships as one of the aspects of palliative care for cancer patients consisted of two sub - themes referred as comprehensive acceptance and psychological support . several studies have mentioned the role of human relationships in caring for cancer patients and those at end - stages of life . the results of a qualitative study in the united kingdom showed that the social relationships are considered as the center of palliative care for patients with life - limiting conditions ( 13 ) . also , in another qualitative study carried out in the usa to examine the factors associated with the palliative care offered to patients at end - stage of life , the dominant theme revealed was the issue of relationship ( 14 ) . the first sub - theme in our findings was the comprehensive acceptance . based on the experiences of iranian nurses over human relationships with cancer patients , the nurses comprehensively accepted and understood the cancer patients under any circumstances because of their specific conditions . as abrahm ( 2012 ) also stated , the palliative care for cancer patients is a comprehensive care ( 15 ) . likewise , in another qualitative study , the degree of understanding over the rehabilitation of cancer patients among 21 korean nurses working at cancer wards was investigated . two major themes found in the study were comprehensive nursing activities and active involvement with patients while sharing feelings was demonstrated to be one of the sub - themes of such engagement ( 16 ) . the results of a study involving 14 cancer patients showed that the palliative care for cancer patients is a process of continuous search to know and understand such patients . in addition , our nurses described that the type of palliative care offered to cancer patients must be specific . similarly , in a hermeneutic phenomenological study by valente and teixeira ( 2009 ) in brazil in which 17 nurses participated , it was concluded that the type of caring given to patients at their end - stage of life should be unique and specific ( 18 ) . our study showed that cancer patients trust nurses and love them like their sisters or advisers . in fact , trust is an important element of nurse - patient relationship in the field of palliative care . the results of a qualitative study in china indicated that when relationships between nurses and cancer patients are formed based on trust ; the patients do not consider the nurses only as care providers but regard them as part of their families or a good friend . in addition , the nurses who build up the relationships based on trust , show a holistic approach towards patient care , get familiarity with their unstated needs , and provide them with comfort without being asked to do so(6 ) . the findings of the present study revealed that the nurses who offered palliative care to cancer patients were good listeners . other researchers described that when providing patients with palliative care , it should be noted to consider their personal and social needs as allocating time to listen and understand the patients could provide the opportunity to know them and make a stand for developing a better communications ( 19 ) . in an ethnographic study , a semi - structured in - depth interviews with 10 sri lankan nurses showed that the cancer patients initially need the nurses support to have their physical pain and complications alleviated(20 ) . also , the results of a study in australia confirmed that offering support to cancer patients improves their communications ( 21 ) . the findings of another study showed that cancer patients need to receive support more than other patients ( 22 ) . moreover , another study reported that support is one of the important approaches to cope with cancer and play a crucial role in improving the patients quality of life ( 23 ) . similar to the experiences of iranian nurses concerning the human relationships in palliative care of cancer patients in which the issue of psychological support was emphasized , the qualitative studies conducted in other cultures have also confirmed these findings . in a qualitative study while the korean nurses expressed their experiences associated with giving care to cancer patients , the importance of clinical support for this group of patients was highlighted ( 16 ) . likewise , the results of another study reported from the state demonstrated that providing the cancer patients with support improves their ability to withstand disease ( 24 ) . in a qualitative study carried out in iran also demonstrated that cancer , as a stressful event , affects patients emotionally therefore , the psychological support of cancer patients by nurses is of prime importance ( 10 ) . other researchers in their study also pointed out the emotional role of nurses in caring of cancer patients in israel ( 25 ) . despite the cultural diversity of the studies , similar results are observed therefore it could be concluded that the issue of psychological support is one of the essential elements of palliative care for cancer patients which is globally a focus of attention in nursing but what makes this essential element in the iranian culture different from other cultures is the strength of human relationships in which the members of the society are considered as the organs of one body who share their agony and happiness . these relationships even become stronger when an individual is suffering from an incurable disease . therefore , it seems that , our nurses in the field of palliative care of cancer patients have a single body and this leads them towards comprehensive acceptance and psychological support of cancer patients . our results provide deep understanding of experiences obtained by the iranian nurses over human relationships in palliative care of cancer patients . these findings also show that the relationship between nurses and cancer patients is a close connection in a supportive atmosphere with comprehensive acceptance of patient . on the other hand , given that this is the first study on the palliative care of cancer patients conducted in the cultural context of iran , the results obtained in this study could reveal some aspects of palliative care of cancer patients in this society . this study was performed on a limited number of nurses . the small sample size and the nature of the study however , as with all qualitative researches , the results were not intended to be generalized . nevertheless , the findings of this study add to the body of knowledge in this area .

background : cancer patients require palliative care . aim : the purpose of this study was to explore the experiences of nurses , who provide palliative care for cancer patients , within the context of iranian culture.methods:we conducted a hermeneutic phenomenological study . semi structured in - depth interviews with 10 nurses were audio taped and transcribed . the transcriptions were then analyzed by van manen s method.results:one of the most important themes that emerged was human relationships , which also contained the subthemes of comprehensive acceptance and psychological support.conclusions:the results provide deep understanding of human relationships in palliative care of cancer patients in iran .

1. INTRODUCTION 2. METHODS 2.1. Participants 2.2. Interviews 2.3. Procedure 2.4. Ethical considerations 3. RESULTS 3.1. Comprehensive acceptance 3.2. Psychological support 4. DISCUSSION 5. CONCLUSION

cancer patients are unable to continue their lives as it used to be and the appearance of problems in all aspects of personal , familial , and social life eventually leads the quality of life to decrease ( 2 ) . therefore , to prevent such events , palliative care is followed for this group of patients . in fact , palliative care is a comprehensive approach that focuses on improving the quality of life in its final stages and usually it is provided for life - threatening diseases with unpleasant prognosis ( 3 ) . palliative care for cancer patients includes physical , psychological , social , and spiritual dimensions ( 4 ) . the main objective of this type of care is to reduce the suffering through early identification of problems in these patients ( 5 ) . on the other hand , human relationships in this type of care for cancer patients are mainly formed in the framework of nurse - patient relationship . in this respect , mok and chiu ( 2004 ) in their qualitative study in china described that one of the key elements of good palliative care is the nurse - patient relationship . these relationships provided by palliative care not only improve the cancer patients physical and emotional conditions but also facilitate their acceptance of disease , lower their pain , and eventually lead them towards a good experience over the last stages of life ( 6 ) . a phenomenological study carried out in cultural texture of iran also showed that the nurse - patient relationships are associated with altruism and support and in general the concept of care is regarded as sacred ( 7 ) . iran is an ancient country located in the middle east with more than 5000 years of civilization and a population of about 70 millions , mostly muslim ( 95% ) and with a life expectancy of 71 years(8 ) . cancer is the third cause of death in iran , after the heart disease and road accidents as the two leading causes of fatality . cancer occurrence in iran is estimated to be around 48 - 112 and 51 - 144 cases per million for women and men , respectively ( 9 ) . as mentioned , the rate of cancer is high in iran however , despite widespread medical centers for treating cancer patients , palliative care centers in this country are very limited and at present there is only one palliative care center located in the capital of iran ( tehran ) which is affiliated to tehran university of medical sciences . unfortunately , the wrong cultural beliefs about cancer are still present among people indicating that the cancer taboo has not yet broken and many iranians believe that cancer is equivalent to death and end of life ( 10 ) . according to our literature review , we found that the number of researches in particular those associated with qualitative studies on palliative care of cancer patients in iran is so limited , therefore it seemed rational to the authors of the present study to perform a research in this field . the purpose of our study was to explore the experiences of nurses providing palliative care to cancer patients within the context of iranian culture . a qualitative approach was chosen because it describes lived experiences , gives meaning to them and increases the understanding of human experiences ( 11 ) . hermeneutic phenomenological approach in our study , would allow participants to focus on their life experiences through description of their personal experiences of giving palliative care to cancer patients . van manen(2001 ) presented a phenomenological method based on elements of husserl and heidegger s philosophies ( 12 ) . he proposed 6 research activities for conducting hermeneutic phenomenology research , which are used in this study : activity 1 : returning to the nature of the lived experience . activity 6 : balancing the research context by considering the parts and the whole . in our study , the research question was raised when the second author was really impressed by the experience of giving care to cancer patients . in the second activity , we searched the literature about the lived experiences of individuals from around the world to be able to guide our participants . in the third activity , we used thematic analysis to identify what signified the experience of giving palliative care to cancer patients . in the fourth activity , we provided an exhaustive description of the findings to present the experience of giving palliative care as it emerged . in the fifth activity , we tried to understand the experience of giving palliative care in a more humanitarian sense and not to settle for superficialities and falsities . in the final activity , we simultaneously considered the whole and the contextual data to comprehend the contribution of each part to the formation of the phenomenon . the nurses ranged from 27 to 44 years of age , and had worked in the cancer wards for 6 - 10 years . the main criterion for inclusion was the experience of giving palliative care to cancer patients , the phenomena under the study . since 2 participants were inter - viewed twice ( due to their long stories or fatigue ) , a total number of 12 interviews were conducted . our interviews were conducted using what is the meaning of giving palliative care to cancer patients ? analysis of data was followed by activities 36 of the methodology suggested by van manen . the transcriptions were hence read repeatedly to identify the statement(s ) that seemed particularly essential to or revealing about the palliative care experience of the nurses . the process of analysis included moving back and forth between the whole and the parts of the transcripts to increase the understanding and compare the text and critical reflection on it . free imaginative variation was employed to differentiate and verify essential and incidental themes . in the activity 6 , using the hermeneutic circle , the researchers repeatedly referred to the whole and the parts of the text to analyze how they were connected . in our study , all the transcripts were read repeatedly and compared with the audio - taped interviews to confirm the accuracy of the data . the credibility of the results and interpretations were assured through prolonged engagement with the data during all stages of the study . this tactic helped to reduce the effect of any particular researcher s preconceived ideas on the final outcome . further , a member check for validation of the interpretation and classification of the interviews was conducted . they had the right to withdraw from the study at any time during or after the interviews and could ask the researchers to return their audio - taped interviews . the interview setting was a quiet location in the palliative care center , on the basis of participant convenience and preference . we recruited a purposive sample of 10 baccalaureate nurses ( 7 women and 3 men ) . the nurses ranged from 27 to 44 years of age , and had worked in the cancer wards for 6 - 10 years . the main criterion for inclusion was the experience of giving palliative care to cancer patients , the phenomena under the study . second author performed all the interviews in persian and translated them into english after transcription . at the end of each interview session , each interview was transcribed verbatim by the jet audio lyric maker and converted to rich text format to be compatible with the maxqda software which was used for data management . our interviews were conducted using what is the meaning of giving palliative care to cancer patients ? , what is the meaning of that notion ? analysis of data was followed by activities 36 of the methodology suggested by van manen . in the activity 3 , each transcription was read several times and a short description was written using a holistic approach ( macro thematic reflection ) . in this interpretive act , we expressed the overall meaning of the text . the transcriptions were hence read repeatedly to identify the statement(s ) that seemed particularly essential to or revealing about the palliative care experience of the nurses . the process of analysis included moving back and forth between the whole and the parts of the transcripts to increase the understanding and compare the text and critical reflection on it . collaborative analysis was then performed by presenting the preliminary findings to the co - researchers . free imaginative variation was employed to differentiate and verify essential and incidental themes . for achieving depth , the researchers tried to deeply understand the phenomenon studied and to avoid superficialities and falsities in the whole process of data analysis . in the activity 6 , using the hermeneutic circle , the researchers repeatedly referred to the whole and the parts of the text to analyze how they were connected . in addition , the interviews transcribed and coded in an iterative fashion because coding in this fashion allows the interviewer to incorporate information learned from previous interviews into subsequent interviews . in our study , all the transcripts were read repeatedly and compared with the audio - taped interviews to confirm the accuracy of the data . the credibility of the results and interpretations were assured through prolonged engagement with the data during all stages of the study . further , a member check for validation of the interpretation and classification of the interviews was conducted . they had the right to withdraw from the study at any time during or after the interviews and could ask the researchers to return their audio - taped interviews . the interview setting was a quiet location in the palliative care center , on the basis of participant convenience and preference . in this study , the main theme of human relationships was consisted of two sub - themes including the comprehensive acceptance and psychological support . in other words , the experiences of participants indicated that the human relationships in palliative care given to cancer patients bear two entities of comprehensive acceptance and psychological support together . here are some examples of the interviews presented to give the readers the opportunity to make a better judgment on the results provided with more details . one important aspect of human relationships in the palliative care of cancer patients was the issue of comprehensive acceptance . this is evident in the narratives of the participants : i have accepted the particular conditions of a cancer patient and agreed to deal with this group of patients ; i have admitted that the patients in this place spend the end - stage of their lives ; i have learned that a patient must be accepted as a human . if as a nurse i reject accepting a cancer patient , i should not expect the other members of the public to accept such patient . a cancer patient , even in the last days of life , has feelings indigenous to man , understands the reactions shown by a nurse , and needs the nurse s acceptance . i think this characteristic is specific to cancer patients ward , where a nurse with all her heart and soul understands a human who is facing the final days of life . these expressions were made by a female nurse with 7 years work experience . by working in cancer patients ward i have accepted that it does nt matter if the cancer patient has just one day left to live in the world but what is important is to extend the useful load of this day into one month and even longer for the patient . another aspect of human relationships in the palliative care of cancer patients was related to psychological support . are some examples of psychological support expressed by our participants in their narratives : sometimes cancer patient are looking for someone to trust in order to decrease the load of their excitement through self - expression . for example , one patient came to me seeking for consultation over a problem and asking what decision is better to make to solve the problem i can tell you that i and my colleagues in this place are sponsors for cancer patients and they do not come to us only as a nurse but they refer to us as a sister or a friend and seek counseling . i personally think that i have to support cancer patients and give them necessary guidance . these expressions were made by a female nurse with 8 years of work experience while talking with great enthusiasm . when i listen to a patient and sit next to him , he realizes that there is someone acting as his patron , a person who is the listener of his problems . although many cancer patients of this place know that they will die soon but i think i can help them . sometimes i come to the conclusion that the patient is just one step away from the cemetery but it does not cause me to leave the patient alone , instead , i try to be with patient under any circumstances to increase the patient s tolerance against cancer . one important aspect of human relationships in the palliative care of cancer patients was the issue of comprehensive acceptance . this is evident in the narratives of the participants : i have accepted the particular conditions of a cancer patient and agreed to deal with this group of patients ; i have admitted that the patients in this place spend the end - stage of their lives ; i have learned that a patient must be accepted as a human . if as a nurse i reject accepting a cancer patient , i should not expect the other members of the public to accept such patient . i think this characteristic is specific to cancer patients ward , where a nurse with all her heart and soul understands a human who is facing the final days of life . by working in cancer patients ward i have accepted that it does nt matter if the cancer patient has just one day left to live in the world but what is important is to extend the useful load of this day into one month and even longer for the patient . another aspect of human relationships in the palliative care of cancer patients was related to psychological support . are some examples of psychological support expressed by our participants in their narratives : sometimes cancer patient are looking for someone to trust in order to decrease the load of their excitement through self - expression . for example , one patient came to me seeking for consultation over a problem and asking what decision is better to make to solve the problem i can tell you that i and my colleagues in this place are sponsors for cancer patients and they do not come to us only as a nurse but they refer to us as a sister or a friend and seek counseling . i personally think that i have to support cancer patients and give them necessary guidance . although many cancer patients of this place know that they will die soon but i think i can help them . sometimes i come to the conclusion that the patient is just one step away from the cemetery but it does not cause me to leave the patient alone , instead , i try to be with patient under any circumstances to increase the patient s tolerance against cancer . in present study , the human relationships in palliative care of cancer patients were revealed as the nurses described their experiences . on the other hand , this study introduces the human relationships as a key aspect of experiences found by the iranian nurses while providing palliative care to cancer patients . the experiences of nurses participating in this study suggest that the human relationships as one of the aspects of palliative care for cancer patients consisted of two sub - themes referred as comprehensive acceptance and psychological support . several studies have mentioned the role of human relationships in caring for cancer patients and those at end - stages of life . the results of a qualitative study in the united kingdom showed that the social relationships are considered as the center of palliative care for patients with life - limiting conditions ( 13 ) . also , in another qualitative study carried out in the usa to examine the factors associated with the palliative care offered to patients at end - stage of life , the dominant theme revealed was the issue of relationship ( 14 ) . the first sub - theme in our findings was the comprehensive acceptance . based on the experiences of iranian nurses over human relationships with cancer patients , the nurses comprehensively accepted and understood the cancer patients under any circumstances because of their specific conditions . as abrahm ( 2012 ) also stated , the palliative care for cancer patients is a comprehensive care ( 15 ) . likewise , in another qualitative study , the degree of understanding over the rehabilitation of cancer patients among 21 korean nurses working at cancer wards was investigated . two major themes found in the study were comprehensive nursing activities and active involvement with patients while sharing feelings was demonstrated to be one of the sub - themes of such engagement ( 16 ) . the results of a study involving 14 cancer patients showed that the palliative care for cancer patients is a process of continuous search to know and understand such patients . in addition , our nurses described that the type of palliative care offered to cancer patients must be specific . similarly , in a hermeneutic phenomenological study by valente and teixeira ( 2009 ) in brazil in which 17 nurses participated , it was concluded that the type of caring given to patients at their end - stage of life should be unique and specific ( 18 ) . our study showed that cancer patients trust nurses and love them like their sisters or advisers . in fact , trust is an important element of nurse - patient relationship in the field of palliative care . the results of a qualitative study in china indicated that when relationships between nurses and cancer patients are formed based on trust ; the patients do not consider the nurses only as care providers but regard them as part of their families or a good friend . in addition , the nurses who build up the relationships based on trust , show a holistic approach towards patient care , get familiarity with their unstated needs , and provide them with comfort without being asked to do so(6 ) . the findings of the present study revealed that the nurses who offered palliative care to cancer patients were good listeners . other researchers described that when providing patients with palliative care , it should be noted to consider their personal and social needs as allocating time to listen and understand the patients could provide the opportunity to know them and make a stand for developing a better communications ( 19 ) . in an ethnographic study , a semi - structured in - depth interviews with 10 sri lankan nurses showed that the cancer patients initially need the nurses support to have their physical pain and complications alleviated(20 ) . also , the results of a study in australia confirmed that offering support to cancer patients improves their communications ( 21 ) . the findings of another study showed that cancer patients need to receive support more than other patients ( 22 ) . moreover , another study reported that support is one of the important approaches to cope with cancer and play a crucial role in improving the patients quality of life ( 23 ) . similar to the experiences of iranian nurses concerning the human relationships in palliative care of cancer patients in which the issue of psychological support was emphasized , the qualitative studies conducted in other cultures have also confirmed these findings . in a qualitative study while the korean nurses expressed their experiences associated with giving care to cancer patients , the importance of clinical support for this group of patients was highlighted ( 16 ) . likewise , the results of another study reported from the state demonstrated that providing the cancer patients with support improves their ability to withstand disease ( 24 ) . in a qualitative study carried out in iran also demonstrated that cancer , as a stressful event , affects patients emotionally therefore , the psychological support of cancer patients by nurses is of prime importance ( 10 ) . other researchers in their study also pointed out the emotional role of nurses in caring of cancer patients in israel ( 25 ) . despite the cultural diversity of the studies , similar results are observed therefore it could be concluded that the issue of psychological support is one of the essential elements of palliative care for cancer patients which is globally a focus of attention in nursing but what makes this essential element in the iranian culture different from other cultures is the strength of human relationships in which the members of the society are considered as the organs of one body who share their agony and happiness . these relationships even become stronger when an individual is suffering from an incurable disease . therefore , it seems that , our nurses in the field of palliative care of cancer patients have a single body and this leads them towards comprehensive acceptance and psychological support of cancer patients . our results provide deep understanding of experiences obtained by the iranian nurses over human relationships in palliative care of cancer patients . these findings also show that the relationship between nurses and cancer patients is a close connection in a supportive atmosphere with comprehensive acceptance of patient . on the other hand , given that this is the first study on the palliative care of cancer patients conducted in the cultural context of iran , the results obtained in this study could reveal some aspects of palliative care of cancer patients in this society . this study was performed on a limited number of nurses . the small sample size and the nature of the study however , as with all qualitative researches , the results were not intended to be generalized . nevertheless , the findings of this study add to the body of knowledge in this area .

due to an ever - aging society , increasing healthcare costs are today a major challenge in many countries . hence , the concept of homecare is becoming increasingly important in medical technology and will play an important role in the future . it is expected that outsourcing of healthcare from clinical facilities to home - delivered care will lead to significant reduction of costs . for this purpose , sensor systems are needed which meet specific requirements , since correct application in a home environment may be difficult to control . obstructive sleep apnea ( osa ) is a sleep disorder in which temporary airway narrowing leads to apnea and a decrease in arterial oxygen saturation \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ ( s_{p}o_{2})$\end{document } during sleep . in western countries , diagnosis is usually performed in sequential steps : anamnesis , physical examination , ambulatory cardiorespiratory screening and stationary polysomnography ( psg ) . standardized diagnosis of osa involves many different physiological parameters including eeg , eog , emg , blood oxygen saturation , respiration , excursion of thorax and abdomen , ecg , body position , etc . however , because many sensors need to be attached to the patient , the quality of sleep is reduced which , in turn , can affect the results of the examination . this complex diagnostic procedure for osa is associated with physical stress for the patients and considerable costs for the healthcare system . therefore , we developed an unobtrusive single - sensor system which is suitable for homecare screening of sleep , taking into account the special requirements of the homecare setting . the technique is based on the principle of pulse oximetry , a multi - wavelength version of photoplethymography ( ppg ) , . in contrast to conventional pulse oximeters the sensor is applied to the inner ear . since pulse oximetry is mainly used for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ s_{p}o_{2}$\end{document } and heart rate ( hr ) measurement in routine clinical care , only a fraction of its capability is used . advancements in measurement efficiency makes the coincidental measurement of multiple vital signs like hr , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ s_{p}o_{2}$\end{document } , respiration rate , vasomotion , thm waves and other possible with a single sensor which is noninvasive , unobtrusive and cheap . taking these aspects into account , ppg is probably the best way to a discreet and cost - effective sleep monitoring at home . such a sleep - monitoring sensor could allow diagnosis and even treatment of osa in a familiar environment . this may increase the reliability of the diagnosis , since the quality of sleep is expected to improve due to a significant reduction of sensors , cables and devices . with pre - diagnosis based on nocturnal oximetry , the number of unnecessary psg screenings in clinical sleep laboratories can be minimized and thereby help to reduce healthcare costs . combined with telemedicine technology , such a system can also be used for long - term nocturnal monitoring of patients at risk ( e.g. , those with chronic cardiovascular disease ) and of the elderly . the sensor has been developed in collaboration with the cis institute for microsystems and photovoltaics ( erfurt , germany ) and was first reported in . during human hypoxia trials , we demonstrated in 2012 that a reliable measurement of arterial oxygen saturation is possible . based on those results , we now want to evaluate the feasibility of the in - ear sensor for cardiovascular monitoring during sleep . therefore , trials with the sensor are being performed in patients who suffer from osa ; the results are compared with standard psg data . although the studies are not yet completed , the preliminary results presented here demonstrate the potential of the system for pre - diagnosis of osa patients and/or for nocturnal monitoring of the elderly and patients at risk . photoplethysmography ( greek : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ photo~plethore~graphein = light~volume~record$\end{document } ) is a noninvasive , optical method to visualize sub dermal blood volume . blood volume fluctuations cause changes in the intensity of the measured light and several rhythmic vital parameters can be observed ( i.e. , heart rate ) . by means of different light wavelengths an additional spectral analysis of the blood can be performed . since the red and infrared absorption / reflection spectra of reduced and oxygenated hemoglobin distinguish significantly , ppg is also often used for an easy measurement of blood oxygen saturation ( e.g. , in pulse oximetry ) . especially for long - term monitoring of vital parameters during sleep , since most sensors are attached to finger or earlobe , measurements are often disrupted by motion artifacts like subsultis during the first stage of sleep ( drifting into sleep ) , movements during light sleep or abrupt awakenings from deep sleep . sensors which are limited to these body parts will always be subject to such problems . therefore , we developed a reflective sensor type , which is not limited to peripheral body parts but can be placed in the auditory channel for reduced motion artifacts and a high level of wearing comfort ( fig . 1 ) . in addition , the proximity to the brain guarantees stable perfusion conditions , since the tragus is supplied by an arterial network coming from the arteries temporalis superficialis and arteria auricularis posterior . while shock induced centralization ( e.g. , sepsis , hypothermia ) is known to inhibit peripheral pulse oximetry due to a lack of peripheral perfusion ( ) , the in - ear pulse oximetry is not likely to be affected by this . the sensor is connected to the sensor interface device via a cable which is taped for artifact reduction . the sensor is connected to the sensor interface device via a cable which is taped for artifact reduction . red and infrared diodes ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \lambda_{1}=760~{\rm nm}$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \lambda_{2}=900~{\rm nm}$\end{document } ) and a photodiode are placed side by side , with 2 mm of distance . the major part of the reflected light is attributed to blood cells since the reflection spectrum of hemoglobin predominates the other components for red and near infrared light . direct light crosstalk ( this signal does not carry any information about the local perfusion ) is prevented by an optical barrier between sources and detector . the sensor is sealed into a biocompatible ear mold and measurements are made in the ear channel ( at the tragus ) . the ear mold is individually customized to the patient 's ear ( fig . 1 ) . this ensures a good mechanical fit and an exceptionally high level of wearing comfort : this is a prerequisite for continuous monitoring over several hours . a preliminary check with 10 people wearing the sensor ( non - stop ) for about 40 min resulted in a reported comfort level of ( on average ) 0.75 on a scale from 0 ( imperceptible ) to 10 ( unbearable ) . during the study , the patients accepted the sensor for the entire night ( 57 hours ) and the overall feedback regarding the wearing comfort was positive . anyhow , since technical feasibility was the main focus of this work , we did not investigate in detail if the patients prefer the new sensor design over the conventional finger sensor ; this should be part of future studies . the alternating part ( ac ) of the ppg signal is up to 10 times smaller than the conventional transmissive measurement method . this requires a high resolution digital conversion of the photocurrent which is converted into a proportional voltage by a transimpedance converter . the developed microcontroller - based sensor interface device ( fig . 2 ) provides 24-bit analog - to - digital conversion and a sampling rate of 200 samples / s without previous analog filtering ( apart from anti - aliasing ) . a block diagram of the sensor interface device is shown in fig . 3 . the sensor - led current is regulated by an h - bridge which is combined with two voltage - controlled current sources . the current is regulated by 16 bit d / a outputs of the microcontroller ( msp430f1611 , texas instruments , inc . ) as a function of the detected light intensity , , . 2.the sensor interface device ( height 8.5 cm , width 4.5 cm , depth 2 cm ) . the microcontroller based sensor - pc interface device provides a usb connection for easy pc connection with an ftdi standard driver . the photocurrent is converted into a proportional voltage and directly digitalized by a 24-bit analog - to - digital converter . a microcontroller ( msp430f1611 texas instruments inc . ) provides digital signal processing ( adopted from ) . the sensor interface device ( height 8.5 cm , width 4.5 cm , depth 2 cm ) . the microcontroller based sensor - pc interface device provides a usb connection for easy pc connection with an ftdi standard driver . the photocurrent is converted into a proportional voltage and directly digitalized by a 24-bit analog - to - digital converter . a microcontroller ( msp430f1611 texas instruments inc . ) provides digital signal processing ( adopted from ) . an r - value is defined which takes the alternating ( ac ) and the static ( dc ) components of the red \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ ( \lambda_{1})$\end{document } and infrared \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ ( \lambda_{2})$\end{document } ppg signals into account.\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ $ r={{{\rm i}_{{\rm ac,}\lambda_{1}}}\over{{\rm i}_{{\rm dc,}\lambda_{1}}}}\bigg/{{{\rm i}_{{\rm ac,}\lambda_{2}}}\over{{\rm i}_{{\rm dc,}\lambda_{2}}}}\eqno{\hbox{(1)}}$$\end{document } since \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ s_{p}o_{2}$\end{document } always has a high variance , the r - values are smoothed by a 20-s moving average filter . the correlation between the r - value and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ s_{p}o_{2}$\end{document } was determined based on our human hypoxia studies .\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ $ s_{p}o_{2}=f(r)=33r^{2}-152.6r+176.4+r_{comp}\eqno{\hbox{(2)}}$$\end{document } although a high correlation was confirmed in , global calibration proved to be challenging due to an unexpected effect , which was probably due to the influence of subdermal venous blood , : because of a slight shift in the calibration curve every time a measurement starts , only relative \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ s_{p}o_{2}$\end{document } measurements can be made . nevertheless , this is not a limitation because , with regard to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ s_{p}o_{2}$\end{document } , sleep diagnosis focuses on temporary periods of apnea with a related drop in oxygen saturation . to meet the calibration requirement , an initial \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ s_{p}o_{2}$\end{document } measurement with a reference device can be performed to compensate for the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ s_{p}o_{2}$\end{document } offset by the parameter \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ r_{comp}$\end{document } in ( 2 ) . if the offset is compensated for , an in - ear \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ s_{p}o_{2}$\end{document } measurement with the current sensor design is feasible \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ ( { \rm mean~squared~error}=3.149 , { \rm r}^{2}=0.959)$\end{document } . this ongoing study is performed at the department of neurology at the rwth aachen university hospital ( aachen , germany ) ( clinicaltrials.gov identifier : nct01626274 ) . after patient education and informed consent , ear molding is performed ( kaulard optik & akustik , germany ) to fit the sensor for each patient . the sensor is manufactured at the cis institute for microsystems and photovoltaic gmbh and at audia akustik gmbh ( soemmerda , germany ) . the patients spent one night in the sleep laboratory of the rwth aachen university hospital for a standardized polysomnographic checkup ( diamedic gmbh , germany ) , including ppg , ecg , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ s_{p}o_{2}$\end{document } , respiratory flux and other tests . the sensor is connected to a usb version of the sensor interface device which is connected to a personal computer . the study was approved by the local ethics committee ( ctc - a 10 - 016 ek 231/10 ) and the federal institute for drugs and medical devices ( bfarm ) . so far , 11 patients ( 10 males and 1 female : age range 4859 years ) were included in the study . of these , two patients were excluded due to logistic difficulties during sensor production , another two were excluded due to technical problems , and one patient was physically unable to participate . therefore , 6 evaluable patients are included in this preliminary feasibility analysis . the first trials indicate excellent nocturnal performance of the in - ear sensor ; this success can probably be attributed to considerably reduced motion and the darkness . the signal quality was quantified by the signal - to - noise ratio ( snr).\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ $ snr_{oxi}=10\log\left\{{{\left({\sum_{0.5hz}^{10hz}{psd(f)}}\right)^{2}}\over{\left({\sum_{10hz}^{100hz}{psd(f)}}\right)^{2}}}\right\}db\eqno{\hbox{(3)}}$$\end{document } the signal quality ranged from 26 db to 36 db ( average : 30 db ) and was comparable to the snr achieved under laboratory conditions , . this ensures adequate sensitivity of the new system , which is a key issue in the application of point - of - care healthcare technologies ( pocht ) . particularly in osa patients , very loud snoring ( up to 90 db ) is a common accessory symptom due to upper airway obstruction of the respiratory system . with respect to sensitivity to motion artifacts in ppg measurement , snoring must be considered a potential contraindication for pulse oximetric measurement in the ear channel since it causes vibration . covering a wide frequency range , the highest signal intensity is usually \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { \leq}{\rm 500}~{\rm hz}$\end{document } . since a capacity in the back coupling path of the transimpedance converter provides a low - pass characteristic for anti - aliasing , spectral noise power \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { > } { \rm 100}~{\rm hz}$\end{document } is eliminated . however , spectral components of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { \leq}{\rm 100}~{\rm hz}$\end{document } may corrupt the ppg signal or can be separated from the cardiac frequency band for indirect determination of snoring . microphone signals were used to indicate the noise level of snoring . in fig . 4 , absolute determination of the noise level is not possible with this method due to the high pass characteristic of conventional microphones and the resulting low sensitivity \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { \leq}{\rm 50}~{\rm hz}$\end{document}. also , no defined distance to the source is given . however , for investigation of the impact of snoring on in - ear ppg measurement , we chose a time span with a maximum intensity of the microphone signal and assumed this to be representative for the lower intensities . 4.upper panel : the respiration ( black line ) and microphone ( blue line ) signals clearly indicate snoring . middle and bottom panel : ppg raw data derived from our in - ear pulse oximeter without filtering . the sensor shows excellent measurement performance , independent from the snoring . despite the proximity of the sensor to the throat and the expected impact of vibrations on the ppg signal upper panel : the respiration ( black line ) and microphone ( blue line ) signals clearly indicate snoring . middle and bottom panel : ppg raw data derived from our in - ear pulse oximeter without filtering . the sensor shows excellent measurement performance , independent from the snoring . despite the proximity of the sensor to the throat and the expected impact of vibrations on the ppg signal since snoring is a typical phenomenon in osa patients , such a contraindication would represent a major limitation for this new technique . in fig . 4 , the selected signal segments contrast the signal quality ( unfiltered raw data ) with and without snoring . it can be seen that snoring - induced vibration does not have any negative impact on the signal quality of the sensors . overall , no significant influence on snr could be found for all patients in this study . heart rate ( hr ) was calculated by means of a 4th order butterworth filter which was applied to the ppg raw data . the cutoff frequencies were 0.8 hz and 6 hz ; this is in accordance with physiological heart rates . the upper cutoff frequency has been chosen to maintain authentic ppg pulse - curve morphology . a threshold was defined at the point of maximal gradient of the pulse curve , and the zero crossings were detected . this approach allows more exact definition of heart beats compared to detection of extreme values , since maximum and minimum values are not always precisely defined ( e.g. , due to noise ) . 5 shows a comparison between hr derived from the in - ear sensor and from psg for a representative time segment ; there obviously is a high correlation between hr obtained with the in - ear sensor and with the gold standard . with regard to quantization of the reference hr up / down to whole numbers which is due to an internal algorithm of the psg , a regression analysis was not performed . however , the possibility of hr measurement with high accuracy by in - ear pulse oximeter is evident for nocturnal measurements . 5.comparison of heart rate ( hr , blue dots ) derived from the in - ear ppg sensor and from polysomnography ( ecg , black solid line ) . comparison of heart rate ( hr , blue dots ) derived from the in - ear ppg sensor and from polysomnography ( ecg , black solid line ) . because a sudden drop in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ s_{p}o_{2}$\end{document } serves to identify an apnea phase , a reliable measurement of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ s_{p}o_{2}$\end{document } plays a significant role in the diagnosis of osa . due to the ongoing trial , the performance of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ s_{p}o_{2}$\end{document } measurements by the in - ear sensor is demonstrated by a case study : in fig . 6 , lower panel , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ s_{p}o_{2}$\end{document } of both the psg and the in - ear sensor is compared with respiration shown in fig . 6 , upper panel . the in - ear \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ s_{p}o_{2}$\end{document } was first calibrated by the reference . the respiration signal shows a pathologic situation : the rhythmic variation in breathing intensity indicates cheyne - stokes respiration , including breathing stops of 30 s each . the resulting drops in oxygen saturation are clearly seen in the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ s_{p}o_{2}$\end{document } of the psg and in - ear sensor . in contrast to our system which provides sub - percentage values of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ s_{p}o_{2}$\end{document } with a sampling rate of 200 samples / second , the sampling rate of the reference pulse oximeter is down - sampled to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { + } { 1}$\end{document } sample / second and the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ s_{p}o_{2}$\end{document } values are rounded up / down to whole numbers . therefore , as mentioned before , we did not perform a more detailed correlation analysis . nevertheless , the high correlation demonstrates the feasibility of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ s_{p}o_{2}$\end{document } measurement in the human ear channel in a clinical situation . 6.upper panel : respiration flow ( breathing air ) obtained by psg ; clearly shown are three successive breathing stops of up to 30 seconds each . lower panel : comparison of spo2 derived from the in - ear ppg sensor ( red solid line ) and polysomnography ( for the same time segment , black dots ) . upper panel : respiration flow ( breathing air ) obtained by psg ; clearly shown are three successive breathing stops of up to 30 seconds each . lower panel : comparison of spo2 derived from the in - ear ppg sensor ( red solid line ) and polysomnography ( for the same time segment , black dots ) . the drops in oxygen saturation are clearly identified by both sensors . in general , all rhythmic physiological processes that cause fluctuations in blood pressure and/or blood volume changes affect the plethysmographic waveform . and it has long been known that respiration effects the ppg as well . anyhow , a robust determination of respiration rate ( rr ) still belongs to the most challenging tasks in ppg signal processing since the energy density of corresponding ( respiration - related ) physiological oscillators is comparatively small . the most prominent way to determine rr is a separation of amplitude fluctuations in ppg signal . we have shown earlier that detection of respiration - related information can be achieved by simple filter segmentation . here , we chose a 2nd order butterworth filter with cutoff frequencies 0.1 hz and 0.33 hz to meet durations of breathing cycles lasting from 3 to 10 seconds . however , besides respiratory - related spectral power , the ppg signal also contains other ( slower ) frequencies which are related to slower physiological processes ( e.g. , traube - hering - mayer waves , thermoregulation ) that can interfere with the respiratory frequency band . in this case , a correct attribution to breathing may be challenging . hence , for an adequate sensitivity regarding clinical purposes this sole method will not be sufficient . therefore , we suggest a combination of this method with two other signal - processing methods for rr determination which are physiologically ( partly ) redundant to each other : 1)in contrast to the blood - pressure related measurement technique described above ( a result of inhalation of air over time ) , variations in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ s_{p}o_{2}$\end{document } mainly depend on the amount of oxygen in blood over time.2)in addition to analysis of the ppg amplitude and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ s_{p}o_{2}$\end{document } variation , respiration rate can also be calculated from respiratory sinus arrhythmia ( rsa ) . rsa describes the effect of an increase in hr due to inspiration and a decrease due to expiration . although rsa is not yet entirely understood , it is supposedly due to the baroreflex control mechanism . in contrast to the blood - pressure related measurement technique described above ( a result of inhalation of air over time ) , variations in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ s_{p}o_{2}$\end{document } mainly depend on the amount of oxygen in blood over time . in addition to analysis of the ppg amplitude and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ s_{p}o_{2}$\end{document } variation , respiration rate can also be calculated from respiratory sinus arrhythmia ( rsa ) . rsa describes the effect of an increase in hr due to inspiration and a decrease due to expiration . although rsa is not yet entirely understood , it is supposedly due to the baroreflex control mechanism . while \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ s_{p}o_{2}$\end{document } variation was separated by butterworth bandpass filtering , rsa was calculated in three steps : 1 ) calculation of hr according to the algorithm described in iii - c and interpolation . 2 ) 2nd order butterworth filtering with cutoff frequencies of 0.1 hz and 0.33 hz . 3 ) detection of zero crossings , and calculation of subsequent distances and interpolation . to quantify the practicability and the diagnostic significance of these three methods for rr determination with the presented in - ear ppg sensor , we performed an initial laboratory experiment with a volunteer ( 29 yrs healthy male ) who breathed with a constant respiration - rate / heart - rate quotient ( rr - hr - quotient , 1:8 , 1:6 , 1:8 , 1:10 ) . 7 shows the results of the experiment . a good correlation for a rr - hr - quotient of 1:8 and 1:6 ( according to breathing cycles of 6.5 resp . 4.5 seconds ) can clearly be identified for all three methods . at respiration rates that are below the physiological standard ( here : rr - hr - quotient of 10 , according to 8 seconds respiration cycle ) the inner harmony especially rr detection by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ s_{p}o_{2}$\end{document } variation shows high variation . although there is a complex mixture of influences , it is supposedly due to desaturation effects . regardless the variance of the methods makes an identification of apnea / hypopnea events possible since an increase in variance seems to correlate with abnormal respiration events . 7.breathing cycle durations were calculated by three different methods and compared with the reference ( grey solid line ) : variance in ppg amplitude ( blue squares ) , respiratory sinus arrhythmia ( black crosses ) , variation in spo2 ( blue circles ) . the respiration - rate - to - heart - rate - quotient breathing cycle durations were calculated by three different methods and compared with the reference ( grey solid line ) : variance in ppg amplitude ( blue squares ) , respiratory sinus arrhythmia ( black crosses ) , variation in spo2 ( blue circles ) . the respiration - rate - to - heart - rate - quotient was fixed to 1:8 , 1:6 , 1:8 , 1:10 , 1:8 . we define the respiration flow derived from the nasal respiration sensor as reference gold standard . the nasal respiration sensor is part of the psg and is based on a thermistor ( temperature sensitive resistor ) which is fixed close to the nostril . since the respiratory flow leads to an increase in temperature during expiration and a decrease during inspiration there is a ( almost ) linear correlation between respiratory air flux and nasal respiration sensor signal . in contrast to the methods introduced above which are predominated by tidal volume induced pressure variation effects ( e.g. , increasing blood pressure due to lung expansion , partial oxygen and carbon dioxide pressure ) , the nasal respiration sensor signal is predominated by air flux and one must consider a phase shift of 90. the phase shift is compensated in this analysis . 8 illustrates the correlation of the methods with the reference for a representative time segment . compared to the respiration flow derived from the nasal respiration sensor of the psg , a clear correlation is seen that demonstrates the high potential of amplitude - related respiration detection for the demonstrated nocturnal setting ( fig . 8.respiration was calculated by three different methods and compared with respiratory reference sensor signal derived from a psg nasal sensor ( dashed line ) : segmentation by ppg amplitude ( upper panel , solid line ) , segmentation by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ s_{p}o_{2}$\end{document } variation ( middle panel , solid line ) and respiratory sinus arrhythmia ( lower panel , solid line ) . \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ s_{p}o_{2}$\end{document } variation is presented in absolute values ( % ) . respiration was calculated by three different methods and compared with respiratory reference sensor signal derived from a psg nasal sensor ( dashed line ) : segmentation by ppg amplitude ( upper panel , solid line ) , segmentation by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ s_{p}o_{2}$\end{document } variation ( middle panel , solid line ) and respiratory sinus arrhythmia ( lower panel , solid line ) . \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ s_{p}o_{2}$\end{document } variation is presented in absolute values ( % ) . \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ s_{p}o_{2}$\end{document } fluctuates up to 2% in amplitude ( middle panel ) and a correlation is also evident . although the compliance is slightly inferior to the amplitude - related detection of respiration , it proved to be a useful redundant alternative . comparing the respiratory signal with rsa ( fig . 8 , bottom panel ) , a correlation can hardly be identified for the selected time segment . in fact , the characteristic of the coupling between hr variation and respiration differs between individuals and also intra - individually over time . sleep apnea ( i.e. , central sleep apnea ) can also be seen as a cardiorespiratory disease and , thus , rsa may serve as an interesting diagnostic parameter for examination of sleep apnea . rsa also depends on physiological status and is deemed to be very prominent at relaxation . for this reason , an indication of sleep phases by rsa is an interesting direction for future research , but was not evaluated in this work . although these methods are not expected to provide reliable information about the depth of breathing , frequency - related information in the form of a breathing rate ( i.e. , mean number of breaths / minute ) can be supported . for the selected time span in fig . 8 , the breathing rate was calculated by means of zero - crossings detection and a correlation analysis was performed ( table i ) . as expected , although the rsa approach provides no reliable determination of the breathing rate for the analyzed situation , a general feasibility is well known . table ithe reference breathing rate is correlated with the presented methods : amplitude segmentation , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ s_{p}o_{2}$\end{document } variation and respiratory sinus arrhythmiamethodamplitude modulation\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ sp{\rm o2}$\end{document } variationrsacorrelation coefficient0.890.12 - 0.09mean absolute deviation [ s]0.140.81.45mean error [ % ] 3.3619.0836.88standard deviation [ s]0.090.671.36 to quantify the intra - individual reliability we adapted the methods to three time segments ( two minutes each ) of different patients in different stages of sleep . the specificity and sensitivity of a detection of breathing events is listed in table ii . table iisensitivity / specificity [ % ] of the proposed methods for respiration - event detectionmethodpatient # 1patient # 3patient # 4amplitude modulation100/ 10081.5/ 85.784.6/ 80.8\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { \rm spo2}$\end{document } variation100/ 10095.7/ 95.788/ 84.6rsa37.5/62.591.3/95.792/80.8 for a more stable and reliable calculation of respiration rate , we suggest an appropriate combination of the three methods . for a model - based data fusion of those redundant determination strategies several methods exist like bayes ' fusion , kalman filter , neural networks and others . a similar neural network supported respiration rate monitoring with ppg signal detection with a forehead sensor has been published by johansson . although a classification of a pathologic breathing pattern , like the cheyne - stokes respiration in fig . 6 is not realistic at present , we think that a determination of respiration rate and identification of apnea phases with adequate specificity and sensitivity is feasible for most situations with the presented approach . this work is based on the preliminary analysis of a clinical study and evaluates the potential of a novel in - ear pulse oximetric sensor for cardiovascular monitoring during sleep . human studies were performed in sleep apnea patients in a sleep laboratory under standard examination conditions . the feasibility of a simultaneous measurement of heart rate , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ s_{p}o_{2}$\end{document } and respiration could be demonstrated with the presented sensor . this might reduce the amount of necessary sensors , cables and devices in the clinical sleep apnea examination ( psg ) which in turn could improve the quality of sleep during psg and might also enable an easy nocturnal cardiovascular monitoring in a non - clinical environment ( homecare ) with a single sensor . the measurement of breathing rate by means of pulse oximetric data is a complex research field . although the ppg amplitude is considered to fluctuate synchronously with respiration , interference from other oscillating physiological procedures often leads to misinterpretation . therefore , the possibility of a three - way detection of respiration is discussed : i.e. , respiration rate was calculated by means of segmentation by ppg amplitude , segmentation by variation in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ s_{p}o_{2}$\end{document } , and by respiratory sinus arrhythmia . the potential of these three methods for respiration measurement was quantified by an initial laboratory human experiment and the sensitivity and the specificity were evaluated by several patients in different sleep stages . we found the best sensitivity and specificity using ppg amplitude and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ s_{p}o_{2}$\end{document } variation . since these methods offer partial redundancy due to the physiological phenomenon upon which they are based , a combination of these three methods might allow more reliable measurement of respiration rate . also , besides hr , respiration rate and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ s_{p}o_{2}$\end{document } , pulse oximetry may allow the measurement of additional vital parameters in the future ( e.g. , thermoregulation , blood pressure variation ) , thereby reducing the number of sensors required . snoring , a typical symptom of osa , was expected to be a potential contraindication . however , we demonstrated that snoring - induced vibration does not affect the signal quality of pulse oximetric measurement in the ear channel . however , motion artifacts caused by subsultis tendinum , or abrupt awakenings , must be taken into account and carefully evaluated since they can decrease the specificity of the system . however , particularly for pocht applications , both sensitivity and specificity are essential features for reliable health monitoring in a non - clinical environment . since ppg is susceptible to movement , identification of motion artifacts and reduction strategies need to be developed . in the future , we will pursue motion detection by means of the integrated 3-axis accelerometer for automatic detection of and compensation for artifacts .

homecare is healthcare based on the principle outpatient before inpatient , with the aim of moving at least some care - delivery to the home . but reliable determination of vital signs at home requires new , smart sensors , which can be used by the patients themselves . we present a novel pulse oximetry sensor worn in the ear channel . it was previously shown that measurement of heart rate , arterial oxygen saturation and related respiratory information can be performed with reliable accuracy under laboratory conditions . the present study explores the clinical feasibility of the sensor system for cardiovascular monitoring during sleep , with the aim to diagnose sleep apnea . for this , human trials were performed in a sleep laboratory including patients with a clinical suspicion of sleep apnea . besides a general analysis of the sensor 's signal quality during sleep , the evaluation focuses on heart rate dynamics and time - variant oxygen saturation . in addition , several methods to derive respiration rate from photoplethysmographic signals are examined and discussed . results from the in - ear sensor are compared with standard polysomnography monitoring and demonstrate that this novel system allows long - term nocturnal measurement of heart rate , oxygen saturation and respiratory rate with sufficient accuracy .

Introduction Materials and Methods Results Conclusion

obstructive sleep apnea ( osa ) is a sleep disorder in which temporary airway narrowing leads to apnea and a decrease in arterial oxygen saturation \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ ( s_{p}o_{2})$\end{document } during sleep . however , because many sensors need to be attached to the patient , the quality of sleep is reduced which , in turn , can affect the results of the examination . therefore , we developed an unobtrusive single - sensor system which is suitable for homecare screening of sleep , taking into account the special requirements of the homecare setting . the technique is based on the principle of pulse oximetry , a multi - wavelength version of photoplethymography ( ppg ) , . advancements in measurement efficiency makes the coincidental measurement of multiple vital signs like hr , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ s_{p}o_{2}$\end{document } , respiration rate , vasomotion , thm waves and other possible with a single sensor which is noninvasive , unobtrusive and cheap . combined with telemedicine technology , such a system can also be used for long - term nocturnal monitoring of patients at risk ( e.g. based on those results , we now want to evaluate the feasibility of the in - ear sensor for cardiovascular monitoring during sleep . therefore , trials with the sensor are being performed in patients who suffer from osa ; the results are compared with standard psg data . especially for long - term monitoring of vital parameters during sleep , since most sensors are attached to finger or earlobe , measurements are often disrupted by motion artifacts like subsultis during the first stage of sleep ( drifting into sleep ) , movements during light sleep or abrupt awakenings from deep sleep . in addition , the proximity to the brain guarantees stable perfusion conditions , since the tragus is supplied by an arterial network coming from the arteries temporalis superficialis and arteria auricularis posterior . , sepsis , hypothermia ) is known to inhibit peripheral pulse oximetry due to a lack of peripheral perfusion ( ) , the in - ear pulse oximetry is not likely to be affected by this . the sensor is sealed into a biocompatible ear mold and measurements are made in the ear channel ( at the tragus ) . the correlation between the r - value and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ s_{p}o_{2}$\end{document } was determined based on our human hypoxia studies .\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ $ s_{p}o_{2}=f(r)=33r^{2}-152.6r+176.4+r_{comp}\eqno{\hbox{(2)}}$$\end{document } although a high correlation was confirmed in , global calibration proved to be challenging due to an unexpected effect , which was probably due to the influence of subdermal venous blood , : because of a slight shift in the calibration curve every time a measurement starts , only relative \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ s_{p}o_{2}$\end{document } measurements can be made . nevertheless , this is not a limitation because , with regard to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ s_{p}o_{2}$\end{document } , sleep diagnosis focuses on temporary periods of apnea with a related drop in oxygen saturation . to meet the calibration requirement , an initial \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ s_{p}o_{2}$\end{document } measurement with a reference device can be performed to compensate for the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ s_{p}o_{2}$\end{document } offset by the parameter \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ r_{comp}$\end{document } in ( 2 ) . the patients spent one night in the sleep laboratory of the rwth aachen university hospital for a standardized polysomnographic checkup ( diamedic gmbh , germany ) , including ppg , ecg , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ s_{p}o_{2}$\end{document } , respiratory flux and other tests . the first trials indicate excellent nocturnal performance of the in - ear sensor ; this success can probably be attributed to considerably reduced motion and the darkness . the signal quality was quantified by the signal - to - noise ratio ( snr).\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ $ snr_{oxi}=10\log\left\{{{\left({\sum_{0.5hz}^{10hz}{psd(f)}}\right)^{2}}\over{\left({\sum_{10hz}^{100hz}{psd(f)}}\right)^{2}}}\right\}db\eqno{\hbox{(3)}}$$\end{document } the signal quality ranged from 26 db to 36 db ( average : 30 db ) and was comparable to the snr achieved under laboratory conditions , . despite the proximity of the sensor to the throat and the expected impact of vibrations on the ppg signal upper panel : the respiration ( black line ) and microphone ( blue line ) signals clearly indicate snoring . despite the proximity of the sensor to the throat and the expected impact of vibrations on the ppg signal since snoring is a typical phenomenon in osa patients , such a contraindication would represent a major limitation for this new technique . it can be seen that snoring - induced vibration does not have any negative impact on the signal quality of the sensors . 5 shows a comparison between hr derived from the in - ear sensor and from psg for a representative time segment ; there obviously is a high correlation between hr obtained with the in - ear sensor and with the gold standard . 5.comparison of heart rate ( hr , blue dots ) derived from the in - ear ppg sensor and from polysomnography ( ecg , black solid line ) . comparison of heart rate ( hr , blue dots ) derived from the in - ear ppg sensor and from polysomnography ( ecg , black solid line ) . due to the ongoing trial , the performance of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ s_{p}o_{2}$\end{document } measurements by the in - ear sensor is demonstrated by a case study : in fig . 6 , lower panel , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ s_{p}o_{2}$\end{document } of both the psg and the in - ear sensor is compared with respiration shown in fig . the in - ear \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ s_{p}o_{2}$\end{document } was first calibrated by the reference . the resulting drops in oxygen saturation are clearly seen in the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ s_{p}o_{2}$\end{document } of the psg and in - ear sensor . nevertheless , the high correlation demonstrates the feasibility of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ s_{p}o_{2}$\end{document } measurement in the human ear channel in a clinical situation . lower panel : comparison of spo2 derived from the in - ear ppg sensor ( red solid line ) and polysomnography ( for the same time segment , black dots ) . lower panel : comparison of spo2 derived from the in - ear ppg sensor ( red solid line ) and polysomnography ( for the same time segment , black dots ) . therefore , we suggest a combination of this method with two other signal - processing methods for rr determination which are physiologically ( partly ) redundant to each other : 1)in contrast to the blood - pressure related measurement technique described above ( a result of inhalation of air over time ) , variations in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ s_{p}o_{2}$\end{document } mainly depend on the amount of oxygen in blood over time.2)in addition to analysis of the ppg amplitude and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ s_{p}o_{2}$\end{document } variation , respiration rate can also be calculated from respiratory sinus arrhythmia ( rsa ) . to quantify the practicability and the diagnostic significance of these three methods for rr determination with the presented in - ear ppg sensor , we performed an initial laboratory experiment with a volunteer ( 29 yrs healthy male ) who breathed with a constant respiration - rate / heart - rate quotient ( rr - hr - quotient , 1:8 , 1:6 , 1:8 , 1:10 ) . this work is based on the preliminary analysis of a clinical study and evaluates the potential of a novel in - ear pulse oximetric sensor for cardiovascular monitoring during sleep . human studies were performed in sleep apnea patients in a sleep laboratory under standard examination conditions . the feasibility of a simultaneous measurement of heart rate , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ s_{p}o_{2}$\end{document } and respiration could be demonstrated with the presented sensor . this might reduce the amount of necessary sensors , cables and devices in the clinical sleep apnea examination ( psg ) which in turn could improve the quality of sleep during psg and might also enable an easy nocturnal cardiovascular monitoring in a non - clinical environment ( homecare ) with a single sensor . also , besides hr , respiration rate and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ s_{p}o_{2}$\end{document } , pulse oximetry may allow the measurement of additional vital parameters in the future ( e.g. however , we demonstrated that snoring - induced vibration does not affect the signal quality of pulse oximetric measurement in the ear channel .

full details of the selection of participants and measurements have been previously reported ( 810 ) . a total of 4,286 women , aged 6079 years , were randomly recruited from 23 british towns . baseline data ( self - completed questionnaire , research nurse interview , physical examination , and medical record review ) were collected between 1999 and 2001 . these women have been followed up for a median of 7.3 years , to september 2007 , by a detailed review of their medical records conducted every 2 years and by a self - completed questionnaire . informed consent was obtained from the women , and the approval of both the local and multicenter ethics committees was obtained for the study . levels of ggt and alt were determined in fresh serum samples using an automated analyzer ( technicon sequential multiple analyzer ; technicon instruments corporation , tarrytown , ny ) . waist and hip circumference , lipids , fasting glucose and insulin , and blood pressure were measured using standard methods as previously described . information on smoking , physical activity , social class , and alcohol consumption was obtained from a self - completed questionnaire and nurse interview ( 9 ) . insulin resistance was estimated according to the homeostasis model assessment ( homa ) as the product of fasting glucose ( millimoles per liter ) and insulin ( microunits per milliliter ) divided by the constant 22.5 ( 11 ) . baseline diabetes ( for exclusion of these cases ) was defined according to the world health organization criteria as any woman with a doctor 's diagnosis of diabetes ( based on medical records review and self - report ) and/or with a fasting glucose concentration 7 incident cases of diabetes were defined as either a self - report of a doctor diagnosis of diabetes or evidence of diabetes in the follow - up medical record reviews . cox proportional hazards regression models were used to examine associations of exposures with incident diabetes in those with no evidence of diabetes at baseline . in the cox proportional hazards regression models , the participant 's age was the time axis and risk was assessed from the date of baseline examination for each woman . contributions to risk were censored at the date of diagnosis or death from any other cause or at the end of the follow - up period ( 30 september 2007 ) for those who remained alive and free of diabetes . pubmed and embase ( excerpta medica database ) were systematically searched by a.f . in february 2008 for all prospective population - based studies evaluating the association among nafld ( diagnosed by any imaging technique ) , alt , ggt , and incident diabetes . abstracts were scanned , relevant full - text publications were obtained , and inclusion criteria were applied ( i.e. , prospective studies conducted in general populations ) . two sets of meta - analyses were undertaken to reflect the two aims of this study . in the first set , a separate meta - analysis was conducted for each nafld marker , maximizing data use regardless of whether they provided data on ultrasound - defined nafld , alt only , ggt only , or any combination of these . in these analyses , the assumption was that all study results for alt ( ggt or ultrasound ) were part of the same distribution but not that the true effect was the same in all studies ; therefore , a random - effects model was used , though we did also check our assumption for this random effect using the i measure , which quantifies the percentage of total variation across studies that is due to heterogeneity rather than chance ( 13 ) . because different studies presented results on different scales ( e.g. , risk ratios [ rrs ] for quantiles of alt or ggt compared with the lowest quantile or per category or logged unit per liter ) , for the first meta - analysis ( of all studies ) a standard statistical method was used to estimate the log hazard ratio ( hr ) per log unit increase in alt or ggt , together with its se ( 14,15 ) . results are presented as the hr per unit change in alt or ggt on a log scale . when data were presented according to quantiles or categories of alt or ggt , the median or mean in each group , when reported , was used . when these were not reported , the mean in each group was estimated based on the distribution of subjects across groups , as outlined by chne and thompson ( 15 ) . all exposure ( ggt and alt ) levels were converted to the log - normal scale if not already presented as such . the log hr per 1 unit / l increase was then estimated using the method of greenland and longnecker ( 14 ) . the objective of the second meta - analysis was to directly compare the associations of alt and ggt with diabetes . we decided a priori to limit this second analysis to results of studies that provided estimates for both alt and ggt in the same population in order to ensure the comparability of the estimates for alt and ggt . furthermore , only studies for which we could obtain results in which alt and ggt had been standardized ( to account for their different distributions ) were included . the most common method used to standardize alt and ggt was to present results per fourths of the alt and ggt distributions . therefore , we combined the rrs in the top versus the bottom fourth of the alt and ggt . a fixed - effects model was used , thus assuming that the true effect in all studies was the same and any difference between study results was due to chance alone , and this assumption was checked using the i measure ( 13 ) . the effect of duration of follow - up on study results was assessed by meta - regression . small - study effects such as publication bias were examined by using a funnel plot ( 16 ) and formally tested using the egger test ( 17 ) . full details of the selection of participants and measurements have been previously reported ( 810 ) . a total of 4,286 women , aged 6079 years , were randomly recruited from 23 british towns . baseline data ( self - completed questionnaire , research nurse interview , physical examination , and medical record review ) were collected between 1999 and 2001 . these women have been followed up for a median of 7.3 years , to september 2007 , by a detailed review of their medical records conducted every 2 years and by a self - completed questionnaire . informed consent was obtained from the women , and the approval of both the local and multicenter ethics committees was obtained for the study . levels of ggt and alt were determined in fresh serum samples using an automated analyzer ( technicon sequential multiple analyzer ; technicon instruments corporation , tarrytown , ny ) . waist and hip circumference , lipids , fasting glucose and insulin , and blood pressure were measured using standard methods as previously described . information on smoking , physical activity , social class , and alcohol consumption was obtained from a self - completed questionnaire and nurse interview ( 9 ) . insulin resistance was estimated according to the homeostasis model assessment ( homa ) as the product of fasting glucose ( millimoles per liter ) and insulin ( microunits per milliliter ) divided by the constant 22.5 ( 11 ) . baseline diabetes ( for exclusion of these cases ) was defined according to the world health organization criteria as any woman with a doctor 's diagnosis of diabetes ( based on medical records review and self - report ) and/or with a fasting glucose concentration 7 incident cases of diabetes were defined as either a self - report of a doctor diagnosis of diabetes or evidence of diabetes in the follow - up medical record reviews . cox proportional hazards regression models were used to examine associations of exposures with incident diabetes in those with no evidence of diabetes at baseline . in the cox proportional hazards regression models , the participant 's age was the time axis and risk was assessed from the date of baseline examination for each woman . contributions to risk were censored at the date of diagnosis or death from any other cause or at the end of the follow - up period ( 30 september 2007 ) for those who remained alive and free of diabetes . pubmed and embase ( excerpta medica database ) were systematically searched by a.f . in february 2008 for all prospective population - based studies evaluating the association among nafld ( diagnosed by any imaging technique ) , alt , ggt , and incident diabetes . abstracts were scanned , relevant full - text publications were obtained , and inclusion criteria were applied ( i.e. , prospective studies conducted in general populations ) . two sets of meta - analyses were undertaken to reflect the two aims of this study . in the first set , a separate meta - analysis was conducted for each nafld marker , maximizing data use regardless of whether they provided data on ultrasound - defined nafld , alt only , ggt only , or any combination of these . in these analyses , the assumption was that all study results for alt ( ggt or ultrasound ) were part of the same distribution but not that the true effect was the same in all studies ; therefore , a random - effects model was used , though we did also check our assumption for this random effect using the i measure , which quantifies the percentage of total variation across studies that is due to heterogeneity rather than chance ( 13 ) . because different studies presented results on different scales ( e.g. , risk ratios [ rrs ] for quantiles of alt or ggt compared with the lowest quantile or per category or logged unit per liter ) , for the first meta - analysis ( of all studies ) a standard statistical method was used to estimate the log hazard ratio ( hr ) per log unit increase in alt or ggt , together with its se ( 14,15 ) . results are presented as the hr per unit change in alt or ggt on a log scale . when data were presented according to quantiles or categories of alt or ggt , the median or mean in each group , when reported , was used . when these were not reported , the mean in each group was estimated based on the distribution of subjects across groups , as outlined by chne and thompson ( 15 ) . all exposure ( ggt and alt ) levels were converted to the log - normal scale if not already presented as such . the log hr per 1 unit / l increase was then estimated using the method of greenland and longnecker ( 14 ) . the objective of the second meta - analysis was to directly compare the associations of alt and ggt with diabetes . we decided a priori to limit this second analysis to results of studies that provided estimates for both alt and ggt in the same population in order to ensure the comparability of the estimates for alt and ggt . furthermore , only studies for which we could obtain results in which alt and ggt had been standardized ( to account for their different distributions ) were included . the most common method used to standardize alt and ggt was to present results per fourths of the alt and ggt distributions . therefore , we combined the rrs in the top versus the bottom fourth of the alt and ggt . a fixed - effects model was used , thus assuming that the true effect in all studies was the same and any difference between study results was due to chance alone , and this assumption was checked using the i measure ( 13 ) . the effect of duration of follow - up on study results was assessed by meta - regression . small - study effects such as publication bias were examined by using a funnel plot ( 16 ) and formally tested using the egger test ( 17 ) . data on diabetes at baseline were available for 3,829 women ( 89% of 4,286 ) , of whom 377 had diabetes and were excluded from the analysis . of the remaining 3,452 women , 3,041 ( 88% of 3,452 ) characteristics of included women by diabetes status at the end of follow - up are presented in table a1 ( available in an online appendix at http://care.diabetesjournals.org/cgi/content/full/dc08-1870/dc1 ) . women with diabetes at the end of follow - up were older ; had higher age - adjusted mean bmi , waist - to - hip ratio , alt , ggt , triglycerides , glucose , and homa ; and had lower hdl cholesterol . more of the women with incident diabetes exercised for < 2 h per week , and more also had a history of smoking . hrs ( 95% ci ) of incident diabetes per sd change of natural logged alt and ggt model 1 , adjusted for age ; model 2 , model 1 adjustment plus alcohol consumption ; model 3 , model 2 adjustments plus childhood and adult social class , physical activity , and smoking ; model 4 , model 3 adjustments plus homa , waist - to - hip ratio , triglycerides , hdl cholesterol , and systolic blood pressure . alp , alkaline phosphatase ; ast , aspartate aminotransferase ; cardia study , coronary artery risk development in young adults study ; chd , cardiovascular heart disease ; crp , c - reactive protein ; cvd , cardiovascular disease ; epic - potsdam study , european prospective investigation into cancer and nutrition potsdam study ; fev1 , forced expiratory volume in 1 s ; fibar study , firenze bagno a ripoli study ; homa - ir , homa of insulin resistance ; iras , insulin resistance atherosclerosis study ; m - low , glucose disposal during a low - dose insulin infusion ; monica study , monitoring of trends and determinants in cardiovascular disease study ; n / a , not available ; sbp , systolic blood pressure ; wbc , white blood cell ; woscops , west of scotland coronary prevention study . during 281,081 person - years of follow - up , 112 women were diagnosed with diabetes , yielding a rate of 5.3 per 1,000 person - years ( 95% ci 4.46.4 ) . both alt and ggt were associated with incident diabetes , even when adjustment was made for alcohol consumption ( table 1 ) and other potential confounders . when components of the metabolic syndrome were added to the model , the associations of both alt and ggt with incident diabetes were attenuated toward the null . however , the magnitude of associations of alt with diabetes and of ggt with diabetes was comparable in all four models , as was the degree of attenuation of age - adjusted estimates . the electronic search ( crossing terms for alt , ggt , liver steatosis , and diabetes ) yielded 236 potentially relevant publications , of which 20 studied prospective population - based cohorts and assessed baseline ultrasonography - diagnosed nafld , alt , and/or ggt and their associations with incident diabetes . three studies ( 1820 ) ( summarized in table 2 ) assessed ultrasound - diagnosed nafld as a determinant of incident diabetes . no studies that used any other diagnostic test to ascertain nafld in its association with incident diabetes were retrieved . all three studies were conducted in asian populations , and in all three there was evidence that ultrasound - diagnosed nafld was associated with diabetes risk . when estimates from these studies were meta - analyzed ( using the estimate of mild vs. no nafld from the study by kim et al . [ ( 18 ) ] ) , the pooled rr was 2.52 ( 95% ci 1.075.96 ) , but there was evidence of considerable heterogeneity between studies ( i = 90% ) . study characteristics of the 18 prospective population - based studies of the associations of alt and/or ggt with incident diabetes are also summarized in table 2 . ten studies ( 46,2128 ) ( including the current analysis of the british women 's heart and health study [ bwhhs ] ) assessed both alt and ggt as predictors of diabetes , an additional three studies looked at alt only ( 2931 ) , and another four studies looked at the association of ggt with diabetes risk ( 3,3234 ) . age - adjusted and fully adjusted study results examining ( natural logged ) alt and ggt as determinants of incident diabetes were pooled ( separately ) . a change in one logged unit in alt was associated with an hr of 3.05 ( 95% ci 2.593.59 , i = 26% , 13 comparisons ) and in ggt 2.56 ( 2.312.84 , i = 32% , 17 comparisons ) ( online appendix fig . a1 ) . the meta - analysis of fully adjusted results for both alt and ggt are presented in fig . the fully adjusted hr for diabetes per increase in one unit of logged alt was 1.85 ( 1.572.18 , i = 19 , 14 comparisons ) , and in one unit of logged ggt the hr was 1.92 ( 1.662.21 , i = 55% , 18 comparisons ) . meta - analysis of fully adjusted results of alt and ggt as a determinant of incident diabetes . no strong evidence was found for the presence of a small - studies effect for alt or ggt . there was also no strong evidence of an association between duration of follow - up and study results ( all p > 0.69 ) . seven studies ( including the bwhhs ) contributed to the analysis of alt and ggt categorized in fourths as predictors of diabetes ( 4,5,21,22,24,25 ) . additional studies assessed both alt and ggt ; however , one used fifths ( 26,27 ) , another used tenths ( 6 ) , and a final study ( 28 ) used fourths but did not present results in full and therefore could not be included in this analysis . age - adjusted pooled analyses of the included studies are presented in online appendix figure a2 . the rr in the top versus the bottom quartile of alt was 4.42 ( 95% ci 3.615.42 , i = 42% ) , and for ggt it was 5.87 ( 4.637.44 , i = 21% ) . the fully adjusted rr for alt was 2.02 ( 1.592.58 , i = 27% ) and for ggt 2.94 ( 1.983.88 , i = 20% ) ( fig . 2 ) . according to a formal statistical test for heterogeneity between estimates for alt and ggt , there was some evidence suggesting that pooled estimates for ggt were larger than those for alt ( age - adjusted results , p = 0.08 , and fully adjusted results , p = 0.05 ) . meta - analysis of fully adjusted results ( rr in top versus bottom fourth ) of alt and ggt as determinants of incident diabetes . however , repeating the analysis after excluding results for women in the desir ( data from the epidemiological study on the insulin resistance syndrome ) cohort ( 4 ) , which was the only study that showed a potential protective effect of alt , resulted in greater pooled estimates for ggt ( hr 2.91 [ 95% ci 2.193.85 ] ) than for alt ( 2.12 [ 1.662.72 ] ) but no strong evidence for heterogeneity between the estimates ( p 0.10 for both age - adjusted and fully adjusted results ) . data on diabetes at baseline were available for 3,829 women ( 89% of 4,286 ) , of whom 377 had diabetes and were excluded from the analysis . of the remaining 3,452 women , 3,041 ( 88% of 3,452 ) characteristics of included women by diabetes status at the end of follow - up are presented in table a1 ( available in an online appendix at http://care.diabetesjournals.org/cgi/content/full/dc08-1870/dc1 ) . women with diabetes at the end of follow - up were older ; had higher age - adjusted mean bmi , waist - to - hip ratio , alt , ggt , triglycerides , glucose , and homa ; and had lower hdl cholesterol . more of the women with incident diabetes exercised for < 2 h per week , and more also had a history of smoking . hrs ( 95% ci ) of incident diabetes per sd change of natural logged alt and ggt model 1 , adjusted for age ; model 2 , model 1 adjustment plus alcohol consumption ; model 3 , model 2 adjustments plus childhood and adult social class , physical activity , and smoking ; model 4 , model 3 adjustments plus homa , waist - to - hip ratio , triglycerides , hdl cholesterol , and systolic blood pressure . alp , alkaline phosphatase ; ast , aspartate aminotransferase ; cardia study , coronary artery risk development in young adults study ; chd , cardiovascular heart disease ; crp , c - reactive protein ; cvd , cardiovascular disease ; epic - potsdam study , european prospective investigation into cancer and nutrition potsdam study ; fev1 , forced expiratory volume in 1 s ; fibar study , firenze bagno a ripoli study ; homa - ir , homa of insulin resistance ; iras , insulin resistance atherosclerosis study ; m - low , glucose disposal during a low - dose insulin infusion ; monica study , monitoring of trends and determinants in cardiovascular disease study ; n / a , not available ; sbp , systolic blood pressure ; wbc , white blood cell ; woscops , west of scotland coronary prevention study . during 281,081 person - years of follow - up , 112 women were diagnosed with diabetes , yielding a rate of 5.3 per 1,000 person - years ( 95% ci 4.46.4 ) . both alt and ggt were associated with incident diabetes , even when adjustment was made for alcohol consumption ( table 1 ) and other potential confounders . when components of the metabolic syndrome were added to the model , the associations of both alt and ggt with incident diabetes were attenuated toward the null . however , the magnitude of associations of alt with diabetes and of ggt with diabetes was comparable in all four models , as was the degree of attenuation of age - adjusted estimates . the electronic search ( crossing terms for alt , ggt , liver steatosis , and diabetes ) yielded 236 potentially relevant publications , of which 20 studied prospective population - based cohorts and assessed baseline ultrasonography - diagnosed nafld , alt , and/or ggt and their associations with incident diabetes . three studies ( 1820 ) ( summarized in table 2 ) assessed ultrasound - diagnosed nafld as a determinant of incident diabetes . no studies that used any other diagnostic test to ascertain nafld in its association with incident diabetes all three studies were conducted in asian populations , and in all three there was evidence that ultrasound - diagnosed nafld was associated with diabetes risk . when estimates from these studies were meta - analyzed ( using the estimate of mild vs. no nafld from the study by kim et al . [ ( 18 ) ] ) , the pooled rr was 2.52 ( 95% ci 1.075.96 ) , but there was evidence of considerable heterogeneity between studies ( i = 90% ) . study characteristics of the 18 prospective population - based studies of the associations of alt and/or ggt with incident diabetes are also summarized in table 2 . ten studies ( 46,2128 ) ( including the current analysis of the british women 's heart and health study [ bwhhs ] ) assessed both alt and ggt as predictors of diabetes , an additional three studies looked at alt only ( 2931 ) , and another four studies looked at the association of ggt with diabetes risk ( 3,3234 ) . age - adjusted and fully adjusted study results examining ( natural logged ) alt and ggt as determinants of incident diabetes were pooled ( separately ) . a change in one logged unit in alt was associated with an hr of 3.05 ( 95% ci 2.593.59 , i = 26% , 13 comparisons ) and in ggt 2.56 ( 2.312.84 , i = 32% , 17 comparisons ) ( online appendix fig . the meta - analysis of fully adjusted results for both alt and ggt are presented in fig . the fully adjusted hr for diabetes per increase in one unit of logged alt was 1.85 ( 1.572.18 , i = 19 , 14 comparisons ) , and in one unit of logged ggt the hr was 1.92 ( 1.662.21 , i = 55% , 18 comparisons ) . meta - analysis of fully adjusted results of alt and ggt as a determinant of incident diabetes . no strong evidence was found for the presence of a small - studies effect for alt or ggt . there was also no strong evidence of an association between duration of follow - up and study results ( all p > 0.69 ) . seven studies ( including the bwhhs ) contributed to the analysis of alt and ggt categorized in fourths as predictors of diabetes ( 4,5,21,22,24,25 ) . additional studies assessed both alt and ggt ; however , one used fifths ( 26,27 ) , another used tenths ( 6 ) , and a final study ( 28 ) used fourths but did not present results in full and therefore could not be included in this analysis . age - adjusted pooled analyses of the included studies are presented in online appendix figure a2 . the rr in the top versus the bottom quartile of alt was 4.42 ( 95% ci 3.615.42 , i = 42% ) , and for ggt it was 5.87 ( 4.637.44 , i = 21% ) . the fully adjusted rr for alt was 2.02 ( 1.592.58 , i = 27% ) and for ggt 2.94 ( 1.983.88 , i = 20% ) ( fig . 2 ) . according to a formal statistical test for heterogeneity between estimates for alt and ggt , there was some evidence suggesting that pooled estimates for ggt were larger than those for alt ( age - adjusted results , p = 0.08 , and fully adjusted results , p = 0.05 ) . meta - analysis of fully adjusted results ( rr in top versus bottom fourth ) of alt and ggt as determinants of incident diabetes . however , repeating the analysis after excluding results for women in the desir ( data from the epidemiological study on the insulin resistance syndrome ) cohort ( 4 ) , which was the only study that showed a potential protective effect of alt , resulted in greater pooled estimates for ggt ( hr 2.91 [ 95% ci 2.193.85 ] ) than for alt ( 2.12 [ 1.662.72 ] ) but no strong evidence for heterogeneity between the estimates ( p 0.10 for both age - adjusted and fully adjusted results ) . ultrasound - diagnosed nafld , alt , and ggt all predicted diabetes risk . nafld defined by ultrasound was associated with more than a doubling of the risk of incident diabetes . in a meta - analysis of fully adjusted results ( albeit variably adjusted ) of all population - based , systematically identified prospective studies , a 1 unit / l increase of natural logged alt was associated with an 85% increase in diabetes risk and a 1 unit / l increase of natural logged ggt was associated with a 92% increase in diabetes risk . a second meta - analysis that assessed the association of both alt and ggt ( using fourths of their distribution ) with incident diabetes in the same populations was performed . belonging to the top fourth of the ggt distribution was associated with a 194% increase in the risk of diabetes compared with risk increase associated with belonging to the bottom fourth . for alt , the corresponding increase in the risk of diabetes was 102% , with formal evidence of heterogeneity between these two estimates ( both based on fully adjusted models ) . there are two possible explanations for the potentially stronger association of ggt with diabetes compared with that of alt . first , both alt and ggt are biomarkers of liver fat , but ggt may simply be the better marker . in light of the paucity of relevant evidence as to which liver enzyme however , ggt is present on the surface of most cell types and is highly active in organs other than the liver , such as the kidney and pancreas ( 35 ) . ggt is the enzyme responsible for the extracellular catabolism of antioxidant glutathione ( 36 ) and may be linked to greater oxidative stress ( 37 ) . because oxidative stress has been implicated in insulin resistance , diabetes , and cardiovascular disease ( 37,38 ) , ggt 's potentially stronger association with diabetes may reflect its associations with several different processes relevant to diabetes pathogenesis . although a stronger association of ggt ( compared with that of alt ) with diabetes is biologically plausible , our results should be interpreted with caution . the exclusion of a single observation substantially reduced the strength of evidence that the associations of alt and ggt with incident diabetes were different . only 6 of the 10 studies that presented results for both alt and ggt could be included in the meta - analyses of the risk estimates in the top fourth of the alt and ggt distributions versus the bottom fourth ( despite our best efforts to contact and obtain relevant information from authors , results in the form necessary to include the additional three studies were not forthcoming ) . an additional limitation is that two of the studies included in the systematic review could not be included in the meta - analysis of all studies as a result of missing information . attempts were made to obtain additional results from study authors , but this was not always possible . limitations of the bwhhs include the lack of oral glucose tolerance testing at baseline , the use of self - reported medical diagnoses of diabetes at follow - up , a less robust measure of insulin resistance ( homa ) , and the inclusion of women only . the latter is also the study 's strength because 6 of the 18 studies do not include any women . in conclusion , nafld and associated elevations in liver enzymes are associated with incident diabetes above and beyond commonly measured diabetes risk factors . results suggest that the association of ggt with diabetes risk may be of greater magnitude than that of alt with diabetes , but further studies are needed to confirm this . in the meantime , raised levels of both enzymes in the context of other clinical features of insulin resistance ( obesity , hypertriglyceridemia , raised fasting glucose , etc . )

objectiveto estimate and compare associations of alanine aminotransferase ( alt ) and -glutamyltransferase ( ggt ) with incident diabetes.research design and methodsalt and ggt were studied as determinants of diabetes in the british women 's heart and health study , a cohort of 4,286 women 6079 years old ( median follow - up 7.3 years ) . a systematic review and a meta - analysis of 21 prospective , population - based studies of ultrasonography , which diagnosed nonalcoholic fatty liver disease ( nafld ) , alt , and ggt as determinants of diabetes , were conducted , and associations of alt and ggt with diabetes were compared.resultsultrasonography-diagnosed nafld was associated with more than a doubling in the risk of incident diabetes ( three studies ) . alt and ggt both predicted diabetes . the fully adjusted hazard ratio ( hr ) for diabetes per increase in one unit of logged alt was 1.83 ( 95% ci 1.572.14 , i2 = 8% ) and for ggt was 1.92 ( 1.662.21 , i2 = 55% ) . to directly compare alt and ggt as determinants of diabetes , the fully adjusted risk of diabetes in the top versus bottom fourth of the alt and ggt distributions was estimated using data from studies that included results for both markers . for alt , the hr was 2.02 ( 1.592.58 , i2 = 27% ) , and for ggt the hr was 2.94 ( 1.983.88 , i2 = 20% ) , suggesting that ggt may be a better predictor ( p = 0.05).conclusionsfindings are consistent with the role of liver fat in diabetes pathogenesis . ggt may be a better diabetes predictor than alt , but additional studies with directly determined liver fat content , alt , and ggt are needed to confirm this finding .

RESEARCH DESIGN AND METHODS British Women's Heart and Health Study Systematic review and meta-analysis RESULTS British Women's Heart and Health Study Systematic review and meta-analysis Ultrasound-diagnosed NAFLD ALT and GGT Comparing associations of ALT and GGT with incident diabetes CONCLUSIONS Supplementary Material

a total of 4,286 women , aged 6079 years , were randomly recruited from 23 british towns . baseline diabetes ( for exclusion of these cases ) was defined according to the world health organization criteria as any woman with a doctor 's diagnosis of diabetes ( based on medical records review and self - report ) and/or with a fasting glucose concentration 7 incident cases of diabetes were defined as either a self - report of a doctor diagnosis of diabetes or evidence of diabetes in the follow - up medical record reviews . cox proportional hazards regression models were used to examine associations of exposures with incident diabetes in those with no evidence of diabetes at baseline . contributions to risk were censored at the date of diagnosis or death from any other cause or at the end of the follow - up period ( 30 september 2007 ) for those who remained alive and free of diabetes . in february 2008 for all prospective population - based studies evaluating the association among nafld ( diagnosed by any imaging technique ) , alt , ggt , and incident diabetes . in the first set , a separate meta - analysis was conducted for each nafld marker , maximizing data use regardless of whether they provided data on ultrasound - defined nafld , alt only , ggt only , or any combination of these . in these analyses , the assumption was that all study results for alt ( ggt or ultrasound ) were part of the same distribution but not that the true effect was the same in all studies ; therefore , a random - effects model was used , though we did also check our assumption for this random effect using the i measure , which quantifies the percentage of total variation across studies that is due to heterogeneity rather than chance ( 13 ) . , risk ratios [ rrs ] for quantiles of alt or ggt compared with the lowest quantile or per category or logged unit per liter ) , for the first meta - analysis ( of all studies ) a standard statistical method was used to estimate the log hazard ratio ( hr ) per log unit increase in alt or ggt , together with its se ( 14,15 ) . the objective of the second meta - analysis was to directly compare the associations of alt and ggt with diabetes . we decided a priori to limit this second analysis to results of studies that provided estimates for both alt and ggt in the same population in order to ensure the comparability of the estimates for alt and ggt . the most common method used to standardize alt and ggt was to present results per fourths of the alt and ggt distributions . therefore , we combined the rrs in the top versus the bottom fourth of the alt and ggt . the effect of duration of follow - up on study results was assessed by meta - regression . a total of 4,286 women , aged 6079 years , were randomly recruited from 23 british towns . baseline diabetes ( for exclusion of these cases ) was defined according to the world health organization criteria as any woman with a doctor 's diagnosis of diabetes ( based on medical records review and self - report ) and/or with a fasting glucose concentration 7 incident cases of diabetes were defined as either a self - report of a doctor diagnosis of diabetes or evidence of diabetes in the follow - up medical record reviews . cox proportional hazards regression models were used to examine associations of exposures with incident diabetes in those with no evidence of diabetes at baseline . contributions to risk were censored at the date of diagnosis or death from any other cause or at the end of the follow - up period ( 30 september 2007 ) for those who remained alive and free of diabetes . in february 2008 for all prospective population - based studies evaluating the association among nafld ( diagnosed by any imaging technique ) , alt , ggt , and incident diabetes . in the first set , a separate meta - analysis was conducted for each nafld marker , maximizing data use regardless of whether they provided data on ultrasound - defined nafld , alt only , ggt only , or any combination of these . in these analyses , the assumption was that all study results for alt ( ggt or ultrasound ) were part of the same distribution but not that the true effect was the same in all studies ; therefore , a random - effects model was used , though we did also check our assumption for this random effect using the i measure , which quantifies the percentage of total variation across studies that is due to heterogeneity rather than chance ( 13 ) . , risk ratios [ rrs ] for quantiles of alt or ggt compared with the lowest quantile or per category or logged unit per liter ) , for the first meta - analysis ( of all studies ) a standard statistical method was used to estimate the log hazard ratio ( hr ) per log unit increase in alt or ggt , together with its se ( 14,15 ) . the objective of the second meta - analysis was to directly compare the associations of alt and ggt with diabetes . we decided a priori to limit this second analysis to results of studies that provided estimates for both alt and ggt in the same population in order to ensure the comparability of the estimates for alt and ggt . the most common method used to standardize alt and ggt was to present results per fourths of the alt and ggt distributions . therefore , we combined the rrs in the top versus the bottom fourth of the alt and ggt . the effect of duration of follow - up on study results was assessed by meta - regression . of the remaining 3,452 women , 3,041 ( 88% of 3,452 ) characteristics of included women by diabetes status at the end of follow - up are presented in table a1 ( available in an online appendix at http://care.diabetesjournals.org/cgi/content/full/dc08-1870/dc1 ) . women with diabetes at the end of follow - up were older ; had higher age - adjusted mean bmi , waist - to - hip ratio , alt , ggt , triglycerides , glucose , and homa ; and had lower hdl cholesterol . more of the women with incident diabetes exercised for < 2 h per week , and more also had a history of smoking . hrs ( 95% ci ) of incident diabetes per sd change of natural logged alt and ggt model 1 , adjusted for age ; model 2 , model 1 adjustment plus alcohol consumption ; model 3 , model 2 adjustments plus childhood and adult social class , physical activity , and smoking ; model 4 , model 3 adjustments plus homa , waist - to - hip ratio , triglycerides , hdl cholesterol , and systolic blood pressure . during 281,081 person - years of follow - up , 112 women were diagnosed with diabetes , yielding a rate of 5.3 per 1,000 person - years ( 95% ci 4.46.4 ) . both alt and ggt were associated with incident diabetes , even when adjustment was made for alcohol consumption ( table 1 ) and other potential confounders . when components of the metabolic syndrome were added to the model , the associations of both alt and ggt with incident diabetes were attenuated toward the null . however , the magnitude of associations of alt with diabetes and of ggt with diabetes was comparable in all four models , as was the degree of attenuation of age - adjusted estimates . the electronic search ( crossing terms for alt , ggt , liver steatosis , and diabetes ) yielded 236 potentially relevant publications , of which 20 studied prospective population - based cohorts and assessed baseline ultrasonography - diagnosed nafld , alt , and/or ggt and their associations with incident diabetes . three studies ( 1820 ) ( summarized in table 2 ) assessed ultrasound - diagnosed nafld as a determinant of incident diabetes . no studies that used any other diagnostic test to ascertain nafld in its association with incident diabetes were retrieved . all three studies were conducted in asian populations , and in all three there was evidence that ultrasound - diagnosed nafld was associated with diabetes risk . [ ( 18 ) ] ) , the pooled rr was 2.52 ( 95% ci 1.075.96 ) , but there was evidence of considerable heterogeneity between studies ( i = 90% ) . study characteristics of the 18 prospective population - based studies of the associations of alt and/or ggt with incident diabetes are also summarized in table 2 . ten studies ( 46,2128 ) ( including the current analysis of the british women 's heart and health study [ bwhhs ] ) assessed both alt and ggt as predictors of diabetes , an additional three studies looked at alt only ( 2931 ) , and another four studies looked at the association of ggt with diabetes risk ( 3,3234 ) . age - adjusted and fully adjusted study results examining ( natural logged ) alt and ggt as determinants of incident diabetes were pooled ( separately ) . a change in one logged unit in alt was associated with an hr of 3.05 ( 95% ci 2.593.59 , i = 26% , 13 comparisons ) and in ggt 2.56 ( 2.312.84 , i = 32% , 17 comparisons ) ( online appendix fig . the meta - analysis of fully adjusted results for both alt and ggt are presented in fig . the fully adjusted hr for diabetes per increase in one unit of logged alt was 1.85 ( 1.572.18 , i = 19 , 14 comparisons ) , and in one unit of logged ggt the hr was 1.92 ( 1.662.21 , i = 55% , 18 comparisons ) . meta - analysis of fully adjusted results of alt and ggt as a determinant of incident diabetes . seven studies ( including the bwhhs ) contributed to the analysis of alt and ggt categorized in fourths as predictors of diabetes ( 4,5,21,22,24,25 ) . additional studies assessed both alt and ggt ; however , one used fifths ( 26,27 ) , another used tenths ( 6 ) , and a final study ( 28 ) used fourths but did not present results in full and therefore could not be included in this analysis . the rr in the top versus the bottom quartile of alt was 4.42 ( 95% ci 3.615.42 , i = 42% ) , and for ggt it was 5.87 ( 4.637.44 , i = 21% ) . the fully adjusted rr for alt was 2.02 ( 1.592.58 , i = 27% ) and for ggt 2.94 ( 1.983.88 , i = 20% ) ( fig . according to a formal statistical test for heterogeneity between estimates for alt and ggt , there was some evidence suggesting that pooled estimates for ggt were larger than those for alt ( age - adjusted results , p = 0.08 , and fully adjusted results , p = 0.05 ) . meta - analysis of fully adjusted results ( rr in top versus bottom fourth ) of alt and ggt as determinants of incident diabetes . however , repeating the analysis after excluding results for women in the desir ( data from the epidemiological study on the insulin resistance syndrome ) cohort ( 4 ) , which was the only study that showed a potential protective effect of alt , resulted in greater pooled estimates for ggt ( hr 2.91 [ 95% ci 2.193.85 ] ) than for alt ( 2.12 [ 1.662.72 ] ) but no strong evidence for heterogeneity between the estimates ( p 0.10 for both age - adjusted and fully adjusted results ) . of the remaining 3,452 women , 3,041 ( 88% of 3,452 ) characteristics of included women by diabetes status at the end of follow - up are presented in table a1 ( available in an online appendix at http://care.diabetesjournals.org/cgi/content/full/dc08-1870/dc1 ) . women with diabetes at the end of follow - up were older ; had higher age - adjusted mean bmi , waist - to - hip ratio , alt , ggt , triglycerides , glucose , and homa ; and had lower hdl cholesterol . more of the women with incident diabetes exercised for < 2 h per week , and more also had a history of smoking . hrs ( 95% ci ) of incident diabetes per sd change of natural logged alt and ggt model 1 , adjusted for age ; model 2 , model 1 adjustment plus alcohol consumption ; model 3 , model 2 adjustments plus childhood and adult social class , physical activity , and smoking ; model 4 , model 3 adjustments plus homa , waist - to - hip ratio , triglycerides , hdl cholesterol , and systolic blood pressure . during 281,081 person - years of follow - up , 112 women were diagnosed with diabetes , yielding a rate of 5.3 per 1,000 person - years ( 95% ci 4.46.4 ) . both alt and ggt were associated with incident diabetes , even when adjustment was made for alcohol consumption ( table 1 ) and other potential confounders . when components of the metabolic syndrome were added to the model , the associations of both alt and ggt with incident diabetes were attenuated toward the null . however , the magnitude of associations of alt with diabetes and of ggt with diabetes was comparable in all four models , as was the degree of attenuation of age - adjusted estimates . the electronic search ( crossing terms for alt , ggt , liver steatosis , and diabetes ) yielded 236 potentially relevant publications , of which 20 studied prospective population - based cohorts and assessed baseline ultrasonography - diagnosed nafld , alt , and/or ggt and their associations with incident diabetes . no studies that used any other diagnostic test to ascertain nafld in its association with incident diabetes all three studies were conducted in asian populations , and in all three there was evidence that ultrasound - diagnosed nafld was associated with diabetes risk . [ ( 18 ) ] ) , the pooled rr was 2.52 ( 95% ci 1.075.96 ) , but there was evidence of considerable heterogeneity between studies ( i = 90% ) . study characteristics of the 18 prospective population - based studies of the associations of alt and/or ggt with incident diabetes are also summarized in table 2 . ten studies ( 46,2128 ) ( including the current analysis of the british women 's heart and health study [ bwhhs ] ) assessed both alt and ggt as predictors of diabetes , an additional three studies looked at alt only ( 2931 ) , and another four studies looked at the association of ggt with diabetes risk ( 3,3234 ) . age - adjusted and fully adjusted study results examining ( natural logged ) alt and ggt as determinants of incident diabetes were pooled ( separately ) . a change in one logged unit in alt was associated with an hr of 3.05 ( 95% ci 2.593.59 , i = 26% , 13 comparisons ) and in ggt 2.56 ( 2.312.84 , i = 32% , 17 comparisons ) ( online appendix fig . the meta - analysis of fully adjusted results for both alt and ggt are presented in fig . the fully adjusted hr for diabetes per increase in one unit of logged alt was 1.85 ( 1.572.18 , i = 19 , 14 comparisons ) , and in one unit of logged ggt the hr was 1.92 ( 1.662.21 , i = 55% , 18 comparisons ) . meta - analysis of fully adjusted results of alt and ggt as a determinant of incident diabetes . seven studies ( including the bwhhs ) contributed to the analysis of alt and ggt categorized in fourths as predictors of diabetes ( 4,5,21,22,24,25 ) . additional studies assessed both alt and ggt ; however , one used fifths ( 26,27 ) , another used tenths ( 6 ) , and a final study ( 28 ) used fourths but did not present results in full and therefore could not be included in this analysis . the rr in the top versus the bottom quartile of alt was 4.42 ( 95% ci 3.615.42 , i = 42% ) , and for ggt it was 5.87 ( 4.637.44 , i = 21% ) . the fully adjusted rr for alt was 2.02 ( 1.592.58 , i = 27% ) and for ggt 2.94 ( 1.983.88 , i = 20% ) ( fig . according to a formal statistical test for heterogeneity between estimates for alt and ggt , there was some evidence suggesting that pooled estimates for ggt were larger than those for alt ( age - adjusted results , p = 0.08 , and fully adjusted results , p = 0.05 ) . meta - analysis of fully adjusted results ( rr in top versus bottom fourth ) of alt and ggt as determinants of incident diabetes . however , repeating the analysis after excluding results for women in the desir ( data from the epidemiological study on the insulin resistance syndrome ) cohort ( 4 ) , which was the only study that showed a potential protective effect of alt , resulted in greater pooled estimates for ggt ( hr 2.91 [ 95% ci 2.193.85 ] ) than for alt ( 2.12 [ 1.662.72 ] ) but no strong evidence for heterogeneity between the estimates ( p 0.10 for both age - adjusted and fully adjusted results ) . ultrasound - diagnosed nafld , alt , and ggt all predicted diabetes risk . nafld defined by ultrasound was associated with more than a doubling of the risk of incident diabetes . in a meta - analysis of fully adjusted results ( albeit variably adjusted ) of all population - based , systematically identified prospective studies , a 1 unit / l increase of natural logged alt was associated with an 85% increase in diabetes risk and a 1 unit / l increase of natural logged ggt was associated with a 92% increase in diabetes risk . a second meta - analysis that assessed the association of both alt and ggt ( using fourths of their distribution ) with incident diabetes in the same populations was performed . belonging to the top fourth of the ggt distribution was associated with a 194% increase in the risk of diabetes compared with risk increase associated with belonging to the bottom fourth . for alt , the corresponding increase in the risk of diabetes was 102% , with formal evidence of heterogeneity between these two estimates ( both based on fully adjusted models ) . first , both alt and ggt are biomarkers of liver fat , but ggt may simply be the better marker . because oxidative stress has been implicated in insulin resistance , diabetes , and cardiovascular disease ( 37,38 ) , ggt 's potentially stronger association with diabetes may reflect its associations with several different processes relevant to diabetes pathogenesis . although a stronger association of ggt ( compared with that of alt ) with diabetes is biologically plausible , our results should be interpreted with caution . the exclusion of a single observation substantially reduced the strength of evidence that the associations of alt and ggt with incident diabetes were different . only 6 of the 10 studies that presented results for both alt and ggt could be included in the meta - analyses of the risk estimates in the top fourth of the alt and ggt distributions versus the bottom fourth ( despite our best efforts to contact and obtain relevant information from authors , results in the form necessary to include the additional three studies were not forthcoming ) . an additional limitation is that two of the studies included in the systematic review could not be included in the meta - analysis of all studies as a result of missing information . limitations of the bwhhs include the lack of oral glucose tolerance testing at baseline , the use of self - reported medical diagnoses of diabetes at follow - up , a less robust measure of insulin resistance ( homa ) , and the inclusion of women only . in conclusion , nafld and associated elevations in liver enzymes are associated with incident diabetes above and beyond commonly measured diabetes risk factors . results suggest that the association of ggt with diabetes risk may be of greater magnitude than that of alt with diabetes , but further studies are needed to confirm this .

diabetes mellitus ( dm ) is characterized by endocrine - induced metabolic disorders with complex etiology . the major pathophysiological issue for dm patients is insufficient insulin ( ins ) secretion associated with the destruction of pancreatic islet -cells and insulin resistance ( ir ) , ultimately resulting in complications in numerous systems , such as the cardiovascular and renal systems [ 2 , 3 ] . dm - associated cardiovascular complications have the highest disability and mortality rates and have caused the greatest damage of all the chronic complications in china . until now , dm patients had to consistently receive injections of exogenous ins or take oral hypoglycemic drugs , and this long - term treatment induces drug side effects and creates a significant economic burden . therefore , it is worth exploring diet composition and nutritional status to prevent and/or identify therapies to treat this chronic disease , as it is seldom studied . polysaccharides , which belong to a class of essential organic compounds that form the base of all living creatures , are generated by combining multiple monosaccharides of the same type or of different types . with advancements in related research , the activity of marine algal polysaccharides has become a hot research topic worldwide [ 6 , 7 ] . marine algae are rich in polysaccharides , which account for more than 50% of their dry weight . studies have demonstrated that marine algal polysaccharides are predominantly sulfated polysaccharides ; the antioxidant activity of sulfated glucans is greater than that of regular glucans . previous studies have shown that many marine algal polysaccharides , including laminarin 13 , exopolysaccharide of porphyridium cruentum 18 , spirulina polysaccharides 22 , and alginic sodium diester 25 , play a role in lowering blood sugar and treating dm complications , such as hyperlipidemia [ 1013 ] . however , different marine algal polysaccharides contain different functional agents ; therefore , they may work to prevent or treat dm by different mechanisms and further studies in this area are warranted . enteromorpha prolifera belongs to the phylum chlorophyta , class chlorophyceae , order ulvales , and genus enteromorpha . it is a large , natural wild green alga that grows in offshore shoals and is widely distributed in rock pools in river mouths and tidal zones . it is highly abundant , and currently , the majority grows and dies without human management or control . enteromorpha prolifera naturally has a strong reproductive capacity , which can have a significant negative impact on the environment and aquaculture . the large - scale outbreak of large green algae , such as enteromorpha prolifera , is now called green tide , which is considered to be a type of marine disaster similar to red tide . however , if these algae could be utilized , then waste could be turned into a valuable resource . previous studies have demonstrated that pep have numerous functional bioactivities , such as strong antibacterial , anti - inflammatory , antitumor , and antioxidant functions , as well as the abilities to reinforce immunity and regulate blood lipid levels [ 1619 ] . however , whether pep has an anti - dm pharmacological effect has been seldom studied . based on these observations , the purpose of this study was to determine the anti - dm effects of pep in a rat model of dm and investigate the possible underlying mechanisms . our study provides experimental evidence for the clinical treatment of dm and the theoretical basis for the development and application of pep . ninety six - week - old male sprague - dawley ( sd ) rats weighting 90 10 g were obtained from the shanghai slac laboratory animal co. ltd . the experimental protocol was approved by the animal care and use committee of fujian medical university . the experimental procedures were carried out in accordance with international guidelines for the care and use of laboratory animals . a high - sugar , high - fat diet was prepared based on the following formula that was slightly modified from a previous study : 65% basal feed , 20% sucrose , 10% lard , 2.5% egg yolk powder , and 2.5% cholesterol . the basal feed was prepared and provided by the laboratory animal center of fujian medical university ; the main components were 30% corn , 21% soybean cake , 10% bran , 28% wheat middlings , 9% fish powder , 1.7% cahpo4 , 0.3% salt , 1.5% yeast , and 1% fish liver oil . pep was extracted and prepared in our laboratory from enteromorpha prolifera that was collected from the coastal region of fujian , china , using the water extraction - alcohol precipitation method . streptozotocin ( stz ) was obtained from sigma chemical co. ( st . louis , mo , usa ) . after one week of acclimatization , the rats were randomly divided into the control group of ten rats and model groups of eighty rats . then , the control group was fed a normal diet , and the model group was fed the above high - sugar , high - fat diet for four weeks . after four weeks , the model group was injected intraperitoneally with 30 mg / kg streptozotocin ( stz ) ; after 72 h , plasma glucose levels were determined , and a glucose concentration 200 mg / dl indicated that the hyperglycemic rat ( dm ) model is established . subsequently , 50 successful dm model rats were divided into five groups of ten rats each : the model , metformin hydrochloride ( metformin hc ) , and low- , medium- , and high - dose pep groups . the metformin hc group received 500 mg / kg metformin hydrochloride intragastrically , and the low- , medium- , and high - dose pep groups were dosed intragastrically with pep at 150 mg / kg , 300 mg / kg , and 600 mg / kg ( distilled water as the solvent ) , respectively , once per day . the doses of pep used in this study were selected based on our previous studies and in accordance with other previous reports [ 23 , 24 ] . , the rats were fasted for 12 hours and then sacrificed by decapitation , and the blood samples , perirenal white adipose tissue , liver , and pancreatic tissue samples were harvested and stored at 80c until use . after the rats were fasted for 12 hours , the blood sample was collected and fbg was determined on days 0 , 7 , 14 , 21 , and 28 after the intervention treatment using a blood glucose meter on the caudal vein . after measuring the fbg on day 28 , the rats in each group were dosed intragastrically with a 50% glucose solution ( 2 g / kg ) ; the blood glucose levels after 0.5 , 1 , and 2 h were measured for the ogtt . after the rats were fasted for 12 hours , the blood sample was collected and the serum fins levels for all rats in each group were measured using the elisa method . the isi is the reciprocal of the product derived from the multiplication of the fbg and ins levels . because this index demonstrated a skewed distribution , the natural logarithm malondialdehyde ( mda ) content was assayed using the tba method of buege and aust . glutathione peroxidase ( gph - px ) activity was assayed using the method of rotruck et al . . these biomolecules were quantified using test kits that were obtained from the nanjing jiancheng bioengineering institute ( nanjing , china ) . pancreatic tissues were fixed in a 10% neutral formalin solution for 24 h. after being washed overnight with running water , the samples were embedded in paraffin , sectioned , baked , and stained with hematoxylin and eosin ( h&e ) . a specific antibody against insulin / proinsulin was utilized to observe the residual -cells in islets by using immunohistochemical method ( ihc ) . the -cells in islets were calculated as integrated optical density per area ( mean integrated optical density ) using the image - pro plus 4.5 image analysis software . the mrna levels of gck , insr , glut-4 , and apn were determined using real - time pcr . total rna was extracted from each sample ( ten samples per group ) using the rnaiso plus reagent ( takara biotechnology co. , ltd . , rna was reverse - transcribed into cdna using the primescript reverse transcriptase reagent ( takara biotechnology co. , ltd . , dalian , china ) according to the manufacturer 's instructions . briefly , the following substrates were placed into a tube : 2.0 g of rna in 2 l , 1.0 l of 50 m oligo - dt primer , 1.0 l of primescript rt enzyme mix , 1.0 l of 100 m random hexamers , 5.0 l of 5 x primescript buffer , and 10.0 l of rnase - free dh2o , for a total volume of 20 l . the mixture was incubated at 37c for 15 min and at 85c for 5 sec . after reverse transcription , the samples were stored at 20c until polymerase chain reaction ( pcr ) was performed . real - time fluorescent quantitative pcr was carried out with the sybr green i fluorescent dye reagent ( sybr premix ex taq ii , takara biotechnology co. , ltd . ) and an abi system sequence detector 7500 ( applied biosystems inc . -actin was used as an internal standard to normalize all samples for potential variations in mrna content . amplifications were performed under the following conditions : stage 1 , 95c for 30 sec , 1 cycle ; stage 2 , 95c for 10 sec and 60c for 34 sec , 40 cycles . the relative expression levels of gck , insr , glut-4 , and apn were normalized by the internal standard -actin and expressed as a percentage of the -actin value . the oligonucleotide sequences of primers used for detecting the mrna expression of these markers are presented below : -actin forward primer : 5-ggagattactgccctggctccta-3 reverse primer : 5-gactcatcgtactcctgcttgctg-3 gck forward primer : 5-agtatgaccggatggtggatgaa-3 reverse primer : 5-ccagcttaagcagcacaagtcgta-3 insr forward primer : 5-tcatggatggaggctatctgga-3 reverse primer : 5-tccttgagcaggttgacgatttc-3 glut-4 forward primer : 5-ccgggacactataccctattca-3 reverse primer : 5-aggaccagtgtcccagtcactc-3 apn forward primer : 5-ctgtctgtacgagtgccagtgga-3 reverse primer : 5-cttcatgactgggcaggattaagag-3 forward primer : 5-ggagattactgccctggctccta-3 reverse primer : 5-gactcatcgtactcctgcttgctg-3 5-ggagattactgccctggctccta-3 5-gactcatcgtactcctgcttgctg-3 forward primer : 5-agtatgaccggatggtggatgaa-3 reverse primer : 5-ccagcttaagcagcacaagtcgta-3 5-agtatgaccggatggtggatgaa-3 5-ccagcttaagcagcacaagtcgta-3 forward primer : 5-tcatggatggaggctatctgga-3 reverse primer : 5-tccttgagcaggttgacgatttc-3 5-tcatggatggaggctatctgga-3 5-tccttgagcaggttgacgatttc-3 forward primer : 5-ccgggacactataccctattca-3 reverse primer : 5-aggaccagtgtcccagtcactc-3 5-ccgggacactataccctattca-3 5-aggaccagtgtcccagtcactc-3 forward primer : 5-ctgtctgtacgagtgccagtgga-3 reverse primer : 5-cttcatgactgggcaggattaagag-3 5-ctgtctgtacgagtgccagtgga-3 5-cttcatgactgggcaggattaagag-3 all data are expressed as the mean standard deviation . an lsd test was used to further analyze the differences between the two groups if equal variances were assumed . pep was separated to four components , pep-1 , pep-2 , pep-3 , and pep-4 , by deae - cellulose methods ( figure 1(a ) ) . pep-2 shows a single symmetric peak when it flows through sephadex-100 ( figure 1(b ) ) . infrared spectrum analysis shows that pep-2 is a sulfate ester - containing polysaccharide ( figure 1(c ) ) . gas chromatographic analysis shows that the monosaccharides of pep-2 were rhamnose , glucuronic acid , arabinose , fucose , xylose , and glucose ( figures 1(d1 ) and 1(d2 ) ) . the proportions of the monosaccharides are 5.12 : 1.32 : 3.38 : 1.62 : 1 : 1.03 , respectively . during the experiment , gut dysfunction and death were not observed in dm rats . however , body weight and daily food consumption were significantly lower in the model group compared with the control group ( p < 0.05 ) . on the contrary , the daily drinking water intake was significantly higher than that in the control group ( p < 0.05 ) . compared with the model group , drinking water intakes were lower in the medium- and high - dose pep groups ( p < 0.05 ) . body weight and food consumption were not significantly different between the low- , medium- , and high - dose pep groups , the metformin hc group , and the model group ( p > 0.05 ) ( table 1 ) . before the intragastric treatment ( on day 0 ) , the fbg was significantly higher in the model , metformin hc , and pep groups than in the control group ( p < 0.05 ) . one week after the intragastric intervention , the fbg levels decreased in all groups ; however , from weeks 1 through 4 , rats in the model group had a higher fbg than rats in the other groups . after four weeks of intragastric treatment , the fbg was lower in the pep and metformin hc groups than in the model group ( p < 0.05 ) . compared with day 0 , the fbg levels in the low- , medium- , and high - dose pep groups and in the metformin hc group decreased ( p < 0.05 ) ( table 2 ) . the ogtt indicated that the blood glucose levels in all the rats peaked 30 min after glucose injection and essentially recovered within 2 h. compared with the model group , the blood glucose levels recovered faster in the pep and metformin hc groups , but the differences between the pep groups were not significant ( figure 2 ) . at the end of the experiment , fins levels were higher and the isi was significantly lower in the model group compared with the control group ( p < 0.05 ) . compared with the model group , fins levels were lower in the high - dose pep and the metformin hc groups ( p < 0.05 ) . the isi was lower in the low- , medium- , and high - dose pep and metformin hc groups than in the model group ( p < 0.05 ) ( table 3 ) . compared with the control group , the mda levels were higher and the gsh - px activity was lower in the model group ( p < 0.05 ) ; although there were no significant differences in sod enzymatic activity ( p > 0.05 ) , it exhibited a decreasing trend . compared with the model group , the mda levels in the low- , medium- , and high - dose pep and metformin hc groups were lower ( p < 0.05 ) . the sod enzymatic activity in the low- , medium- , and high - dose pep groups increased . the gsh - px activity increased in the medium- and high - dose pep and metformin hc groups ( table 4 ) . light microscopy following h&e staining indicated that the pancreatic acini and islets were intact in the control group . in the model group , the islets were scattered , the islet volume was significantly decreased , most islet cells were atrophic and more lightly stained , there were fewer cells , the cytoplasm was significantly decreased , the nuclei exhibited large - scale condensation , and lymphocytic filtration was extensive around and invading into the islets . in the metformin hc group , the morphological changes were not as severe as those in the model group . in the medium- and high - dose pep groups , the number of islets and -cells was significantly increased , the morphology and structure were essentially normal , the cytoplasm was homogenous and in greater quantity , the nucleoli were clear , and occasionally a small amount of lymphocytic infiltration was observed ( figure 3 ) . the immunohistochemistry results indicated that , in the pancreatic islets , the positively stained cells containing cytoplasmic bright red granules were -cells . in the control group , the islets were predominantly round or oval with different sizes , defined edges , and intact morphology ; the islets were full of copious bright red granules , and the staining was dark . in the model group , the islet structure was damaged , the edges were irregular , the islet area was decreased , there were significantly fewer bright red granules , and the staining was light . compared with the model group , islets with darker staining were present in the metformin hc group , and there were significantly more bright red granules ; similar results were observed for the low- , medium- , and high - dose pep groups ( figure 4(a ) ) . the mean integrated optical density of -cells in islets was significantly lower in the model group compared with the control group ( p < 0.05 ) . compared with the model group , the mean integrated optical densities of -cells were significant increased in the low- , medium- , and high - dose groups ( p < 0.05 ) . however , the differences between the model group and metformin hc group were not significant ( figure 4(b ) ) . compared with the model group , insr mrna expression in the liver in the low- , medium- , and high - dose pep groups significantly increased ( p < 0.05 ) , and gck mrna expression in the liver in the low- , medium- , and high - dose pep and the metformin hc groups increased ( p < 0.05 ) . apn mrna expression in adipose tissue was higher in the high - dose pep group than in the model group ( p < 0.05 ) . glut-4 mrna expression in adipose tissue in the low- , medium- , and high - dose pep and the metformin hc groups was not significantly different from that in the model group ( p > 0.05 ) , but glut-4 expression exhibited an increasing trend wherein the expression level increased with increasing pep concentration ( figure 5 ) . it is primarily considered to result from a combination of genetic factors and various environmental factors . there is no drug or method for the prevention or treatment of dm that has a significant therapeutic effect , few toxic side effects , and an acceptable safety profile [ 3234 ] . therefore , studying nutritious compounds that have minimal toxic side effects and can have long - term use is important for the prevention and treatment of dm . due to the different adsorption capacities of polysaccharides by the deae cellulose method and the different retention capabilities of polysaccharides by the sephadex-100 gel method , we separated and highly purified polysaccharides from enteromorpha prolifera . by infrared spectrum analysis , the main components of the polysaccharides were carbohydrates , which not only are rich in sulfate esters but also contain ether compounds and unsaturated carbon chains , which is in accord with previous studies . the sulfate content of polysaccharides from enteromorpha prolifera was 19.2% in our study , which was higher than that of previous studies . gas chromatographic analysis showed that the monosaccharides of pep were rhamnose , glucuronic acid , arabinose , fucose , xylose , and glucose , which is inconsistent with a previous study in which galactose and mannose were observed . the discrepancies in these results may be due to differences in the species , origin , and season of enteromorpha . high blood glucose levels , hyperinsulinemia ( in the early stages ) , and low insulin sensitivity are the salient features of dm . impaired glucose tolerance ( igt ) occurs in the middle of the transition from normal to dm and is a primary feature of glucose metabolic disorders . the mechanism by which igt occurs may be associated with ir and impaired islet -cell function . the rapid changes in glucose tolerance and/or blood sugar levels are prone to induce dm ; therefore , ogtts and blood glucose levels can be utilized to monitor and control the occurrence and development of dm . this study confirmed that pep reduced blood glucose levels in dm rats and significantly improved igt with similar effects as the hypoglycemic agent metformin hc ; in particular , prominent effects were observed in the medium - dose pep group . serum insulin levels were significantly reduced in the high - dose pep group , and insulin sensitivity was significantly higher in all the pep intervention groups . furthermore , pep intervention alleviated the diabetes symptom of drinking more water without changing caloric intake and inducing gut dysfunction for food consumption and body weight had no changed . these results indicated that pep affected the regulation of glucose metabolism by improving igt , promoting insulin secretion by islet -cells , and enhancing insulin sensitivity . chronic exposure to high glucose levels increases the oxidation of glucose itself , which produces enediol and dihydroxy compounds as well as a significant amount of ros . therefore , in the model used in the study , the high - glucose , high - fat diet that was fed to the rats stimulated oxidative stress ; moreover , in combination with the stz injection that damaged the pancreas , the oxidative damage in the pancreas as well as liver was exacerbated , resulting in dm . gsh - px is a scavenger that protects the structure and functional integrity of the cell membrane by reducing hydrogen peroxide and lipid hydroperoxides . mda levels reflect the degree of lipid peroxidation in the body and indirectly reflect the degree of cellular damage . in this study , compared with the control group , mda levels increased , gsh - px activity decreased , and sod activity exhibited a decreasing trend in the model group , indicating that there was more severe oxidative damage in the dm rats , potentially attributable to the oxidative stress induced by the high - sugar , high - fat diet and stz . after four weeks of pep intervention , mda levels significantly decreased and sod and gsh - px activities increased in a dose - dependent manner in all the pep treatment groups compared with the model group . these results indicated that pep inhibited lipid peroxidation in the cell membrane , relieved cellular oxidative damage , and promoted the functional recovery of tissues by enhancing antioxidant activity and promoting the clearance of free radicals and peroxidation products , such as mda . the pathological examination of the pancreas revealed that the number of pancreatic islet cells in the rats in the high- and medium - dose pep groups significantly increased compared with the model group , whereas low - dose pep had a slightly weaker effect . the cellular morphology of the pancreatic islet cells was nearly normal in the rats in the high- and medium - dose pep groups . this result confirmed that pep facilitated the repair of tissue damage and exerted a protective function in the pancreas . it is currently understood that insulin acts by interacting with the insr on the cell membrane ( predominantly in hepatocytes , adipocytes , and muscle cells ) and that its functions rely on receptor density and affinity in the cell membrane . therefore , insr is the first key point of action for insulin , and the insr gene is critical for studies of ir . our study demonstrated that mrna level of insr was significantly higher in the low- , medium- , and high - dose pep groups than that in the model group ; this result was consistent with the majority of previous studies that have reported a reduction in both the number and affinity for insulin of membrane insr in liver and muscle tissue of dm rats with ir . the blood sugar and insulin levels in the model group remained high , indicating that pep reversed ir by upregulating the number of insr . gck is a key enzyme in the regulation of glucose metabolism and insulin secretion and is the first rate - limiting enzyme in glycolysis . it catalyzes the phosphorylation of glucose to glucose-6-phosphate , and this process is a prerequisite for liver glycogen synthesis . the functional loss or decreased expression of gck suppresses glucose utilization in the liver and decreases insulin secretion from islet -cells . studies have reported that gck is critical for beta cell hyperplasia under the condition of high - fat diet - induced ir . our study demonstrated that significantly higher gck mrna expression levels in the livers of rats in the low- , medium- , and high - dose pep groups compared with those in the model group ; the difference of gck mrna expression levels between the high - dose group and the metformin hc group was most dramatic . previous study showed that dietary phenolic compounds altered glucose metabolism and decreased the risk of type 2 diabetes by increasing the level of glucokinase mrna . therefore , it is possible that pep improved glucose metabolism in dm rats by upregulating gck mrna expression . glut-4 is a glycoprotein of 509 amino acids that is highly expressed in insulin - sensitive tissues , including muscle , adipose , and liver . these tissues also have high levels of glucose uptake and utilization ; these processes are regulated by blood glucose and insulin . 90% of glut-4 molecules are distributed on the inner cell membrane , and only a small portion is on the outer cell membrane . when the insulin stimulation signal is transmitted into cells , glut-4 translocates from the inner to the outer membrane , therefore increasing glucose transport . when glut-4 function is impaired , glucose uptake as well as utilization in peripheral tissues decreases , particularly in skeletal muscle and adipose tissue , leading to postreceptor ir . this study determined that glut-4 mrna expression in adipose tissue decreased in the rat models ; despite the fact that the differences between the intervention groups and the model group were not significant , pep intervention induced glut-4 mrna expression to various extents . previous study showed that the alterations in glut-4 mrna level in adipose tissue contributed to the physiological activities of n-3 pufa in preventing body fat accumulation and in regulating glucose metabolism in rats . therefore , it is possible that pep improved glucose metabolism by upregulating the glut-4 mrna expression . apn is composed of 247 amino acids ; the apn gene is located on 3q27 and encodes susceptibility genes for dm and metabolic syndrome . apn is a protective factor for ir , and therefore it is highly correlated with insulin sensitivity and plays an important role in ir . similar to insulin , apn promotes glucose and fat utilization and energy consumption in muscle and inhibits gluconeogenesis and the synthesis of fatty acids and cholesterol in the liver . apn is predominantly expressed in adipocytes , and the apn gene is only expressed in white adipose tissue ; therefore , changes in adipocyte number can affect apn expression . it has been demonstrated in animal models that injecting apn into lipoatrophic mice partially reversed ir and that the ability to control blood sugar levels with insulin in transgenic rats with high apn expression significantly increased . in humans , the direct injection of apn reduces blood sugar levels , and increased apn increases insulin sensitivity in obese , dm , and ir patients . therefore , adipocytokines , such as apn , reach target organs via various pathways to induce ir - related diseases , and the downregulation of related factors might prevent ir by improving insulin sensitivity . in this study , we determined that the difference in apn gene expression between the high - dose pep group and the model group was significant , whereas apn gene expression was higher in the low- and medium - dose pep groups than in the model group but did not reach significance . these results indicated that pep improved ir and enhanced insulin sensitivity by upregulating apn gene expression in rat white adipose tissue and that it therefore has potential for the prevention of dm . previous study had showed that enteromorpha compressa and polysaccharide fraction from enteromorpha prolifera exhibit potent antioxidant activity [ 17 , 18 ] . these studies indicate that pep might directly exhibit antioxidant activity to alleviate oxidative damage in the pancreas , and finally improve glucose metabolism . however , pep might improve glucose metabolism by upregulating the insr , gck , apn , and glut-4 mrna expression in our present study , which may alleviate oxidative damage for high glucose , ultimately result in nonenzymatic oxidation of proteins , and induce oxidative stress . therefore , in our present study , it is unclear whether the improvements in glucose drive the improvements in oxidative measures or vice versa . . the mechanism may relate to the antioxidant activity of pep and its ability to regulate the mrna level of insr , gck , apn , and glut-4 gene in liver and adipose tissue . these findings may suggest that pep could be useful for the therapy of diabetes mellitus .

this study investigated the effects of polysaccharides from enteromorpha prolifera ( pep ) on glucose metabolism in a rat model of diabetes mellitus ( dm ) . pep ( 0 , 150 , 300 , and 600 mg / kg ) was administered intragastrically to rats for four weeks . after treatment , fasting blood glucose ( fbg ) and insulin ( ins ) levels were measured , and the insulin sensitivity index ( isi ) was calculated . the morphopathological changes in the pancreas were observed . serum samples were collected to measure the oxidant - antioxidant status . the mrna expression levels of glucokinase ( gck ) and insulin receptor ( insr ) in liver tissue and glucose transporter type 4 ( glut-4 ) and adiponectin ( apn ) in adipose tissue were determined . compared with the model group , the fbg and ins levels were lower , the isi was higher , and the number of islet -cells was significantly increased in all the pep groups . in the medium- and high - dose pep groups , mda levels decreased , and the enzymatic activities of sod and gsh - px increased . the mrna expression of insr and gck increased in all the pep groups ; apn mrna expression increased in the high - dose pep group , and glut-4 mrna expression increased in adipose tissue . these findings suggest that pep is a potential therapeutic agent that can be utilized to treat dm .

1. Introduction 2. Material and Methods 3. Results 4. Discussion 5. Conclusion

diabetes mellitus ( dm ) is characterized by endocrine - induced metabolic disorders with complex etiology . the major pathophysiological issue for dm patients is insufficient insulin ( ins ) secretion associated with the destruction of pancreatic islet -cells and insulin resistance ( ir ) , ultimately resulting in complications in numerous systems , such as the cardiovascular and renal systems [ 2 , 3 ] . based on these observations , the purpose of this study was to determine the anti - dm effects of pep in a rat model of dm and investigate the possible underlying mechanisms . then , the control group was fed a normal diet , and the model group was fed the above high - sugar , high - fat diet for four weeks . after four weeks , the model group was injected intraperitoneally with 30 mg / kg streptozotocin ( stz ) ; after 72 h , plasma glucose levels were determined , and a glucose concentration 200 mg / dl indicated that the hyperglycemic rat ( dm ) model is established . subsequently , 50 successful dm model rats were divided into five groups of ten rats each : the model , metformin hydrochloride ( metformin hc ) , and low- , medium- , and high - dose pep groups . the metformin hc group received 500 mg / kg metformin hydrochloride intragastrically , and the low- , medium- , and high - dose pep groups were dosed intragastrically with pep at 150 mg / kg , 300 mg / kg , and 600 mg / kg ( distilled water as the solvent ) , respectively , once per day . , the rats were fasted for 12 hours and then sacrificed by decapitation , and the blood samples , perirenal white adipose tissue , liver , and pancreatic tissue samples were harvested and stored at 80c until use . after the rats were fasted for 12 hours , the blood sample was collected and fbg was determined on days 0 , 7 , 14 , 21 , and 28 after the intervention treatment using a blood glucose meter on the caudal vein . after measuring the fbg on day 28 , the rats in each group were dosed intragastrically with a 50% glucose solution ( 2 g / kg ) ; the blood glucose levels after 0.5 , 1 , and 2 h were measured for the ogtt . after the rats were fasted for 12 hours , the blood sample was collected and the serum fins levels for all rats in each group were measured using the elisa method . the isi is the reciprocal of the product derived from the multiplication of the fbg and ins levels . pancreatic tissues were fixed in a 10% neutral formalin solution for 24 h. after being washed overnight with running water , the samples were embedded in paraffin , sectioned , baked , and stained with hematoxylin and eosin ( h&e ) . the mrna levels of gck , insr , glut-4 , and apn were determined using real - time pcr . after reverse transcription , the samples were stored at 20c until polymerase chain reaction ( pcr ) was performed . the relative expression levels of gck , insr , glut-4 , and apn were normalized by the internal standard -actin and expressed as a percentage of the -actin value . the oligonucleotide sequences of primers used for detecting the mrna expression of these markers are presented below : -actin forward primer : 5-ggagattactgccctggctccta-3 reverse primer : 5-gactcatcgtactcctgcttgctg-3 gck forward primer : 5-agtatgaccggatggtggatgaa-3 reverse primer : 5-ccagcttaagcagcacaagtcgta-3 insr forward primer : 5-tcatggatggaggctatctgga-3 reverse primer : 5-tccttgagcaggttgacgatttc-3 glut-4 forward primer : 5-ccgggacactataccctattca-3 reverse primer : 5-aggaccagtgtcccagtcactc-3 apn forward primer : 5-ctgtctgtacgagtgccagtgga-3 reverse primer : 5-cttcatgactgggcaggattaagag-3 forward primer : 5-ggagattactgccctggctccta-3 reverse primer : 5-gactcatcgtactcctgcttgctg-3 5-ggagattactgccctggctccta-3 5-gactcatcgtactcctgcttgctg-3 forward primer : 5-agtatgaccggatggtggatgaa-3 reverse primer : 5-ccagcttaagcagcacaagtcgta-3 5-agtatgaccggatggtggatgaa-3 5-ccagcttaagcagcacaagtcgta-3 forward primer : 5-tcatggatggaggctatctgga-3 reverse primer : 5-tccttgagcaggttgacgatttc-3 5-tcatggatggaggctatctgga-3 5-tccttgagcaggttgacgatttc-3 forward primer : 5-ccgggacactataccctattca-3 reverse primer : 5-aggaccagtgtcccagtcactc-3 5-ccgggacactataccctattca-3 5-aggaccagtgtcccagtcactc-3 forward primer : 5-ctgtctgtacgagtgccagtgga-3 reverse primer : 5-cttcatgactgggcaggattaagag-3 5-ctgtctgtacgagtgccagtgga-3 5-cttcatgactgggcaggattaagag-3 all data are expressed as the mean standard deviation . gas chromatographic analysis shows that the monosaccharides of pep-2 were rhamnose , glucuronic acid , arabinose , fucose , xylose , and glucose ( figures 1(d1 ) and 1(d2 ) ) . however , body weight and daily food consumption were significantly lower in the model group compared with the control group ( p < 0.05 ) . on the contrary , the daily drinking water intake was significantly higher than that in the control group ( p < 0.05 ) . compared with the model group , drinking water intakes were lower in the medium- and high - dose pep groups ( p < 0.05 ) . body weight and food consumption were not significantly different between the low- , medium- , and high - dose pep groups , the metformin hc group , and the model group ( p > 0.05 ) ( table 1 ) . before the intragastric treatment ( on day 0 ) , the fbg was significantly higher in the model , metformin hc , and pep groups than in the control group ( p < 0.05 ) . one week after the intragastric intervention , the fbg levels decreased in all groups ; however , from weeks 1 through 4 , rats in the model group had a higher fbg than rats in the other groups . after four weeks of intragastric treatment , the fbg was lower in the pep and metformin hc groups than in the model group ( p < 0.05 ) . compared with day 0 , the fbg levels in the low- , medium- , and high - dose pep groups and in the metformin hc group decreased ( p < 0.05 ) ( table 2 ) . the ogtt indicated that the blood glucose levels in all the rats peaked 30 min after glucose injection and essentially recovered within 2 h. compared with the model group , the blood glucose levels recovered faster in the pep and metformin hc groups , but the differences between the pep groups were not significant ( figure 2 ) . at the end of the experiment , fins levels were higher and the isi was significantly lower in the model group compared with the control group ( p < 0.05 ) . compared with the model group , fins levels were lower in the high - dose pep and the metformin hc groups ( p < 0.05 ) . the isi was lower in the low- , medium- , and high - dose pep and metformin hc groups than in the model group ( p < 0.05 ) ( table 3 ) . compared with the control group , the mda levels were higher and the gsh - px activity was lower in the model group ( p < 0.05 ) ; although there were no significant differences in sod enzymatic activity ( p > 0.05 ) , it exhibited a decreasing trend . compared with the model group , the mda levels in the low- , medium- , and high - dose pep and metformin hc groups were lower ( p < 0.05 ) . the sod enzymatic activity in the low- , medium- , and high - dose pep groups increased . the gsh - px activity increased in the medium- and high - dose pep and metformin hc groups ( table 4 ) . in the model group , the islets were scattered , the islet volume was significantly decreased , most islet cells were atrophic and more lightly stained , there were fewer cells , the cytoplasm was significantly decreased , the nuclei exhibited large - scale condensation , and lymphocytic filtration was extensive around and invading into the islets . in the metformin hc group , the morphological changes were not as severe as those in the model group . in the medium- and high - dose pep groups , the number of islets and -cells was significantly increased , the morphology and structure were essentially normal , the cytoplasm was homogenous and in greater quantity , the nucleoli were clear , and occasionally a small amount of lymphocytic infiltration was observed ( figure 3 ) . in the control group , the islets were predominantly round or oval with different sizes , defined edges , and intact morphology ; the islets were full of copious bright red granules , and the staining was dark . in the model group , the islet structure was damaged , the edges were irregular , the islet area was decreased , there were significantly fewer bright red granules , and the staining was light . compared with the model group , islets with darker staining were present in the metformin hc group , and there were significantly more bright red granules ; similar results were observed for the low- , medium- , and high - dose pep groups ( figure 4(a ) ) . the mean integrated optical density of -cells in islets was significantly lower in the model group compared with the control group ( p < 0.05 ) . compared with the model group , the mean integrated optical densities of -cells were significant increased in the low- , medium- , and high - dose groups ( p < 0.05 ) . however , the differences between the model group and metformin hc group were not significant ( figure 4(b ) ) . compared with the model group , insr mrna expression in the liver in the low- , medium- , and high - dose pep groups significantly increased ( p < 0.05 ) , and gck mrna expression in the liver in the low- , medium- , and high - dose pep and the metformin hc groups increased ( p < 0.05 ) . apn mrna expression in adipose tissue was higher in the high - dose pep group than in the model group ( p < 0.05 ) . glut-4 mrna expression in adipose tissue in the low- , medium- , and high - dose pep and the metformin hc groups was not significantly different from that in the model group ( p > 0.05 ) , but glut-4 expression exhibited an increasing trend wherein the expression level increased with increasing pep concentration ( figure 5 ) . due to the different adsorption capacities of polysaccharides by the deae cellulose method and the different retention capabilities of polysaccharides by the sephadex-100 gel method , we separated and highly purified polysaccharides from enteromorpha prolifera . the sulfate content of polysaccharides from enteromorpha prolifera was 19.2% in our study , which was higher than that of previous studies . gas chromatographic analysis showed that the monosaccharides of pep were rhamnose , glucuronic acid , arabinose , fucose , xylose , and glucose , which is inconsistent with a previous study in which galactose and mannose were observed . high blood glucose levels , hyperinsulinemia ( in the early stages ) , and low insulin sensitivity are the salient features of dm . the rapid changes in glucose tolerance and/or blood sugar levels are prone to induce dm ; therefore , ogtts and blood glucose levels can be utilized to monitor and control the occurrence and development of dm . this study confirmed that pep reduced blood glucose levels in dm rats and significantly improved igt with similar effects as the hypoglycemic agent metformin hc ; in particular , prominent effects were observed in the medium - dose pep group . serum insulin levels were significantly reduced in the high - dose pep group , and insulin sensitivity was significantly higher in all the pep intervention groups . these results indicated that pep affected the regulation of glucose metabolism by improving igt , promoting insulin secretion by islet -cells , and enhancing insulin sensitivity . therefore , in the model used in the study , the high - glucose , high - fat diet that was fed to the rats stimulated oxidative stress ; moreover , in combination with the stz injection that damaged the pancreas , the oxidative damage in the pancreas as well as liver was exacerbated , resulting in dm . gsh - px is a scavenger that protects the structure and functional integrity of the cell membrane by reducing hydrogen peroxide and lipid hydroperoxides . in this study , compared with the control group , mda levels increased , gsh - px activity decreased , and sod activity exhibited a decreasing trend in the model group , indicating that there was more severe oxidative damage in the dm rats , potentially attributable to the oxidative stress induced by the high - sugar , high - fat diet and stz . after four weeks of pep intervention , mda levels significantly decreased and sod and gsh - px activities increased in a dose - dependent manner in all the pep treatment groups compared with the model group . these results indicated that pep inhibited lipid peroxidation in the cell membrane , relieved cellular oxidative damage , and promoted the functional recovery of tissues by enhancing antioxidant activity and promoting the clearance of free radicals and peroxidation products , such as mda . the pathological examination of the pancreas revealed that the number of pancreatic islet cells in the rats in the high- and medium - dose pep groups significantly increased compared with the model group , whereas low - dose pep had a slightly weaker effect . the cellular morphology of the pancreatic islet cells was nearly normal in the rats in the high- and medium - dose pep groups . it is currently understood that insulin acts by interacting with the insr on the cell membrane ( predominantly in hepatocytes , adipocytes , and muscle cells ) and that its functions rely on receptor density and affinity in the cell membrane . our study demonstrated that mrna level of insr was significantly higher in the low- , medium- , and high - dose pep groups than that in the model group ; this result was consistent with the majority of previous studies that have reported a reduction in both the number and affinity for insulin of membrane insr in liver and muscle tissue of dm rats with ir . the blood sugar and insulin levels in the model group remained high , indicating that pep reversed ir by upregulating the number of insr . gck is a key enzyme in the regulation of glucose metabolism and insulin secretion and is the first rate - limiting enzyme in glycolysis . the functional loss or decreased expression of gck suppresses glucose utilization in the liver and decreases insulin secretion from islet -cells . our study demonstrated that significantly higher gck mrna expression levels in the livers of rats in the low- , medium- , and high - dose pep groups compared with those in the model group ; the difference of gck mrna expression levels between the high - dose group and the metformin hc group was most dramatic . therefore , it is possible that pep improved glucose metabolism in dm rats by upregulating gck mrna expression . these tissues also have high levels of glucose uptake and utilization ; these processes are regulated by blood glucose and insulin . this study determined that glut-4 mrna expression in adipose tissue decreased in the rat models ; despite the fact that the differences between the intervention groups and the model group were not significant , pep intervention induced glut-4 mrna expression to various extents . previous study showed that the alterations in glut-4 mrna level in adipose tissue contributed to the physiological activities of n-3 pufa in preventing body fat accumulation and in regulating glucose metabolism in rats . therefore , it is possible that pep improved glucose metabolism by upregulating the glut-4 mrna expression . apn is predominantly expressed in adipocytes , and the apn gene is only expressed in white adipose tissue ; therefore , changes in adipocyte number can affect apn expression . therefore , adipocytokines , such as apn , reach target organs via various pathways to induce ir - related diseases , and the downregulation of related factors might prevent ir by improving insulin sensitivity . in this study , we determined that the difference in apn gene expression between the high - dose pep group and the model group was significant , whereas apn gene expression was higher in the low- and medium - dose pep groups than in the model group but did not reach significance . these results indicated that pep improved ir and enhanced insulin sensitivity by upregulating apn gene expression in rat white adipose tissue and that it therefore has potential for the prevention of dm . these studies indicate that pep might directly exhibit antioxidant activity to alleviate oxidative damage in the pancreas , and finally improve glucose metabolism . however , pep might improve glucose metabolism by upregulating the insr , gck , apn , and glut-4 mrna expression in our present study , which may alleviate oxidative damage for high glucose , ultimately result in nonenzymatic oxidation of proteins , and induce oxidative stress . the mechanism may relate to the antioxidant activity of pep and its ability to regulate the mrna level of insr , gck , apn , and glut-4 gene in liver and adipose tissue . these findings may suggest that pep could be useful for the therapy of diabetes mellitus .

to date no treatment can completely reverse the clinical outcomes associated with spinal cord injury ( sci ) . tissue injury following sci occurs in two phases : the first involves the primary mechanical injury itself , whereas the second is progressive , mediated over the course of days to weeks after sci by a variety of cytotoxic factors present within the injury environment . host cellular efforts to restrict progressive tissue injury , including glial reactivity , extracellular matrix deposition , and scar formation around the lesion site , subsequently give rise to a nonpermissive environment for axon regeneration and the endogenous recovery of function . this extrinsic antagonism of neurorepair is compounded by a reduction in the intrinsic growth capacity of adult central neurons . finding novel therapeutics that combat secondary injury , overcome the growth - inhibiting environment of the lesion site , and/or enhance the intrinsic capacity of neurons to regenerate is imperative for restoring function after sci . strategies combining cellular with pharmacological , molecular , or biomaterial approaches have shown the most promise in overcoming these obstacles to attain meaningful anatomical and functional repair . one encouraging adjunctive strategy for both surmounting the growth - inhibitory environment of the lesion and stimulating intrinsic regeneration has been the elevation of cyclic adenosine monophosphate ( cyclic amp ) , a second messenger molecule . sustained elevation of cyclic amp has been shown to facilitate the extension of neurites across inhibitory substrates such as myelin and myelin - associated glycoprotein , to promote neuroprotection by elevating antiapoptotic proteins , and to suppress immune cell activation and ensuing inflammation . the primary target of the cyclic amp pathway is the dna - bound , constitutive transcription factor creb , which upon phosphorylation by the cyclic amp - activated protein kinase a ( pka ) initiates the expression of numerous genes , including those that encode certain neuropeptides , neurotrophins , and arginase 1 ( a crucial enzyme in the synthesis of polyamines ; figure 1 ) . downstream products of arginase 1 , including the polyamine putrescine , have been shown to mediate several of cyclic amp 's effects on neurite outgrowth . supplementing glial - derived neurotrophic factor gene delivery with the substrate of arginase 1 , l - arginine , demonstrated significant reductions in contusion size in traumatic brain injury models , although without improvement in cognitive function . in paradigms of sci , augmenting peripheral nerve grafts with acidic fibroblast growth factor supplementation increased arginase 1 expression and polyamine synthesis and was associated with favorable m2 macrophage responses and axonal regeneration [ 8 , 9 ] . additionally , human mesenchymal stromal cell implants that overexpressed a key enzyme in the synthesis of polyamines , arginine decarboxylase , resulted in improved locomotor recovery and reduced scar formation following sci . based upon our previous studies demonstrating the ability of cyclic amp elevation to improve the effectiveness of sc implants after sci and the growing evidence that polyamine synthesis may mediate several of cyclic amp 's downstream effects on axon regeneration [ 3 , 4 , 6 ] , we investigated whether the supplementation of sc implants with putrescine , given either within 30 minutes or following a 1-week delay after contusive sci , could improve sc implant - host integration , axon growth support , and locomotor recovery in an experimental contusive sci paradigm . scs were obtained from the sciatic nerves of adult female fischer rats ( harlan co. , indianapolis , in ) as previously described [ 11 , 12 ] . scs were grown to confluence and thrice passaged onto new dishes . following previous methods , sc purity for implantation adult female fischer rats ( harlan co. , n = 34 ; 180200 g ) were housed according to the guidelines recommended by the nih and the guide for the care and use of animals . all animal procedures were approved by the institutional animal care and use committee of the university of miami . prior to surgery , rats were anesthetized ( 70 mg / kg ketamine , 5 mg / kg xylazine ) by intraperitoneal injection . the mascis weight drop device was used to induce a moderate thoracic ( t9 ) contusion injury . a laminectomy was performed at the t8 thoracic vertebra prior to injury in order to expose the dorsal surface of the underlying spinal cord ( t9 ) without perturbing the dura mater . the exposed spinal cord was then subjected to a moderate injury by dropping a 10 g rod from a height of 12.5 mm . those which had height or velocity errors exceeding 6% or a compression distance that was not within a range of 1.251.75 mm were immediately excluded [ 3 , 15 ] . according to these injury parameter criteria no animals required exclusion from the study . following injury , the muscles were sutured in layers and the skin was closed with metal wound clips . animals were allowed to recover in warmed cages with easy access to food and water . gentamicin ( 5 mg / kg ; abbott laboratories , north chicago , il ) was intramuscularly administered immediately after the surgery and repeated daily for 7 days . mg / kg ; reckitt benckiser , richmond , va ) was delivered subcutaneously following surgery and then daily for 2 days , accompanied by postoperative treatment as described previously . prior to sci induction , animals were randomly assigned into one of the following three treatment groups : ( i ) sc implant with 0.9% physiological saline ( vehicle ) control ( n = 11 ) , ( ii ) sc implant with acute putrescine ( administered within 30 minutes of injury , n = 14 ) , and ( iii ) sc implant with delayed putrescine ( administered 1 week after injury at time of implantation , n = 9 ) . putrescine ( 100 mm ) was delivered at a rate of 0.5 l / hr using two sequentially implanted subcutaneous alzet minipumps ( model 2001 ; durect corp . , cupertino , ca ) for a 2-week administration period ; emptied pumps were replaced after 1 week . , scs were trypsinized , harvested , counted , resuspended in dmem / f12 media as aliquots and placed on ice prior to surgery . one week after injury the rats were anesthetized with 2% halothane and the injured spinal cord was reexposed . , all animals received 2 10 scs in 6 l of dmem - f12 media into the epicenter of the contused area , injected at the midline to a depth of 1 mm . injections were performed at a rate of 2 l / min using a 10 l siliconized hamilton syringe with a pulled , beveled glass pipette tip ( 120 m diameter ) , which was held in a micromanipulator with an attached microinjector ( world precision instruments , sarasota , fl ) . the injection pipette was held in place for an additional 3 min postinjection to minimize leakage upon withdrawal . one animal was excluded from the study due to a malfunction of the microinjector during cell transplantation . following the injection , the muscle layers and the skin rats were euthanized at 10 weeks after injury ( 9 weeks after implantation ; 100 mg / kg ketamine , 10 mg / kg xylazine ) and transcardially perfused with 4% paraformaldehyde ( pfa , 0.1 m , ph 7.4 ) . the tissue was then dissected and sectioned with a freezing microtome for immunohistochemical analysis according to previous procedures . spinal cords ( 2 cm ) encompassing the injury epicenter were sectioned sagittally at 40 m into five series free - floating . as previously described , every fifth sagittal section was immunochemically stained for subsequent fluorescent microscopic analysis using a double labeling procedure involving monoclonal and polyclonal antibodies . these antibodies were raised against the low - affinity neurotrophin receptor ( p75 ; 1 : 5,000 ; abcam , cambridge , ma ) , serotonin ( 5ht ; 1 : 5,000 ; immunostar , hudson , wi ) , or calcitonin gene related peptide ( cgrp ; 1 : 1,000 ; peninsula laboratories , san carlos , ca ) for identifying the implanted scs or delineating the growth of specific descending or ascending axonal populations , respectively , within the p75 sc implants . sections were then washed and incubated with corresponding fluorescent secondary antibodies ( alexa 488- or alexa 594-conjugated goat anti - rabbit or anti - mouse antibodies , 1 : 200 ; molecular probes , eugene , or ) . sections were mounted onto snowcoat x - tra slides ( surgipath , richmond , il ) and cover - slipped with vectashield mounting medium ( vector laboratories , burlingame , ca ) containing the nuclear dye hoechst for storage at 4c . in one series of sections , the average area of p75-positive cellular immunoreactivity was quantified across three sections containing the center of the sc implant . expression of p75 permits delineation of the implant and identification of the implant - host cord interface . for this analysis , stained sections were visualized and quantitatively assessed using an unbiased method employing computer - assisted fluorescence microscopy and neurolucida software ( version 4.5 ; microbrightfield bioscience , williston , vt ) . the p75 area for each of the three sections that comprised the center of the sc implant was traced under a 20x objective , quantified using neurolucida , and then the sc implant area calculated for each animal as the average p75 area across the three sections . quantification of 5-hydroxytryptophan ( 5ht ) labeled axon growth along the rostral - caudal axis of the spinal cord at distances of 1000 , 500 , 100 , and 0 m rostral to the center of the implant ( as identified using p75 immunoreactivity ) was performed on the second series of sections using 63x objective under oil immersion . calcitonin gene related peptide ( cgrp ) labeled axons were quantified similarly , but at 1000 , 500 , 100 , and 0 m caudal to the center of the implant . the average number of fibers per section was obtained by counting those immunostained axons that crossed imaginary lines perpendicular to the rostral - caudal axis at the 1000 , 500 , 100 , and 0 m intervals from the center of the implant . the total number of axons counted for a given animal was summated across the sections analyzed ( ~1012 sections per animal ) and then divided by the number of sections to determine the number of fibers per section ( f / s ) at each distance . the open field bbb locomotor test developed by basso et al in addition , a subscore analysis of the bbb that allows for evaluation of hind limb positioning and placement as well as balance and tail use on a 013 point scale was also used as described previously by our group . lastly , deficits in descending motor control were examined by counting footfall errors on an irregularly spaced horizontal grid walk at the end of the study , prior to animal perfusion . a one - way anova and subsequent bonferroni post hoc test were used to compare counts of immunostained axons among groups . for analysis of weekly functional recovery patterns following implantation ( bbb and bbb subscore ) , a mixed factorial ( repeated measures ) anova followed by the tukey - kramer posttest was employed . differences were acknowledged to be statistically significant at p < 0.05 , < 0.01 , and < 0.001 , compared to the sc only control or the other temporal treatment group , as indicated . all errors are given as standard errors of the mean . statistical comparison of sc implant size among control and treatment cohorts showed a significant effect of treatment ( f2,31 = 5.784 , p < 0.05 ) . compared to the implant size in the sc only group ( 1.23 0.07 mm ) , those animals receiving putrescine administration beginning at the time of implantation exhibited 1.8-fold increase in implant area ( 2.24 0.34 mm ; t33 = 3.391 , p < 0.01 ; figures 3(a ) , 3(c ) , and 3(d ) ) as identified using p75 immunochemistry ( a marker highly expressed on scs ) . in contrast , putrescine administration beginning at the time of injury was without effect ( 1.56 0.19 mm , p < 0.05 ) . serotonergic axons descending from the reticular formation and raphe nuclei of the brainstem were identified penetrating the sc implant using immunostaining for 5ht ( figures 4(a)4(c ) ; white arrows ) . for all animal groups there were few serotonergic axons that were able to penetrate beyond the center of the sc implant and no treatment effect was observed at this location ( f2,31 = 1.414 , p > 0.05 ; figure 3(c ) ) . however , statistical comparison of 5ht axon numbers within the rostral part of the sc implant showed a significant treatment effect ( f2,31 = 4.104 , p < 0.05 at 1000 m , f2,31 = 6.200 , p < 0.01 at 500 m , and f2,31 = 41.15 , p < 0.001 at 100 m from the center of the implant ) . acute putrescine supplementation significantly increased the number of serotonergic fibers within the proximal sc implant compared to the vehicle control : an average of 20 1.2 5ht fibers per section ( f / s ) at 500 m ( t33 = 4.829 , p < 0.01 ) and 9 0.6 f / s at 100 m ( t33 = 12.82 , p < 0.001 ) was quantified rostral to the center of the sc implant , while in vehicle controls 13 1.8 5ht f / s and 2 0.4 f / s , respectively , were observed at these distances ( figures 4(a)-4(b ) ) . in addition , many more 5ht axons were observed crossing the host - sc implant interface in acute putrescine treated animals ( figures 4(a)-4(b ) ; white arrows ) . delayed putrescine supplementation produced a significant increase in 5ht axon numbers only at 100 m rostral to the center of the sc implant ( 5.6 0.8 f / s , t33 = 6.011 , p < 0.001 ) . small diameter sensory axons originating from dorsal root ganglia that were immunoreactive for cgrp were quantified within the sc implant ( figures 5(a)5(c ) ; white arrows ) . statistical comparison of cgrp axon numbers among groups showed a significant treatment effect at both 100 m caudal to ( f2,31 = 35.17 , p < 0.001 ) and within the center of the sc implant ( f2,31 = 16.16 , p < 0.001 ) . at 100 m caudal to the center of the sc implant , acute and delayed putrescine supplementation resulted in 2.3-fold ( 19 1.0 f / s , t33 = 11.86 , p < 0.001 ) and 1.7-fold ( 14 0.8 f / s , t33 = 5.941 , p < 0.001 ) increases in cgrp axon numbers , respectively , compared to the vehicle control ( 8.3 0.77 f / s ) . in addition , acute putrescine administration ( 11 1.2 f / s ) significantly increased cgrp axon ingrowth within the center of the sc implant compared to the vehicle control ( 2.8 0.4 f / s , t33 = 9.491 , p < 0.001 , a 3.9-fold increase ) . acute putrescine was also significantly more effective in promoting cgrp axon growth than delayed delivery at both 100 m caudal to ( 14 0.8 f / s , t33 = 4.935 , p < 0.01 , a 1.4-fold increase ) and within the center of the sc implant ( 5.3 0.7 f / s , t33 = 6.257 , p < 0.001 , a 2.1-fold increase ) . locomotor performance and functional recovery following sci , sc implantation , and putrescine treatment were assessed using the bbb score , bbb subscore , and the irregularly spaced grid walk test . statistical comparison of bbb scores among groups showed a significant treatment effect both at endpoint ( f2,31 = 4.303 , p < 0.05 ) and at various time points after implantation ( f2,31 = 8.520 , p < 0.01 at week 3 ; f2,31 = 4.771 , p < 0.05 at week 5 ; figure 6(a ) ) . compared to the sc implant , vehicle controls ( 11.3 0.3 , 10 weeks after sci ) , open field locomotor performance was significantly greater at endpoint in both the acute ( 12.1 0.1 , t33 = 4.079 , p < 0.05 ) and delayed ( 11.9 0.3 , t33 = 3.929 , p < 0.05 ) putrescine with sc implant treatment groups . statistical comparison of bbb subscores among groups showed a significant effect of treatment at all weeks after implantation ( f2,31 = 4.320 , p < 0.05 , 10 weeks after sci ; figure 6(b ) ) . compared to sc implant , vehicle controls ( 3.0 0.3 , 10 weeks post - sci ) , hind paw placement and tail positioning were significantly superior in both acute ( 6.6 1.1 , t33 = 3.631 , p < 0.05 ) and delayed ( 7.0 1.4 , t33 = 3.574 , p < 0.05 ) putrescine with sc implant treatment groups . statistical comparison of the number of footfall errors on the grid walk among groups showed a significant effect of treatment ( f2,31 = 16.76 , p < 0.001 ; figure 6(c ) ) . acute putrescine administration with sc implantation resulted in significantly better hind paw placement performance as evidenced by a lower number of footfalls on the grid walk ( 5.9 0.2 footfalls ) compared to both delayed administration ( 7.3 0.2 footfalls , t33 = 3.832 , p < 0.01 ) and the sc implant , vehicle controls ( 7.8 0.4 footfalls , t33 = 5.538 , p < 0.001 ) . bridging the hostile environment of the injured spinal cord with implanted scs has been shown to be an effective foundation approach for a number of combinatorial repair strategies in a diverse array of experimental sci models [ 3 , 4 , 19 ] . in addition to ensheathing and myelinating axons , scs provide growth - promoting neurotrophins , which may limit tissue loss and encourage axons to overcome the growth - inhibitory injury milieu . importantly , scs can be safely harvested from a patient 's own peripheral nerve and autologously implanted back into his spinal cord , a major advantage to their use in the clinical setting . however , sc implants by themselves can not entice significant numbers of axons to exit the injury site , are unable to support robust growth of most supraspinal axon populations except under specific conditions , and provide only modest improvements in functional outcome [ 13 , 15 , 21 ] . in order to mediate significant anatomical repair and/or functional improvements , sc implants must be augmented with various pharmacological , molecular , or biomaterial approaches that overcome intrinsic or extrinsic inhibitors of axon growth , such as neurotrophin supplementation , chondroitinase abc , polysialic acid , matrix suspension , or cyclic amp elevation [ 4 , 19 , 22 ] . in the current study we have demonstrated that the administration of the initial polyamine , putrescine , when combined with sc implants , significantly improved the size of the implant ~2-fold and thus potentially the number of surviving , implanted scs . in addition , acute putrescine administration enhanced the sprouting and/or sparing of both descending serotonergic and ascending peptidergic axons within the implant / lesion site by up to 400% . delaying the administration of putrescine mirrored the effects of the acute model , but generally with a reduced effectiveness . the improvements in implant size and axon growth support following putrescine administration were accompanied by enhanced functional recovery , including significant gains in open field locomotor ability and hind paw placement compared to animals receiving sc implants alone . we have previously shown the therapeutic potential of combining sc implantation with cyclic amp elevation . elevated levels of cyclic amp are critical in facilitating the growth of axons over inhibitory substrates , such as myelin in vitro , and across the growth - retarding milieu of the injured spinal cord . the upregulation of arginase 1 , a downstream effector of cyclic amp that synthesizes the precursor for polyamines , has similarly been shown to enhance axonal regeneration , whereas blocking ornithine decarboxylase , the rate limiting enzyme which decarboxylates the product of arginase 1 into putrescine , attenuates the ability of dibutyryl cyclic amp and brain - derived neurotrophic factor to overcome growth antagonism by myelin - associated inhibitors [ 610 ] . the polyamine putrescine can further be converted into the polyamines spermidine and spermine ; however the reverse reactions release toxic aldehyde byproducts , making putrescine the polyamine of choice for experimental or clinical application of polyamines [ 23 , 24 ] . in addition to its axon growth - promoting abilities , putrescine has also been shown to act as a neuroprotectant , preventing neuronal cell death following ischemia or trauma , likely through its antioxidant and free radical scavenging actions [ 25 , 26 ] . in addition , polyamines are thought to be incorporated into the elf5a factor , which has been reported to mediate the neurotrophic and neuroprotective effects of nerve growth factor . taken together , these studies evidence the therapeutic benefit of using polyamines to enhance the functionality of cellular implants after sci . in the current study , sc implants were visualized immunohistochemically using an antibody against p75 , the low - affinity nerve growth factor receptor highly expressed in scs both in culture and following spinal cord implantation . we show that delayed , but not acute , supplementation of sc implants with putrescine resulted in an 80% increase in the size of the implant . from the overlap in administration time between the acute and delayed paradigms and the absence of an effect with acute putrescine , it is unlikely that the observed enhancement in implant size is due to a direct stimulation of sc survival or proliferation by putrescine but instead requires the presence of additional host - supplied factors . as both endogenous sc in - migration and axonal ingrowth occur with a 13-week delay after sci , the later delivery of putrescine may enhance endogenous sc in - migration and/or work in concert with signals from in - growing axons to stimulate proliferation of implanted scs [ 3 , 28 ] . following traumatic sci , endogenous scs have been shown to dedifferentiate in the spinal nerve roots , migrate into the injury site , proliferate , and significantly upregulate p75 expression [ 28 , 29 ] . migration and proliferation of endogenous scs occur between 1 and 3 weeks following injury , which temporally corresponds with our delayed putrescine supplementation paradigm . in addition , axon presence is one of the strongest signals for inducing sc mitogenesis and proliferation , and there may be insufficient growth of axons within the implant / lesion site during the first week after sci to stimulate sc proliferation acutely . therefore , implanted sc proliferation and an ensuing increase in implant size may be best benefited by the combination of putrescine and axonal growth signals during the delayed administration period . whether an increased area of p75 immunoreactivity denotes an enhanced proliferation of implanted scs or an enhanced proliferation and/or migration of endogenous scs into the lesion is difficult to determine unequivocally without cell tracking methods . however , depletion of polyamines in a variety of cell types has been shown to impair cell migration by reorganizing the f - actin of lamellipodia , impeding microtubule reformation , and preventing the accumulation of -actin and -tubulin mrna [ 3032 ] . on the other hand , scs are largely mitotically quiescent under normal physiological conditions , proliferating only during development , wallerian degeneration , or a demyelinating condition or insult . the enhanced proliferative capacity of scs during wallerian degeneration has been closely associated with an elevation of ornithine decarboxylase activity , suggesting that putrescine supplementation may promote the proliferation of either endogenous or exogenous scs within the lesion [ 33 , 34 ] . amplified activity of ornithine decarboxylase and polyamines and the elf5a translation factor derived from polyamines have all been shown to upregulate proliferation of a number of cell types [ 35 , 36 ] . further investigations are needed to ascertain the exact mechanism by which p75 and sc implant size are enhanced following sci , implantation , and delayed supplementation of putrescine . in addition to mediating cell migration and proliferation , putrescine and other downstream polyamines have also been shown to promote cell survival . deficiencies in putrescine levels cause swelling of the endoplasmic reticulum and golgi bodies , as well as the disappearance of stress fibers , all of which are hallmarks associated with necrosis . intracellular polyamines can alter ion transport , either blocking k channels to prevent neuron excitotoxicity or promoting ca influxes [ 38 , 39 ] . polyamine - induced ion influxes have been shown to be required to induce the release of 5ht synaptosomes , which may help restore serotonergic axon functionality after sci . sc implants , when used in combinatory approaches , were previously shown to improve the regeneration of serotonergic fibers following sci . here we observed that supplementing sc implants with putrescine significantly increased the number of serotonergic fibers proximal to the center of the implant / lesion , an effect strengthened by administering the putrescine acutely . serotonergic axon projections within the spinal cord are believed to be important for the recovery of locomotion following injury . while it is difficult to determine whether the increase in the numbers of serotonergic fibers is due to the regeneration of axotomized axons , collateral sprouting from existing serotonergic fibers , or sparing of axons , an enhanced presence of serotonergic axons following putrescine administration may provide important stimulatory effects for improved locomotor functioning . future use of a complete spinal cord transection paradigm with sc grafting would provide a means for identifying whether such 5ht axon growth is due to regeneration , sprouting , or sparing and , when combined with neuroanatomical tracing , whether the serotonergic axons are from supraspinal or intraspinal neurons . the growth of primary sensory afferents , immunoreactive for cgrp , which have terminations in the superficial lamina of the spinal cord and are associated with transmission of pain , also benefitted from the delivery of putrescine , with enhanced axon numbers within the center of the sc implant . cgrp - positive neurons have also been implicated in the modulation of fine motor control and the control of posture and thus their growth could be indicative of changes in locomotor function . primary dorsal horn afferent fibers are known to sprout into more medial lamina of the spinal cord following sci , again making it challenging to ascertain whether the increasing number of cgrp - positive fibers was due to regenerating sensory afferents or collateral sprouts . like 5ht axon growth , acute administration of putrescine following sci and sc implantation generated the greatest amount of cgrp fiber growth , almost four times as many fibers compared to scs alone and more than twice as many fibers as delayed supplementation . these cgrp axons may have arisen from drgns present at the level of the lesion or from drgns located more distally . further investigation is needed to explore the mechanisms by which putrescine encourages primary sensory afferents to surmount the inhibitory environment of the host cord - implant interface and penetrate deeply into the sc implants as well as the use of tracing techniques to identify the level(s ) from which responding drgns are located . putrescine supplementation of sc implants also resulted in significant functional improvement , as measured in the open field with the bbb score and on the grid walk test . irrespective of the timing of putrescine delivery , enhanced locomotor ability , as measured by using the bbb score and subscore , was obtained with treatment compared to sc implanted , vehicle controls . overall , both acute and delayed putrescine administration generated similar improvements in hind limb performance and tail posturing , including a one - point higher bbb score and five - point higher bbb subscore . only the acute delivery of putrescine , however , reduced footfall errors on the horizontal grid walk test , by 25% compared to the vehicle control . the effectiveness of acute putrescine only on this outcome measure would indicate that tissue preservation would be a likely mechanistic explanation for this effect . the observed functional improvements following supplementation of sc implants with putrescine are in addition to those that are known to be provided when sc implants alone are used after sci [ 4 , 13 ] . the most pronounced behavioral improvement was displayed by the bbb subscore , in which a persistent improvement in outcome was observed starting at 1 week after implantation . this highly acute benefit may imply that these effects acted through putrescine - mediated neuro- or axopreservation . the later improvements in bbb score and the continuous improvement in subscore may in turn be due to the reparative actions instigated by the initial delivery of putrescine . in conclusion , we supply evidence that modulating polyamines in conjunction with cell implantation is an effective approach for enhancing the functionality of the implant as well as locomotor recovery after sci . future research should seek to gain a greater understanding of the mechanisms underlying putrescine 's effects on sc function , migration , and survival , as well as its ability to promote axon sparing , growth , and/or sprouting .

schwann cell ( sc ) transplantation exhibits significant potential for spinal cord injury ( sci ) repair and its use as a therapeutic modality has now progressed to clinical trials for subacute and chronic human sci . although sc implants provide a receptive environment for axonal regrowth and support functional recovery in a number of experimental sci models , axonal regeneration is largely limited to local systems and the behavioral improvements are modest without additional combinatory approaches . in the current study we investigated whether the concurrent delivery of the polyamine putrescine , started either 30 min or 1 week after sci , could enhance the efficacy of scs when implanted subacutely ( 1 week after injury ) into the contused rat spinal cord . polyamines are ubiquitous organic cations that play an important role in the regulation of the cell cycle , cell division , cytoskeletal organization , and cell differentiation . we show that the combination of putrescine with scs provides a significant increase in implant size , an enhancement in axonal ( sensory and serotonergic ) sparing and/or growth , and improved open field locomotion after sci , as compared to sc implantation alone . these findings demonstrate that polyamine supplementation can augment the effectiveness of scs when used as a therapeutic approach for subacute sci repair .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Conclusions

to date no treatment can completely reverse the clinical outcomes associated with spinal cord injury ( sci ) . host cellular efforts to restrict progressive tissue injury , including glial reactivity , extracellular matrix deposition , and scar formation around the lesion site , subsequently give rise to a nonpermissive environment for axon regeneration and the endogenous recovery of function . finding novel therapeutics that combat secondary injury , overcome the growth - inhibiting environment of the lesion site , and/or enhance the intrinsic capacity of neurons to regenerate is imperative for restoring function after sci . the primary target of the cyclic amp pathway is the dna - bound , constitutive transcription factor creb , which upon phosphorylation by the cyclic amp - activated protein kinase a ( pka ) initiates the expression of numerous genes , including those that encode certain neuropeptides , neurotrophins , and arginase 1 ( a crucial enzyme in the synthesis of polyamines ; figure 1 ) . downstream products of arginase 1 , including the polyamine putrescine , have been shown to mediate several of cyclic amp 's effects on neurite outgrowth . in paradigms of sci , augmenting peripheral nerve grafts with acidic fibroblast growth factor supplementation increased arginase 1 expression and polyamine synthesis and was associated with favorable m2 macrophage responses and axonal regeneration [ 8 , 9 ] . based upon our previous studies demonstrating the ability of cyclic amp elevation to improve the effectiveness of sc implants after sci and the growing evidence that polyamine synthesis may mediate several of cyclic amp 's downstream effects on axon regeneration [ 3 , 4 , 6 ] , we investigated whether the supplementation of sc implants with putrescine , given either within 30 minutes or following a 1-week delay after contusive sci , could improve sc implant - host integration , axon growth support , and locomotor recovery in an experimental contusive sci paradigm . a laminectomy was performed at the t8 thoracic vertebra prior to injury in order to expose the dorsal surface of the underlying spinal cord ( t9 ) without perturbing the dura mater . prior to sci induction , animals were randomly assigned into one of the following three treatment groups : ( i ) sc implant with 0.9% physiological saline ( vehicle ) control ( n = 11 ) , ( ii ) sc implant with acute putrescine ( administered within 30 minutes of injury , n = 14 ) , and ( iii ) sc implant with delayed putrescine ( administered 1 week after injury at time of implantation , n = 9 ) . one week after injury the rats were anesthetized with 2% halothane and the injured spinal cord was reexposed . , all animals received 2 10 scs in 6 l of dmem - f12 media into the epicenter of the contused area , injected at the midline to a depth of 1 mm . following the injection , the muscle layers and the skin rats were euthanized at 10 weeks after injury ( 9 weeks after implantation ; 100 mg / kg ketamine , 10 mg / kg xylazine ) and transcardially perfused with 4% paraformaldehyde ( pfa , 0.1 m , ph 7.4 ) . the p75 area for each of the three sections that comprised the center of the sc implant was traced under a 20x objective , quantified using neurolucida , and then the sc implant area calculated for each animal as the average p75 area across the three sections . quantification of 5-hydroxytryptophan ( 5ht ) labeled axon growth along the rostral - caudal axis of the spinal cord at distances of 1000 , 500 , 100 , and 0 m rostral to the center of the implant ( as identified using p75 immunoreactivity ) was performed on the second series of sections using 63x objective under oil immersion . calcitonin gene related peptide ( cgrp ) labeled axons were quantified similarly , but at 1000 , 500 , 100 , and 0 m caudal to the center of the implant . the average number of fibers per section was obtained by counting those immunostained axons that crossed imaginary lines perpendicular to the rostral - caudal axis at the 1000 , 500 , 100 , and 0 m intervals from the center of the implant . the open field bbb locomotor test developed by basso et al in addition , a subscore analysis of the bbb that allows for evaluation of hind limb positioning and placement as well as balance and tail use on a 013 point scale was also used as described previously by our group . differences were acknowledged to be statistically significant at p < 0.05 , < 0.01 , and < 0.001 , compared to the sc only control or the other temporal treatment group , as indicated . statistical comparison of sc implant size among control and treatment cohorts showed a significant effect of treatment ( f2,31 = 5.784 , p < 0.05 ) . compared to the implant size in the sc only group ( 1.23 0.07 mm ) , those animals receiving putrescine administration beginning at the time of implantation exhibited 1.8-fold increase in implant area ( 2.24 0.34 mm ; t33 = 3.391 , p < 0.01 ; figures 3(a ) , 3(c ) , and 3(d ) ) as identified using p75 immunochemistry ( a marker highly expressed on scs ) . however , statistical comparison of 5ht axon numbers within the rostral part of the sc implant showed a significant treatment effect ( f2,31 = 4.104 , p < 0.05 at 1000 m , f2,31 = 6.200 , p < 0.01 at 500 m , and f2,31 = 41.15 , p < 0.001 at 100 m from the center of the implant ) . acute putrescine supplementation significantly increased the number of serotonergic fibers within the proximal sc implant compared to the vehicle control : an average of 20 1.2 5ht fibers per section ( f / s ) at 500 m ( t33 = 4.829 , p < 0.01 ) and 9 0.6 f / s at 100 m ( t33 = 12.82 , p < 0.001 ) was quantified rostral to the center of the sc implant , while in vehicle controls 13 1.8 5ht f / s and 2 0.4 f / s , respectively , were observed at these distances ( figures 4(a)-4(b ) ) . delayed putrescine supplementation produced a significant increase in 5ht axon numbers only at 100 m rostral to the center of the sc implant ( 5.6 0.8 f / s , t33 = 6.011 , p < 0.001 ) . statistical comparison of cgrp axon numbers among groups showed a significant treatment effect at both 100 m caudal to ( f2,31 = 35.17 , p < 0.001 ) and within the center of the sc implant ( f2,31 = 16.16 , p < 0.001 ) . at 100 m caudal to the center of the sc implant , acute and delayed putrescine supplementation resulted in 2.3-fold ( 19 1.0 f / s , t33 = 11.86 , p < 0.001 ) and 1.7-fold ( 14 0.8 f / s , t33 = 5.941 , p < 0.001 ) increases in cgrp axon numbers , respectively , compared to the vehicle control ( 8.3 0.77 f / s ) . in addition , acute putrescine administration ( 11 1.2 f / s ) significantly increased cgrp axon ingrowth within the center of the sc implant compared to the vehicle control ( 2.8 0.4 f / s , t33 = 9.491 , p < 0.001 , a 3.9-fold increase ) . locomotor performance and functional recovery following sci , sc implantation , and putrescine treatment were assessed using the bbb score , bbb subscore , and the irregularly spaced grid walk test . compared to the sc implant , vehicle controls ( 11.3 0.3 , 10 weeks after sci ) , open field locomotor performance was significantly greater at endpoint in both the acute ( 12.1 0.1 , t33 = 4.079 , p < 0.05 ) and delayed ( 11.9 0.3 , t33 = 3.929 , p < 0.05 ) putrescine with sc implant treatment groups . statistical comparison of bbb subscores among groups showed a significant effect of treatment at all weeks after implantation ( f2,31 = 4.320 , p < 0.05 , 10 weeks after sci ; figure 6(b ) ) . compared to sc implant , vehicle controls ( 3.0 0.3 , 10 weeks post - sci ) , hind paw placement and tail positioning were significantly superior in both acute ( 6.6 1.1 , t33 = 3.631 , p < 0.05 ) and delayed ( 7.0 1.4 , t33 = 3.574 , p < 0.05 ) putrescine with sc implant treatment groups . statistical comparison of the number of footfall errors on the grid walk among groups showed a significant effect of treatment ( f2,31 = 16.76 , p < 0.001 ; figure 6(c ) ) . acute putrescine administration with sc implantation resulted in significantly better hind paw placement performance as evidenced by a lower number of footfalls on the grid walk ( 5.9 0.2 footfalls ) compared to both delayed administration ( 7.3 0.2 footfalls , t33 = 3.832 , p < 0.01 ) and the sc implant , vehicle controls ( 7.8 0.4 footfalls , t33 = 5.538 , p < 0.001 ) . bridging the hostile environment of the injured spinal cord with implanted scs has been shown to be an effective foundation approach for a number of combinatorial repair strategies in a diverse array of experimental sci models [ 3 , 4 , 19 ] . importantly , scs can be safely harvested from a patient 's own peripheral nerve and autologously implanted back into his spinal cord , a major advantage to their use in the clinical setting . however , sc implants by themselves can not entice significant numbers of axons to exit the injury site , are unable to support robust growth of most supraspinal axon populations except under specific conditions , and provide only modest improvements in functional outcome [ 13 , 15 , 21 ] . in order to mediate significant anatomical repair and/or functional improvements , sc implants must be augmented with various pharmacological , molecular , or biomaterial approaches that overcome intrinsic or extrinsic inhibitors of axon growth , such as neurotrophin supplementation , chondroitinase abc , polysialic acid , matrix suspension , or cyclic amp elevation [ 4 , 19 , 22 ] . in the current study we have demonstrated that the administration of the initial polyamine , putrescine , when combined with sc implants , significantly improved the size of the implant ~2-fold and thus potentially the number of surviving , implanted scs . delaying the administration of putrescine mirrored the effects of the acute model , but generally with a reduced effectiveness . the improvements in implant size and axon growth support following putrescine administration were accompanied by enhanced functional recovery , including significant gains in open field locomotor ability and hind paw placement compared to animals receiving sc implants alone . elevated levels of cyclic amp are critical in facilitating the growth of axons over inhibitory substrates , such as myelin in vitro , and across the growth - retarding milieu of the injured spinal cord . the upregulation of arginase 1 , a downstream effector of cyclic amp that synthesizes the precursor for polyamines , has similarly been shown to enhance axonal regeneration , whereas blocking ornithine decarboxylase , the rate limiting enzyme which decarboxylates the product of arginase 1 into putrescine , attenuates the ability of dibutyryl cyclic amp and brain - derived neurotrophic factor to overcome growth antagonism by myelin - associated inhibitors [ 610 ] . the polyamine putrescine can further be converted into the polyamines spermidine and spermine ; however the reverse reactions release toxic aldehyde byproducts , making putrescine the polyamine of choice for experimental or clinical application of polyamines [ 23 , 24 ] . in addition , polyamines are thought to be incorporated into the elf5a factor , which has been reported to mediate the neurotrophic and neuroprotective effects of nerve growth factor . taken together , these studies evidence the therapeutic benefit of using polyamines to enhance the functionality of cellular implants after sci . in the current study , sc implants were visualized immunohistochemically using an antibody against p75 , the low - affinity nerve growth factor receptor highly expressed in scs both in culture and following spinal cord implantation . we show that delayed , but not acute , supplementation of sc implants with putrescine resulted in an 80% increase in the size of the implant . from the overlap in administration time between the acute and delayed paradigms and the absence of an effect with acute putrescine , it is unlikely that the observed enhancement in implant size is due to a direct stimulation of sc survival or proliferation by putrescine but instead requires the presence of additional host - supplied factors . as both endogenous sc in - migration and axonal ingrowth occur with a 13-week delay after sci , the later delivery of putrescine may enhance endogenous sc in - migration and/or work in concert with signals from in - growing axons to stimulate proliferation of implanted scs [ 3 , 28 ] . following traumatic sci , endogenous scs have been shown to dedifferentiate in the spinal nerve roots , migrate into the injury site , proliferate , and significantly upregulate p75 expression [ 28 , 29 ] . in addition , axon presence is one of the strongest signals for inducing sc mitogenesis and proliferation , and there may be insufficient growth of axons within the implant / lesion site during the first week after sci to stimulate sc proliferation acutely . therefore , implanted sc proliferation and an ensuing increase in implant size may be best benefited by the combination of putrescine and axonal growth signals during the delayed administration period . however , depletion of polyamines in a variety of cell types has been shown to impair cell migration by reorganizing the f - actin of lamellipodia , impeding microtubule reformation , and preventing the accumulation of -actin and -tubulin mrna [ 3032 ] . amplified activity of ornithine decarboxylase and polyamines and the elf5a translation factor derived from polyamines have all been shown to upregulate proliferation of a number of cell types [ 35 , 36 ] . further investigations are needed to ascertain the exact mechanism by which p75 and sc implant size are enhanced following sci , implantation , and delayed supplementation of putrescine . deficiencies in putrescine levels cause swelling of the endoplasmic reticulum and golgi bodies , as well as the disappearance of stress fibers , all of which are hallmarks associated with necrosis . sc implants , when used in combinatory approaches , were previously shown to improve the regeneration of serotonergic fibers following sci . here we observed that supplementing sc implants with putrescine significantly increased the number of serotonergic fibers proximal to the center of the implant / lesion , an effect strengthened by administering the putrescine acutely . while it is difficult to determine whether the increase in the numbers of serotonergic fibers is due to the regeneration of axotomized axons , collateral sprouting from existing serotonergic fibers , or sparing of axons , an enhanced presence of serotonergic axons following putrescine administration may provide important stimulatory effects for improved locomotor functioning . future use of a complete spinal cord transection paradigm with sc grafting would provide a means for identifying whether such 5ht axon growth is due to regeneration , sprouting , or sparing and , when combined with neuroanatomical tracing , whether the serotonergic axons are from supraspinal or intraspinal neurons . the growth of primary sensory afferents , immunoreactive for cgrp , which have terminations in the superficial lamina of the spinal cord and are associated with transmission of pain , also benefitted from the delivery of putrescine , with enhanced axon numbers within the center of the sc implant . cgrp - positive neurons have also been implicated in the modulation of fine motor control and the control of posture and thus their growth could be indicative of changes in locomotor function . primary dorsal horn afferent fibers are known to sprout into more medial lamina of the spinal cord following sci , again making it challenging to ascertain whether the increasing number of cgrp - positive fibers was due to regenerating sensory afferents or collateral sprouts . like 5ht axon growth , acute administration of putrescine following sci and sc implantation generated the greatest amount of cgrp fiber growth , almost four times as many fibers compared to scs alone and more than twice as many fibers as delayed supplementation . further investigation is needed to explore the mechanisms by which putrescine encourages primary sensory afferents to surmount the inhibitory environment of the host cord - implant interface and penetrate deeply into the sc implants as well as the use of tracing techniques to identify the level(s ) from which responding drgns are located . putrescine supplementation of sc implants also resulted in significant functional improvement , as measured in the open field with the bbb score and on the grid walk test . irrespective of the timing of putrescine delivery , enhanced locomotor ability , as measured by using the bbb score and subscore , was obtained with treatment compared to sc implanted , vehicle controls . only the acute delivery of putrescine , however , reduced footfall errors on the horizontal grid walk test , by 25% compared to the vehicle control . the effectiveness of acute putrescine only on this outcome measure would indicate that tissue preservation would be a likely mechanistic explanation for this effect . the observed functional improvements following supplementation of sc implants with putrescine are in addition to those that are known to be provided when sc implants alone are used after sci [ 4 , 13 ] . the most pronounced behavioral improvement was displayed by the bbb subscore , in which a persistent improvement in outcome was observed starting at 1 week after implantation . the later improvements in bbb score and the continuous improvement in subscore may in turn be due to the reparative actions instigated by the initial delivery of putrescine . in conclusion , we supply evidence that modulating polyamines in conjunction with cell implantation is an effective approach for enhancing the functionality of the implant as well as locomotor recovery after sci . future research should seek to gain a greater understanding of the mechanisms underlying putrescine 's effects on sc function , migration , and survival , as well as its ability to promote axon sparing , growth , and/or sprouting .

on february 4 , 2009 , a 62-cent increase in the federal cigarette tax was enacted , along with increases in other tobacco taxes , to fund expansion of the state children 's health insurance program ( schip ) . the federal cigarette tax increased to $ 1.01 per pack on april 1 , 2009 . increasing the price of tobacco through excise taxes is an effective way to encourage quit attempts and thus to decrease the prevalence of smoking [ 2 , 3 ] . it is estimated that a 10% increase in cigarette prices leads to a 4% decrease in cigarette consumption in high - income countries and about 8% in low - to - middle income countries [ 2 , 4 ] . a 70% increase in current tobacco prices could prevent 25% of all smoking - related deaths globally and higher taxes have a greater impact on the young and low income smokers by deterring smoking initiation and encouraging smokers to quit . telephone quitlines are an effective population - based form of smoking cessation treatment and their utilization has been shown to be responsive to tobacco control policies [ 59 ] . therefore , the study aims were to ( 1 ) describe call volumes to 16 state quitlines before and after the tax increase ; ( 2 ) examine the characteristics of tobacco users who enrolled with quitlines before and after the tax increase and ( 3 ) examine the outcomes ( quit rates ) of tobacco users who enrolled with state quitlines before and after the tax increase . analyses were conducted to determine whether implementation of the federal tax on april 1 , 2009 coincided with ( 1 ) increased calls to state quitlines ; ( 2 ) increased calls from people with low education levels ; ( 3 ) increased quit rates ( the higher cigarette prices may motivate those attempting to quit to remain quit ) . it was expected that the increase in calls may begin prior to april 1 , 2009 and as early as february 2009 as people become aware of the passage of the federal tax increase and in response to preemptive cigarette price increases instituted by the tobacco industry in december 2008 and march 2009 [ 10 , 11 ] . two different data sources were used : one is based on administrative data collected from all callers and the other is a seven - month follow - up interview with a random sample of quitline participants . administrative data comes from the free & clear database for state tobacco quitlines that tracks call volumes , completed counseling calls and caller characteristics obtained during registration with the quitline . this data comes from 16 of the 17 state quitlines operated by free & clear , inc . the one state that was not included in the analysis had incomplete data for the time period before the tax increase . participating states include alaska , connecticut , georgia , hawaii , indiana , maine , maryland , missouri , north carolina , oklahoma , oregon , south carolina , utah , virginia , washington and wisconsin , whose smokers represented 24% of smokers in the united states in 2009 . data from the seven - month followup comes from four state quitlines and is based on random samples of quitline participants in each state timed to occur seven months from enrollment with the quitline . all 16 states agreed to participate in this study and to contribute their data to the pooled dataset . the 16 state quitlines represent different geographic regions and states with varying state laws ( e.g. , tobacco control programs with varying resources and programmatic activities ) . as well , they have a variety of cessation services such as offering free nicotine replacement therapy ( nrt ) through their quitline during the study period . since states regularly change their services , we chose to portray a snapshot of offerings during the study period ( table 1 ) . all quitlines provided mailed support materials ( quit guides ) , a single reactive ( inbound ) counseling call to all tobacco users , and three or four additional outbound calls to select groups ( e.g. , those ready to quit within 30-days ) . some state quitlines refer insured tobacco users to cessation benefits offered through their health plan or employer . all but four states offered at least some free nrt ( patch or gum ) depending on the state - approved eligibility criteria ( e.g. , insurance status ) . all of the participating states used the same data collection methods and a common questionnaire to collect demographic and tobacco use data at intake and follow - up thus enhancing data comparability across state quitlines and across study years . analyses examined both pooled monthly call volume ( total calls to quitlines ) and pooled daily call volume . state - level data was not examined as this was a descriptive study to assess whether a volume change would be observed in aggregate data from callers to the quitline in 16 states . however , in statistical models of outcomes , state was included as a fixed effect to account for unmeasured variability within and between states . monthly data was examined from december 2008 through august 2009 and from a similar time period the year before for comparison ( december 2007 through august 2008 ) in order to show call volume prior to the tax increase ( december 2008 , january 2009 , february 2009 , march 2009 ) , during the months that the tobacco industry increased prices in anticipation of the tax increase ( december 2008 through march 2009 ) , during the month the tax increase was passed ( february 2009 ) , and after the tax increase took effect ( april 2009 , may 2009 anticipated to have heavy call volumes ) . daily call volume was then examined directly before and after the april 1 , 2009 tax increase to determine when the calls began to increase in anticipation of the tax increase and how soon the calls returned to previous levels . the time period selected for this analysis was march - april 2009 ( march - april 2008 was also examined for comparative purposes ) . a comprehensive set of variables collected when a person enrolls with a state quitline was used to describe caller characteristics . variables included participant demographics ( age , gender , race / ethnicity , insurance status , educational level ) , current tobacco use ( tobacco type , amount used ) , duration of smoking , time to first cigarette upon waking , living or working with smokers , and how they heard about the quitline . chronic disease status was assessed by asking : have you been diagnosed with any of the following conditions ; asthma , chronic obstructive pulmonary disease or emphysema , coronary artery disease or heart disease or diabetes ? responses were captured with a yes or no to each chronic disease . data from the seven - month followup survey was obtained for persons who enrolled from march 2009may 2009 ( and for comparison march 2008may 2008 ) . this time period was selected for comparisons of demographics and quit rates because this was the period in which the impact of the tax would most likely be observed . the four states with available data ( i.e. , some did not conduct the seven - month survey during these time periods and others used another organization to conduct the seven - month survey and their data was unavailable ) used similar survey sampling protocols and similar questionnaires . information collected at the seven - month follow - up included use of medications since enrolling with the quitline and current smoking status . successful cessation was defined as seven - day and 30-day abstinence by asking participants : when was the last time you smoked a cigarette , even a puff ? our questionnaire included the standard battery of questions used in the minimum data set ( mds ) instrument recommended by the north american quitline consortium ( naqc ) . data across all states was pooled and presented in aggregate form in graphs and tables . first , the total number of monthly calls to the 16 quitlines was collected and presented in a figure as well as the number of tobacco users who received one or more counseling calls from december 2007 through august 2008 and december 2008 through august 2009 . rao - scott chi - square and t - test statistics were used to compare characteristics of callers during the time of the 2009 tax increase and for the same months in the prior year and included state as a fixed variable to account for the variability in services provided across quitlines . a p value of 0.01 was used as a cut - off value for the hypotheses tests because of the large sample size . for the four states with data from the seven - month follow - up , multivariate logistic regression analyses were used to examine tobacco abstinence outcomes ( 7-day point prevalence and 30-day point prevalence ) comparing callers during the time of the 2009 tax increase to callers during the same time period in the prior year . again , state was included as a fixed variable , as well as case - mix covariates that differed before versus after the tax increase ( age , race , education , chronic conditions , how they heard about the quitline , and amount smoked at intake ) , and gender because it is associated with cessation outcomes [ 14 , 15 ] . for those who enrolled with the multicall program , utilization of services ( number of counseling calls completed ) and quit outcomes was also assessed in multivariate and logistic regression analyses . outcomes were reported in two ways : first among those who completed the survey ( respondent analysis ) and second using the intent - to - treat ( itt ) analysis whereby persons with missing outcomes data are assumed to be smoking . the logistic model was logit(probability of abstinence ( yes / no ) ) = overall mean+time indicator variable+individual covariates + state + callprogram + howheard , where time indicator variable = before or after ( 0 or 1 ) , callprogram = one versus multicall program and howheard = how participant heard about the quitline figure 1 presents the number of calls to the 16 quitlines over time and shows the spike in calls during march - april 2009 . overall , there was a 23.5% increase in total call volume when comparing december 2007may 2008 ( 84,541 calls ) to december 2008may 2009 ( 104,452 calls ) . in 2009 , calls increased beginning in march and began to taper off in may ( a 59.1% increase in call volumes comparing march 2008may 2008 ( 38,919 calls ) to march 2009may 2009 ( 61,935 calls ) ) . comparing each month in 2008 - 2009 with the corresponding month from the prior year , increases in call volume were observed in december 2008 and february through may 2009 , with the largest percent increase ( 94.1% ) occurring in march 2009 . increases during march and april 2009 occurred both in total call volume ( calls from tobacco users , friends , family , health care professionals , and the general public seeking information ) , as well as in the number of tobacco users per month who received at least one counseling call . data for june , july , and august are not shown in figure 1 since the tax effect on call volumes had returned to the before tax levels in may . note that the observed increase in quitline calls ( and enrollments ) around january 1 for both time periods was expected and is often attributed to new years ' resolutions . this was the case in january 2008 whereby the spike in calls corresponds to promotional activities of one large state quitline . in post hoc analyses , omitting this state from the sample resulted in a similar pattern ( although a lower number of calls ) as that shown in figure 1 . figure 2 shows a more detailed analysis of call volumes around the april 2009 tax increase ; the daily call volumes from march through april 2009 compared to daily call volumes from march through april 2008 . daily call volume was higher in 2009 than 2008 , particularly from march 8 through april 26 . table 2 shows results of the comparisons of demographic and other characteristics between tobacco users who enrolled with the quitline before and after the announcement and implementation of the april 2009 federal tax increase . the time periods for this analysis were march 1 , 2008 through may 31 , 2008 versus march 1 , 2009 through may 31 , 2009 ( the window of time showing the peak activity in call volumes ) . results reveal differences in callers between the two time periods . in the after tax period , although the mean age of callers was slightly younger ( 41.9 versus 41.2 ) , fewer callers were aged 1824 years ( 11.5% after tax versus 13.6% before tax ) . more callers in 2009 ( compared with the prior year ) were white , had less than a high school education , were more likely to live with a smoker , had shorter durations of cigarettes smoking , and were more likely to report hearing about the quitline from family or friends or their health care provider , rather than from the media . although fewer callers enrolled in the multicall program ( 4 - 5 counseling calls ) after tax , they completed slightly more counseling sessions compared with those who enrolled for the multiple calls before tax ( 1.9 versus 2.2 , respectively , p < 0.0001 ) . although there were differences in the prevalence of chronic disease ( copd and cad ) and use of other tobacco products when comparing callers after the tax increase to those before the tax increase , these differences between the callers in these two time periods were small . table 3 shows results of analyses of seven - month outcomes data and suggests that participant quit rates did not differ significantly before versus after the tax . these results held for unadjusted and adjusted analyses of seven - day and 30-day respondent and intent - to - treat analyses . for example , seven - day respondent quit rates were 30.7% before and 28.7% after the tax ( o.r . analyses of the subgroup that participated in the multicall program showed a similar lack of change in smoking status after tax compared with a similar period before the tax . this study 's results are consistent with prior research showing that implementing an increase in excise taxes on tobacco will drive calls to the state tobacco control programs ' free quitline services [ 3 , 17 ] . reported an increase in call volumes to the montana quitline following an increase in the state 's cigarette taxes . they also reported that the tax attracted younger smokers to call the quitline , as well as more female and white smokers and heavier smokers . in the current study , although smokers who called the quitline around the time of the federal tax increase were more likely to be white , no significant differences were found for gender or amount smoked . however , fewer young tobacco users ( age 1824 ) and fewer smokers with smoking durations of 20 years called around the time of the tax increase . because tax increases tend to decrease the prevalence of smoking among younger persons and persons with lower incomes more than older persons and those with higher incomes , it was expected that differences would emerge in these demographic characteristics in callers during the time of the tax increase compared to those who called the year before . although persons with lower education levels were more likely to call after the tax increase , young adults were slightly less likely to call . however , for all characteristics , the magnitude of the differences before and after the tax increase was small . observed changes in who called the quitline around the time of the tax increase versus the year before could be due to multiple factors such as state quitline promotional efforts that were timed to correspond to the tax increase as well as the local increases in actual cigarette prices themselves . this study is descriptive in nature and did not address the myriad of changes in tobacco control policies and interventions that may have occurred at the state and local levels during this time period and how those changes would have influenced both the number of calls to the quitline and demographic and other characteristics of quitline callers . for example , in addition to the federal excise tax increase in april 2009 , 13 states in this study increased their cigarette excise taxes between november 2008 and november 2009 . more research would be needed to estimate the effect of the federal tax increase apart from these and other changes that were occurring at the state and local levels . interestingly , in terms of caller characteristics , the variable that changed the most among callers around the time of the federal tax increase compared to the year before was how the caller heard about the quitline . callers after the tax increase were more likely to report that friends and family told them about the quitline than those who called before the tax increase . future research could explore how cigarette tax increases influence friends ' and families ' interest in encouraging and assisting smokers with cessation . additional research is also needed to determine the synergistic effects on call volumes and treatment outcomes of state promotional events that may have coincided with implementation of the tax increase . the lack of higher quit rates after the tax is not surprising since the quitlines did not provide additional counseling or other services for tobacco users after the tax and in fact may have reduced the availability of more intensive cessation treatments ( see table 1 ) . although the quit rates were similar before and after the federal tax increase , the number of tobacco users who enrolled in the quitlines was larger after the tax increase . therefore , in terms of absolute numbers , more persons successfully quit after the tax increase . in these 16 states , of the 19,911 additional tobacco users who called during the time of the tax an additional 5,714 would quit smoking ( 19,911 more callers after tax 28.7% quit rate ) . however , it is important to remember that only 1%5% of smokers in the united states call quitlines each year and tobacco users often quit without the use of cessation services or medications . increasing the price of cigarettes is associated with increase quitting ( 1 ) and future research could examine the effects of the increase in the federal excise tax on more general population - based measures of cessation . one limitation is that the number and types of callers to quitlines vary within and between states over time and are a function of promotional events ( e.g. , offering free nrt ) and eligibility criteria ( e.g. , nrt for uninsured only ) that were not examined in this analysis . note that the services provided did not change before / after in the four states with 7-month data . furthermore , since the primary intention of this paper is to describe the populations using the quitlines around the tax increase , it is likely that the data accurately portrays the types of callers who were calling around that time . although analyses of individual states ' promotional activities or other tobacco control initiatives were not conducted , state was included in the statistical models to control for such variability . note that the pattern of calls was similar in graphs with and without one outlier state that had paired the normal january increase in calls with a state tax increase and promotional activities around the quitline . future studies should consider including a more detailed analysis of promotional efforts as well as state - specific tax increases . unfortunately , there is no data source currently available that tracks the amount , content , and timing of state antitobacco promotional efforts [ 20 , 21 ] . another consideration is that data were missing for both time periods for over 10% of enrolled callers at intake for four variables ( education , presence of other household smokers , years of tobacco use , and whether they were mailed nrt by the quitline ) . this is a reasonable amount of missing responses for these specific measures obtained during quitline enrollment . however , results for those variables may have been influenced by this nonresponse although it is difficult to predict the magnitude and direction of how the nonresponse would affect the relationship to the tax increase . in addition , only four states had seven - month quit rate data that spanned the study period ; therefore , these quit rate results might not generalize to other quitlines . analyses compared cessation rates among those who enrolled around the time of the tax increase compared to persons who enrolled during the same months during the prior year but there were no questions to determine if success was due to the individual 's interaction with the quitline . also , seven - month survey response rates tend to be fairly low . low response rates are a common finding in phone - based follow - up surveys with individuals seeking treatments . response rates in the 3040% range are reasonable and consistent with other studies ( naqc 2009 ) . although analyses controlled for response rates by reporting the intent to treat quit rates , the assumption that nonrespondents are continued smokers has been challenged as being too conservative . cessation rates may have differed significantly between time periods if higher response rates had been obtained . the true quit rates will lie somewhere between the responder and intent - to - treat results . because of the above limitations , conclusions about changes in quit rates among quitline callers after the federal tax increase should be interpreted with caution . evidence - based cessation services combined with tax and price increases , smoke - free laws , antitobacco advertising , and bans on tobacco advertising and promotion increase cessation and decrease tobacco use prevalence . frieden and colleagues found that intensive tobacco control measures decreased the prevalence of smoking by 11% among new york city adults from 2002 to 2003 and estimated that 59% of that reduction in smoking was due to price increases . further , the interactive effects of multiple policies are more effective and have a greater public health impact when combined with other evidence - based components of tobacco control programs . however , in a recent survey of quitline service providers , 89% reported that reduced funding had a direct effect on provision of services ( e.g. , limiting eligibility for services , reducing the number of counseling sessions , or eliminating provision of nrt ) . this is unfortunate since offering free nrt through the quitline can increase calls and increase cessation [ 5 , 7 , 9 ] . in the current study , variability in the type and intensity of cessation services ( e.g. , number of counseling sessions , amount of nrt offered ) provided by each state over the two time periods may have been due to budgetary constraints . through best practices for comprehensive tobacco control programs , cdc recommends funding levels for comprehensive tobacco control programs , including effective interventions such as quitlines . if all states met cdc 's recommended annual levels of funding for tobacco control programs ( $ 9$18 per capita ) , in five years , an estimated five million fewer persons would smoke and hundreds of thousands of premature tobacco - related deaths could be prevented .

background . on april 1 , 2009 , the federal cigarette excise tax increased from 39 cents to $ 1.01 per pack . methods . this study describes call volumes to 16 state quitlines , characteristics of callers and cessation outcomes before and after the tax . results . calls to the quitlines increased by 23.5% in 2009 and more whites , smokers 25 years of age , smokers of shorter duration , those with less education , and those who live with smokers called after ( versus before ) the tax . quit rates at 7 months did not differ before versus after tax . conclusions . descriptive analyses revealed that the federal excise tax on cigarettes was associated with increased calls to quitlines but multivariate analyses revealed no difference in quit rates . however , more callers at the same quit rate indicates an increase in total number of successful quitters . if revenue obtained from increased taxation on cigarettes is put into cessation treatment , then it is likely future excise taxes would have an even greater effect .

1. Introduction 2. Methods 3. Measures 4. Analyses 5. Results 6. Discussion 7. Limitations 8. Conclusions

on february 4 , 2009 , a 62-cent increase in the federal cigarette tax was enacted , along with increases in other tobacco taxes , to fund expansion of the state children 's health insurance program ( schip ) . the federal cigarette tax increased to $ 1.01 per pack on april 1 , 2009 . increasing the price of tobacco through excise taxes is an effective way to encourage quit attempts and thus to decrease the prevalence of smoking [ 2 , 3 ] . it is estimated that a 10% increase in cigarette prices leads to a 4% decrease in cigarette consumption in high - income countries and about 8% in low - to - middle income countries [ 2 , 4 ] . a 70% increase in current tobacco prices could prevent 25% of all smoking - related deaths globally and higher taxes have a greater impact on the young and low income smokers by deterring smoking initiation and encouraging smokers to quit . telephone quitlines are an effective population - based form of smoking cessation treatment and their utilization has been shown to be responsive to tobacco control policies [ 59 ] . therefore , the study aims were to ( 1 ) describe call volumes to 16 state quitlines before and after the tax increase ; ( 2 ) examine the characteristics of tobacco users who enrolled with quitlines before and after the tax increase and ( 3 ) examine the outcomes ( quit rates ) of tobacco users who enrolled with state quitlines before and after the tax increase . analyses were conducted to determine whether implementation of the federal tax on april 1 , 2009 coincided with ( 1 ) increased calls to state quitlines ; ( 2 ) increased calls from people with low education levels ; ( 3 ) increased quit rates ( the higher cigarette prices may motivate those attempting to quit to remain quit ) . it was expected that the increase in calls may begin prior to april 1 , 2009 and as early as february 2009 as people become aware of the passage of the federal tax increase and in response to preemptive cigarette price increases instituted by the tobacco industry in december 2008 and march 2009 [ 10 , 11 ] . two different data sources were used : one is based on administrative data collected from all callers and the other is a seven - month follow - up interview with a random sample of quitline participants . administrative data comes from the free & clear database for state tobacco quitlines that tracks call volumes , completed counseling calls and caller characteristics obtained during registration with the quitline . this data comes from 16 of the 17 state quitlines operated by free & clear , inc . the one state that was not included in the analysis had incomplete data for the time period before the tax increase . participating states include alaska , connecticut , georgia , hawaii , indiana , maine , maryland , missouri , north carolina , oklahoma , oregon , south carolina , utah , virginia , washington and wisconsin , whose smokers represented 24% of smokers in the united states in 2009 . data from the seven - month followup comes from four state quitlines and is based on random samples of quitline participants in each state timed to occur seven months from enrollment with the quitline . all 16 states agreed to participate in this study and to contribute their data to the pooled dataset . the 16 state quitlines represent different geographic regions and states with varying state laws ( e.g. , tobacco control programs with varying resources and programmatic activities ) . as well , they have a variety of cessation services such as offering free nicotine replacement therapy ( nrt ) through their quitline during the study period . all quitlines provided mailed support materials ( quit guides ) , a single reactive ( inbound ) counseling call to all tobacco users , and three or four additional outbound calls to select groups ( e.g. , those ready to quit within 30-days ) . some state quitlines refer insured tobacco users to cessation benefits offered through their health plan or employer . all of the participating states used the same data collection methods and a common questionnaire to collect demographic and tobacco use data at intake and follow - up thus enhancing data comparability across state quitlines and across study years . analyses examined both pooled monthly call volume ( total calls to quitlines ) and pooled daily call volume . state - level data was not examined as this was a descriptive study to assess whether a volume change would be observed in aggregate data from callers to the quitline in 16 states . however , in statistical models of outcomes , state was included as a fixed effect to account for unmeasured variability within and between states . monthly data was examined from december 2008 through august 2009 and from a similar time period the year before for comparison ( december 2007 through august 2008 ) in order to show call volume prior to the tax increase ( december 2008 , january 2009 , february 2009 , march 2009 ) , during the months that the tobacco industry increased prices in anticipation of the tax increase ( december 2008 through march 2009 ) , during the month the tax increase was passed ( february 2009 ) , and after the tax increase took effect ( april 2009 , may 2009 anticipated to have heavy call volumes ) . daily call volume was then examined directly before and after the april 1 , 2009 tax increase to determine when the calls began to increase in anticipation of the tax increase and how soon the calls returned to previous levels . the time period selected for this analysis was march - april 2009 ( march - april 2008 was also examined for comparative purposes ) . a comprehensive set of variables collected when a person enrolls with a state quitline was used to describe caller characteristics . variables included participant demographics ( age , gender , race / ethnicity , insurance status , educational level ) , current tobacco use ( tobacco type , amount used ) , duration of smoking , time to first cigarette upon waking , living or working with smokers , and how they heard about the quitline . chronic disease status was assessed by asking : have you been diagnosed with any of the following conditions ; asthma , chronic obstructive pulmonary disease or emphysema , coronary artery disease or heart disease or diabetes ? data from the seven - month followup survey was obtained for persons who enrolled from march 2009may 2009 ( and for comparison march 2008may 2008 ) . this time period was selected for comparisons of demographics and quit rates because this was the period in which the impact of the tax would most likely be observed . the four states with available data ( i.e. , some did not conduct the seven - month survey during these time periods and others used another organization to conduct the seven - month survey and their data was unavailable ) used similar survey sampling protocols and similar questionnaires . information collected at the seven - month follow - up included use of medications since enrolling with the quitline and current smoking status . successful cessation was defined as seven - day and 30-day abstinence by asking participants : when was the last time you smoked a cigarette , even a puff ? data across all states was pooled and presented in aggregate form in graphs and tables . first , the total number of monthly calls to the 16 quitlines was collected and presented in a figure as well as the number of tobacco users who received one or more counseling calls from december 2007 through august 2008 and december 2008 through august 2009 . rao - scott chi - square and t - test statistics were used to compare characteristics of callers during the time of the 2009 tax increase and for the same months in the prior year and included state as a fixed variable to account for the variability in services provided across quitlines . for the four states with data from the seven - month follow - up , multivariate logistic regression analyses were used to examine tobacco abstinence outcomes ( 7-day point prevalence and 30-day point prevalence ) comparing callers during the time of the 2009 tax increase to callers during the same time period in the prior year . again , state was included as a fixed variable , as well as case - mix covariates that differed before versus after the tax increase ( age , race , education , chronic conditions , how they heard about the quitline , and amount smoked at intake ) , and gender because it is associated with cessation outcomes [ 14 , 15 ] . for those who enrolled with the multicall program , utilization of services ( number of counseling calls completed ) and quit outcomes was also assessed in multivariate and logistic regression analyses . outcomes were reported in two ways : first among those who completed the survey ( respondent analysis ) and second using the intent - to - treat ( itt ) analysis whereby persons with missing outcomes data are assumed to be smoking . the logistic model was logit(probability of abstinence ( yes / no ) ) = overall mean+time indicator variable+individual covariates + state + callprogram + howheard , where time indicator variable = before or after ( 0 or 1 ) , callprogram = one versus multicall program and howheard = how participant heard about the quitline figure 1 presents the number of calls to the 16 quitlines over time and shows the spike in calls during march - april 2009 . overall , there was a 23.5% increase in total call volume when comparing december 2007may 2008 ( 84,541 calls ) to december 2008may 2009 ( 104,452 calls ) . in 2009 , calls increased beginning in march and began to taper off in may ( a 59.1% increase in call volumes comparing march 2008may 2008 ( 38,919 calls ) to march 2009may 2009 ( 61,935 calls ) ) . comparing each month in 2008 - 2009 with the corresponding month from the prior year , increases in call volume were observed in december 2008 and february through may 2009 , with the largest percent increase ( 94.1% ) occurring in march 2009 . increases during march and april 2009 occurred both in total call volume ( calls from tobacco users , friends , family , health care professionals , and the general public seeking information ) , as well as in the number of tobacco users per month who received at least one counseling call . data for june , july , and august are not shown in figure 1 since the tax effect on call volumes had returned to the before tax levels in may . note that the observed increase in quitline calls ( and enrollments ) around january 1 for both time periods was expected and is often attributed to new years ' resolutions . in post hoc analyses , omitting this state from the sample resulted in a similar pattern ( although a lower number of calls ) as that shown in figure 1 . figure 2 shows a more detailed analysis of call volumes around the april 2009 tax increase ; the daily call volumes from march through april 2009 compared to daily call volumes from march through april 2008 . daily call volume was higher in 2009 than 2008 , particularly from march 8 through april 26 . table 2 shows results of the comparisons of demographic and other characteristics between tobacco users who enrolled with the quitline before and after the announcement and implementation of the april 2009 federal tax increase . the time periods for this analysis were march 1 , 2008 through may 31 , 2008 versus march 1 , 2009 through may 31 , 2009 ( the window of time showing the peak activity in call volumes ) . results reveal differences in callers between the two time periods . in the after tax period , although the mean age of callers was slightly younger ( 41.9 versus 41.2 ) , fewer callers were aged 1824 years ( 11.5% after tax versus 13.6% before tax ) . more callers in 2009 ( compared with the prior year ) were white , had less than a high school education , were more likely to live with a smoker , had shorter durations of cigarettes smoking , and were more likely to report hearing about the quitline from family or friends or their health care provider , rather than from the media . although fewer callers enrolled in the multicall program ( 4 - 5 counseling calls ) after tax , they completed slightly more counseling sessions compared with those who enrolled for the multiple calls before tax ( 1.9 versus 2.2 , respectively , p < 0.0001 ) . although there were differences in the prevalence of chronic disease ( copd and cad ) and use of other tobacco products when comparing callers after the tax increase to those before the tax increase , these differences between the callers in these two time periods were small . table 3 shows results of analyses of seven - month outcomes data and suggests that participant quit rates did not differ significantly before versus after the tax . for example , seven - day respondent quit rates were 30.7% before and 28.7% after the tax ( o.r . analyses of the subgroup that participated in the multicall program showed a similar lack of change in smoking status after tax compared with a similar period before the tax . this study 's results are consistent with prior research showing that implementing an increase in excise taxes on tobacco will drive calls to the state tobacco control programs ' free quitline services [ 3 , 17 ] . reported an increase in call volumes to the montana quitline following an increase in the state 's cigarette taxes . they also reported that the tax attracted younger smokers to call the quitline , as well as more female and white smokers and heavier smokers . in the current study , although smokers who called the quitline around the time of the federal tax increase were more likely to be white , no significant differences were found for gender or amount smoked . however , fewer young tobacco users ( age 1824 ) and fewer smokers with smoking durations of 20 years called around the time of the tax increase . because tax increases tend to decrease the prevalence of smoking among younger persons and persons with lower incomes more than older persons and those with higher incomes , it was expected that differences would emerge in these demographic characteristics in callers during the time of the tax increase compared to those who called the year before . although persons with lower education levels were more likely to call after the tax increase , young adults were slightly less likely to call . however , for all characteristics , the magnitude of the differences before and after the tax increase was small . observed changes in who called the quitline around the time of the tax increase versus the year before could be due to multiple factors such as state quitline promotional efforts that were timed to correspond to the tax increase as well as the local increases in actual cigarette prices themselves . this study is descriptive in nature and did not address the myriad of changes in tobacco control policies and interventions that may have occurred at the state and local levels during this time period and how those changes would have influenced both the number of calls to the quitline and demographic and other characteristics of quitline callers . for example , in addition to the federal excise tax increase in april 2009 , 13 states in this study increased their cigarette excise taxes between november 2008 and november 2009 . more research would be needed to estimate the effect of the federal tax increase apart from these and other changes that were occurring at the state and local levels . interestingly , in terms of caller characteristics , the variable that changed the most among callers around the time of the federal tax increase compared to the year before was how the caller heard about the quitline . callers after the tax increase were more likely to report that friends and family told them about the quitline than those who called before the tax increase . future research could explore how cigarette tax increases influence friends ' and families ' interest in encouraging and assisting smokers with cessation . additional research is also needed to determine the synergistic effects on call volumes and treatment outcomes of state promotional events that may have coincided with implementation of the tax increase . the lack of higher quit rates after the tax is not surprising since the quitlines did not provide additional counseling or other services for tobacco users after the tax and in fact may have reduced the availability of more intensive cessation treatments ( see table 1 ) . although the quit rates were similar before and after the federal tax increase , the number of tobacco users who enrolled in the quitlines was larger after the tax increase . therefore , in terms of absolute numbers , more persons successfully quit after the tax increase . in these 16 states , of the 19,911 additional tobacco users who called during the time of the tax an additional 5,714 would quit smoking ( 19,911 more callers after tax 28.7% quit rate ) . however , it is important to remember that only 1%5% of smokers in the united states call quitlines each year and tobacco users often quit without the use of cessation services or medications . increasing the price of cigarettes is associated with increase quitting ( 1 ) and future research could examine the effects of the increase in the federal excise tax on more general population - based measures of cessation . one limitation is that the number and types of callers to quitlines vary within and between states over time and are a function of promotional events ( e.g. , nrt for uninsured only ) that were not examined in this analysis . note that the services provided did not change before / after in the four states with 7-month data . furthermore , since the primary intention of this paper is to describe the populations using the quitlines around the tax increase , it is likely that the data accurately portrays the types of callers who were calling around that time . although analyses of individual states ' promotional activities or other tobacco control initiatives were not conducted , state was included in the statistical models to control for such variability . note that the pattern of calls was similar in graphs with and without one outlier state that had paired the normal january increase in calls with a state tax increase and promotional activities around the quitline . future studies should consider including a more detailed analysis of promotional efforts as well as state - specific tax increases . unfortunately , there is no data source currently available that tracks the amount , content , and timing of state antitobacco promotional efforts [ 20 , 21 ] . another consideration is that data were missing for both time periods for over 10% of enrolled callers at intake for four variables ( education , presence of other household smokers , years of tobacco use , and whether they were mailed nrt by the quitline ) . this is a reasonable amount of missing responses for these specific measures obtained during quitline enrollment . however , results for those variables may have been influenced by this nonresponse although it is difficult to predict the magnitude and direction of how the nonresponse would affect the relationship to the tax increase . in addition , only four states had seven - month quit rate data that spanned the study period ; therefore , these quit rate results might not generalize to other quitlines . analyses compared cessation rates among those who enrolled around the time of the tax increase compared to persons who enrolled during the same months during the prior year but there were no questions to determine if success was due to the individual 's interaction with the quitline . response rates in the 3040% range are reasonable and consistent with other studies ( naqc 2009 ) . although analyses controlled for response rates by reporting the intent to treat quit rates , the assumption that nonrespondents are continued smokers has been challenged as being too conservative . cessation rates may have differed significantly between time periods if higher response rates had been obtained . the true quit rates will lie somewhere between the responder and intent - to - treat results . because of the above limitations , conclusions about changes in quit rates among quitline callers after the federal tax increase should be interpreted with caution . evidence - based cessation services combined with tax and price increases , smoke - free laws , antitobacco advertising , and bans on tobacco advertising and promotion increase cessation and decrease tobacco use prevalence . further , the interactive effects of multiple policies are more effective and have a greater public health impact when combined with other evidence - based components of tobacco control programs . however , in a recent survey of quitline service providers , 89% reported that reduced funding had a direct effect on provision of services ( e.g. , limiting eligibility for services , reducing the number of counseling sessions , or eliminating provision of nrt ) . , number of counseling sessions , amount of nrt offered ) provided by each state over the two time periods may have been due to budgetary constraints .

their unique amphiphilic properties have been exploited in a wide range of industrial and consumer applications . pfaas are exceptionally stable chemicals that have a very long degradation half - life in the environment . they can also be formed in the environment from the transformation of other industrially produced precursor compounds . , perfluorooctanesulfonic acid ( pfos ) and perfluorooctanoic acid ( pfoa ) have been associated with a wide range of adverse effects on both mammalian and aquatic organisms . the baltic sea is one of the world s largest brackish water systems with a highly populated and industrialized catchment . the long water residence time of 20 years makes the baltic sea particularly susceptible to pollution , and high levels of a range of persistent organic pollutants such as polychlorinated biphenyls , dibenzo - p - dioxins , and dibenzofurans have been found in baltic sea sediments and food webs . in recent years pfos concentrations in the eggs of baltic sea guillemots are among the highest ever reported in birds , and they are close to the levels that have been shown to cause adverse effects in other bird species . furthermore , temporal trend monitoring in baltic sea herring ( 19802010 ) and sea eagle eggs ( 19662010 ) showed increasing concentrations of pfos and long - chain perfluoroalkylcarboxylic acids ( pfcas , c8c15 ) ( personal communication , anders bignert ) . there is clearly a need to control pfaa levels in the baltic sea environment . in order to develop effective management strategies , the major sources ( both emissions to the environment and transport pathways ) of pfaas to the baltic sea must be identified . different conclusions have been drawn about the major transport pathways of pfaa contamination to surface waters . in a study of several small rivers in germany , it was concluded that the major part of pfaas entered the rivers via municipal wastewater treatment plants ( wwtps ) . in the first attempt at a pfaa mass balance of a large lake , boulanger et al . concluded that wwtp discharges represented a minor input pathway of pfoa to lake ontario compared to inflow from the other great lakes . scott et al . did a similar study for lake superior , a large lake with a much lower population density in the watershed and no known pfaa manufacturing sites . in contrast to boulanger et al . , this study included even precipitation , and it was concluded that wet deposition and riverine discharges represented the dominant pfaa inputs to the lake . to identify the major input pathways of pfaas to the baltic sea , a pfaa mass balance was assembled . available monitoring data were used to estimate pfaa inputs ( from river inflow , atmospheric deposition , wastewater discharges directly to the baltic sea , and inflow from the north sea via the danish straits ) and outputs ( to sediment burial , transformation of the chemical , and outflow via the danish straits to the north sea ) as well as the pfaa inventory in the baltic sea . in addition , a pfaa mass balance was conducted for the oder river watershed that compared the atmospheric deposition to the watershed with the inputs via wwtps throughout the watershed and the output from the watershed via oder river s discharge to the baltic sea . mass balances were conducted for four pfaas : perfluorohexanoic acid ( pfhxa ) , pfoa , perfluorodecanoic acid ( pfda ) and pfos ( see abbreviation list in the supporting information for further frequently used abbreviations and table s1 in supporting information for structures of the target chemicals ) . the data used were reported after 2006 . some of the data sets contained data points that were below the method detection limit ( mdl ; see table s2 in the supporting information ) . this introduced uncertainty into the calculation of the chemical fluxes and inventories . to set bounds on the uncertainty , two estimates were calculated : high - bound estimate ( hbe ) , for which all of the data points reported as nondetected were set to the mdl ; and low - bound estimate ( lbe ) , for which all of the data points reported as nondetected were set to 0 . calculated pfaa fluxes and inventories are given as ranges ( lbe hbe ) throughout the paper and the supporting information . despite this and other uncertainties , the fluxes and inventories are reported with three significant digits due to the need for aggregation for the overall mass balance . two mass balances were conducted , one for the baltic sea and one for the oder river catchment area . the rivers for which pfaa data were available are marked in the figure , and the oder river is highlighted . the baltic sea catchment has a population of 85 million , whereby approximately 15 million live within 10 km of the baltic sea coast . sweden , germany , denmark , and finland have high per capita gross domestic products ( gdp ) ( > u.s . $ 37 000 ) compared to russia , poland , latvia , estonia , and lithuania ( < u.s . there are no pfaa manufacturing sites in the baltic sea catchment to our knowledge . the five basins of the baltic sea are shown separated by gray lines ( bb , bothnian bay ; bs , bothnian sea ; gof , gulf of finland ; gor , gulf of riga ; bp , baltic proper ) . the colored landmasses represent the watersheds of the basins . the black dots are coastal cities with population > 35 000 , and the blue lines represent the rivers for which measurements of pfaa concentrations were available . the oder river is highlighted within the red box . the properties of the baltic sea of most relevance for the mass balance models are summarized in table 1 . although a one - box model was used for the mass balance of the baltic sea , it was divided into five basins for interpolation of the riverine input ( see figure 1 and table s3 in supporting information ) . each of the basins contained rivers for which pfaa concentration data were available : vindellven and kalixlven in the bothnian bay ( bb ) , kokemen and dallven in the bothnian sea ( bs ) , narva , purtse , and vantaa in the gulf of finland ( gof ) , daugava in the gulf of riga ( gor ) , and vistula , oder , eman , nemunas , norrstrm , and motala in the baltic proper ( bp ) . the processes considered in the mass balances for the baltic sea and the oder river catchment are illustrated in figure 2 . the system boundary for the baltic sea mass balance enclosed water and surface sediment . the input pathways ( hereafter also called sources ) treated were river inflow , atmospheric deposition ( which also includes pfaas that were formed from precursors in the atmosphere ) , coastal wastewater discharges , and inflow from the north sea via the danish straits , while the loss processes considered were sediment burial , transformation of the pfaas , and outflow via the danish straits to the north sea . the system boundary for the oder catchment mass balance enclosed the nonatmospheric portions of the catchment ( i.e. , land , surface water , groundwater , and associated solids ) . the input processes treated were atmospheric deposition and wastewater discharges , while the outflow from the oder to the baltic sea was the only loss process considered . schematic illustration of the processes included in pfaa mass balances of the baltic sea and the oder river catchment . the riverine input of pfaas , nriver ( kilograms per year ) , was calculated according to1where criver is the pfaa concentration in river water ( kilograms per cubic meter ) and qriver is the river discharge rate ( cubic meters per year ) . criver was compiled from two studies that analyzed water samples collected close to the mouths of major european rivers during autumn 2005 ( three rivers ) and 2007 ( 11 rivers ) . the detection frequencies of pfhxa , pfoa , pfda , and pfos in the rivers were 14% , 71% , 14% , and 71% , respectively . the data were scaled up from the river to the basin level by use of the river discharge weighted average concentration ( see section 2.1 in the supporting information for details ) . the input of pfaas via atmospheric deposition , ndeposition ( kilograms per year ) , was calculated according to2where cprecip is the pfaa concentration in precipitation ( kilograms per cubic meter of water ) and qprecip is the amount of precipitation deposited directly on the baltic sea ( cubic meters of water per year ) . cprecip was estimated from the mean pfaa concentrations ( see section 2.2 in the supporting information ) measured in 20 bulk deposition samples that were collected continuously between october 1 , 2007 , and may 8 , 2008 , at a site close to the southwestern corner of the baltic sea . concentrations in a similar range have been reported in wet deposition from northern finland , the swedish west coast , the united states , and asia.qprecip was taken from table 1 . the pfaa input from wwtp discharges directly into the baltic sea , nwwtp ( kilograms per year ) , was estimated according to3where cwwtp is the pfaa concentration in the wwtp effluent ( kilograms per cubic meter ) , qwwtp is the amount of wastewater discharged annually per population equivalent [ cubic meters per year per population equivalent ( pe ) ] , and pequiv is the number of population equivalents connected to wwtps discharging directly into the baltic sea . cwwtp was estimated from recent measurements of pfaa concentrations in effluents from wwtps discharging directly to the baltic sea . two to four wwtps were studied for six of the nine countries bordering on the baltic sea . two to six samples were collected from each wwtp between october 9 , 2009 , and august 10 , 2010 . the values of qwwtp were taken from the literature , while pequiv was estimated by selecting coastal cities with populations greater than 35 000 inhabitants ( which serve > 80% of the population in the coastal region ) . input of pfaas from the north sea through the danish straits , nns ( kilograms per year ) , was estimated as4where cns is the pfaa concentration in north sea water ( kilograms per cubic meter ) and qns is the annual inflow of water from the north sea through the danish straits ( cubic meters per year ) . cns was estimated from pfaa concentrations measured in surface water samples collected at three stations along the southern coast of norway in 2007 ( table s11 in supporting information ) . for annual water inflow qns , an average value of 475 10 m / year was used ( table 1 ) . the lower limit for the phototransformation half - life of pfoa in coastal oceans was recently estimated to be 25 600 years . we are not aware of any evidence showing pfaa transformation in water via other mechanisms . hence the transformation half - life t0.5 of pfoa in baltic sea water was assumed to be 25 600 years and the loss due to transformation , ntran ( kilograms per year ) , was calculated according to5where vbaltic is the volume of water in the baltic sea ( cubic kilometers ) and cbaltic is the pfaa concentration in the baltic sea ( kilograms of pfaa per cubic kilometer ) . vbaltic is given in table 1 and cbaltic is given in table s16 ( supporting information ) . in the absence of information on the transformation of other pfaas besides pfoa , t0.5 was assumed to be 25 600 years for all four chemicals . outflow of pfaas through the danish straits , nds ( kilograms per year ) , was determined according to6where cbp is the pfaa concentration in water in the baltic proper ( kilograms per cubic meter ) and qds is the annual outflow of water from the baltic sea through the danish straits ( cubic meters per year ) . the estimation of cbp is described in section 2.4.1 , and qds is given in table 1 . cbp and nds are listed in table s14 ( supporting information ) . the rate of loss of pfaa due to sediment burial , nburial ( kilograms per year ) , was calculated according to7where qburial is the rate at which sediment is buried in the baltic sea ( kilograms of sediment dry weight per year ; see table 1 ) and csed is the pfaa concentration in the sediment being buried ( kilograms of pfaa per kilogram of sediment dry weight ) . the estimation of csed is made from pfaa concentrations measured in baltic sea water and kd values taken from the literature as described in section 2.4.2 . kd , csed , and nburial are listed in table s15 ( supporting information ) . the pfaa inventory in baltic sea water , mwater ( kilograms ) , was calculated according to8where cwater , basin is the pfaa concentration in water in the basin ( kilograms per cubic meter ) and vbasin is the volume of water in the basin ( cubic meters ) . the basins and their water volumes are listed in table s3 ( supporting information ) . cwater , basin was estimated from pfaa concentrations measured in 62 water samples collected at depths of 23 and 5 m on two cruises in the baltic sea during the summer of 2008 . the number of samples collected , detection frequency of the pfaas , average pfaa concentrations , and pfaa inventories are given for each basin in tables s16 and s17 ( supporting information ) . the lack of strong spatial gradients supports the use of the simple box model to assess the chemical mass balance . the pfaa inventory in baltic sea sediment , msed ( kilograms ) , was calculated according to9where csed , basin is the pfaa concentration in the surface sediment of the basin ( kilograms of pfaa per kilogram of sediment dry weight ) , abasin is the surface area of the basin ( square meters ) , dsed is the average depth of the surface sediment ( meters ) , sed is the porosity of the sediment ( cubic meters of nonsolids per cubic meter of bulk sediment ) , and sed is the density of the dry sediment ( kilograms per cubic meter of solids ) . sed was set to 0.89 , while sed was assumed to equal 2500 kg / m . the pfaa concentration in sediment , csed , basin ( kilograms of pfaa per kilogram of sediment dry weight ) , was calculated from the pfaa concentration in water , cwater , basin , with the assumption of equilibrium partitioning:10where kd is the equilibrium partition coefficient ( cubic meters per kilogram of sediment dry weight ) . cwater , basin ( kilograms of pfaa per cubic meter ) was determined as in section 2.4.1 , and kd values measured for baltic sea sediments ( 168 , 423 , 4795 , and 1185 m / kgsediment dry weight for pfhxa , pfoa , pfda , and pfos , respectively ) were used . the pfaa inventories in sediment are given in table s18 ( supporting information ) . the mass balance of the oder catchment considered pfaa inputs from wwtps and atmospheric deposition , while riverine discharge was the only output included ( transformation and sedimentation were assumed to be negligible ) . input from wwtps was calculated by multiplying the annual pfaa input per population equivalent for poland ( see section 2.2.3 ) by the total population within the catchment . atmospheric deposition was calculated from eq 2 , where qprecip was the amount of precipitation to the oder catchment ( cubic meters per year ) , which was taken from the data supporting the nest decision support system ( erik smedberg , personal communication ) . the riverine discharge was equal to the riverine input of the oder to the baltic sea ( see table s4 in supporting information ) . as with the baltic sea model , this was an input / output mass balance that made no assumptions about how the chemicals behaved within the model domain . the results of the mass balance for the baltic sea are summarized in table 2 . detailed results per basin for the lbe and hbe input scenarios are given in tables s12 and s13 , respectively ( supporting information ) . ranges are delineated by low - bound estimates ( lbe ) and high - bound estimates ( hbe ) . rivers were a major source of the four pfaas to the baltic sea ( table 2 ) . they accounted for 1073% of the total input of pfhxa , 4859% of pfoa , 2867% of pfda , and 77% of pfos ( tables s12 and s13 , supporting information ) . it accounted for 1137% of the total input of pfhxa , 3443% of pfoa , 3172% of pfda , and 2021% of pfos . the north sea made a significant contribution to the lbe scenario for pfhxa ( 49% ) , but otherwise its contribution to the mass balance was insignificant . wwtp discharges directly to the baltic sea made a negligible contribution to the overall mass balance . they accounted for 4% of the lbe of the pfhxa mass balance ; otherwise their contribution was 2% . the mean concentrations of pfhxa , pfoa , and pfos in effluent waters were significantly higher for the group of countries with higher per capita gdp ( > u.s . $ 37 000 ) ( i.e. , 4.97 , 8.98 , and 8.45 ng / l , respectively ) in comparison to the group with lower per capita gdp ( < u.s . $ 20 500 ) ( i.e. , 1.07 , 5.33 , and 1.67 ng / l , respectively ) ( p < 0.05 , t - test ) . in tables s12 and s13 ( supporting information ) , the inputs from rivers , atmospheric deposition , and wwtps are compared on a basin basis . the lbe estimates suggest that wwtps could make a significant contribution to the total input of pfhxa , pfoa , and pfos to the gulf of finland ( 20% , 15% , and 13% , respectively ) . the differences between lbe and hbe in table 2 show that a large fraction of nonquantifiable data points imparts considerable uncertainty to some of the input estimates . this is particularly true for the riverine inputs of pfhxa and pfda and the north sea inputs of pfda and pfos . however , this uncertainty does not affect the major observation that rivers and atmospheric deposition are much more important sources of pfaas to the baltic sea than wwtps . outflow through the danish straits is the most important output pathway for pfaas in the baltic sea ( table 2 ) . it accounts for 2432% of the total output of pfda and 9% for pfos , while it is an insignificant sink for pfhxa and pfoa ( 1% and 3% , respectively ) . the inventory of most of the pfaas is largely stored in the water column , not in the sediment . water was estimated to contain 96% , 91% , 46% , and 78% of the baltic sea inventory of pfhxa , pfoa , pfda , and pfos , respectively . the results of the pfaa mass balance of the oder catchment are presented in table 3 . wwtps are an insignificant source of all four pfaas except for pfhxa , for which they contribute 9% . the total inputs and the total outputs are similar for pfhxa and pfos . for pfoa and pfda , the inputs are 3.7 and > 2.7 times greater , respectively , than the outputs . ranges are delineated by low - bound estimates ( lbe ) and high - bound estimates ( hbe ) . a major observation in this study is that wwtps make a minor contribution to the total pfaa input to the baltic sea . the results for the oder catchment mass balance show that this is also true when the indirect inputs of wwtp discharges to rivers flowing into the baltic sea are considered . cohiba , a recent project on the control of hazardous substances in the baltic sea region , estimated the total discharges of pfoa and pfos via wwtps to the baltic sea catchment to be 200 and 100 kg / year , respectively . these are similar to our estimates when they are scaled up to the whole baltic sea catchment . the small contribution of pfaas from wwtp effluents is also consistent with the observations of boulanger et al . , who estimated wwtp discharges of pfoa to lake ontario to be considerably smaller than riverine input via the niagara river . it is also in agreement with the findings of scott et al . , who found that wwtps contributed just 6% and 8% of the pfoa and pfos input , respectively , to lake superior . a second major observation is that atmospheric deposition is a dominant source of pfaas to the baltic sea . this conclusion is reinforced by the indirect contribution of atmospheric deposition through riverine input as indicated by the oder catchment mass balance . other emissions from the technosphere may contribute to pfaas at the catchment level . for instance , the cohiba project estimated that other sources besides wwtp effluent ( largely the use of firefighting foams on land ) result in emissions of 160 kg / year pfos and 330 kg / year pfoa to the baltic sea watershed . this is < 25% of our estimates of the total input to the baltic sea and < 25% of the atmospheric deposition to the baltic sea catchment ( scaled up from the oder catchment ) . the dominant impact of atmospheric deposition is consistent with the work of scott et al . , who concluded that this is the dominant source of pfaas to lake superior . the fact that atmospheric deposition also dominates in the highly populated baltic sea watershed provides stronger evidence that atmospheric deposition is currently a more important source of pfaa contamination of the environment than known anthropogenic emissions to land and water . the importance of atmospheric deposition observed in this study is consistent with the results of a mass balance conducted on a smaller spatial scale . a study of two rain events in a 10 km watershed in switzerland showed that rain , wwtp effluent , and surface runoff in stormwater all contributed significantly to the fluxes of a range of pfaas . an explanation for the greater importance of wwtp effluent in the swiss study is the higher population density of 2100 persons / km compared with 49 and 136 persons / km for the baltic sea and oder catchments , respectively . the dominance of atmospheric deposition as a pfaa source to a highly developed region such as the baltic sea has consequences for the environmental risk management of pfaas . focusing management efforts on reducing municipal wwtp emissions of pfaas will not lead to marked reductions of pfaa inputs to the baltic sea . this can only be achieved by measures that lead to a reduction of atmospheric deposition of pfaas . it is currently not clear what the sources of the pfaas in the atmosphere are . one explanation is direct emissions of the chemicals . for instance , emissions from fluoropolymer production facilities have been proposed as a major source of pfoa to the atmosphere . a third possibility is that pfaas are being transferred via sea spray to the atmosphere , and that the levels in atmospheric deposition are thus coming ( at least partly ) from environmental recirculation and not from primary emissions . armitage et al . used multimedia models and available data to assess the contributions of different sources to pfoa levels in the atmosphere , but they were unable to clearly identify the dominant source(s ) due to large uncertainties in key parameters . the understanding of the sources of pfos in the atmosphere is also incomplete , whereby a major contributor is believed to be the transformation of precursor compounds emitted from production facilities and fluorochemical products . a third major observation of the present study was that the input of the pfaas to the baltic sea exceeds the output . this was the case for all four chemicals , regardless of whether the lbe or hbe results were used . this mass balance excess indicates that the concentrations of all chemicals are increasing in the baltic sea . when the difference between pfaa input and output is compared with the pfaa inventory , the doubling time for the concentration in the baltic sea can be estimated . this amounts to 894 years for pfhxa , 1216 years for pfoa , 35 years for pfda , and 4 years for pfos . general trends of increasing concentrations of pfoa , pfda , and pfos have been observed in biomonitoring data for baltic sea herring up to 2010 ( anders bignert , personal communication ) . although the year - to - year variability is considerable , these biomonitoring data suggest doubling times on the order of 1015 years for pfoa , 48 years for pfda , and 510 years for pfos during the first decade of the millennium . a positive mass balance for the baltic sea suggests that inputs during the period when the samples were collected ( 20052011 ) must have been higher than the average input during the previous 20 years ( the water residence time in the baltic sea ) . it is notable that the contribution of direct atmospheric deposition to the total input to the baltic sea was smaller for pfos than for the other substances ; the dominant source was the rivers ( table 2 ) . there are significant nonatmospheric sources of pfos to river water , such as the use of firefighting foam . firefighting foam is known to have been a major source of pfos to the norrstrm river , which has elevated pfos concentrations compared to the bulk of the rivers ( see table s4 in supporting information ) . the input from such sources could have increased over time . after firefighting training exercises , pfos is known to be transferred into groundwater , from which it will eventually be discharged to surface water . the transfer of pfos from training sites to groundwater has been shown to continue for years after pfos release has ceased . this can result in the peak in pfos release to surface water being delayed compared to the peak in firefighting foam usage . another factor that could have caused an increase in riverine inputs of pfos and other pfaas during the 2000s is release of residues stored in the watershed . the median pfos concentration in the world s background soils was recently estimated to be 0.47 ng / g dry weight , whereby one - sixth of the soil samples included in the international survey were from norway . assuming that the baltic sea watershed is covered with a 10 cm deep layer of soil with this pfos concentration , this soil would contain 60 000 kg of pfos . this is more than an order of magnitude greater than the pfos inventory in the baltic sea ( see table 2 ) . this illustrates that soil potentially is a large reservoir of pfos that will buffer pfos inputs from atmospheric deposition , delaying and modulating their transfer via surface runoff and groundwater to surface water . research is needed to overcome this knowledge gap if we are to be able to develop effective strategies for reducing pfos levels in surface waters and to predict how pfos concentrations in surface waters will change as a result of management strategies . in 2012 the european commission proposed that concentrations of pfos should not exceed 0.13 ng / l in marine waters . there is clearly a need to reduce the inputs of pfos and other pfaas to the baltic sea . this will require identifying and controlling the sources of pfaas in the atmosphere and obtaining a better understanding of how they travel through the terrestrial environment to surface water .

a mass balance was assembled for perfluorohexanoic acid ( pfhxa ) , perfluorooctanoic acid ( pfoa ) , perfluorodecanoic acid ( pfda ) , and perfluorooctanesulfonic acid ( pfos ) in the baltic sea . inputs ( from riverine discharge , atmospheric deposition , coastal wastewater discharges , and the north sea ) and outputs ( to sediment burial , transformation of the chemical , and the north sea ) , as well as the inventory in the baltic sea , were estimated from recently published monitoring data . formation of the chemicals in the water column from precursors was not considered . river inflow and atmospheric deposition were the dominant inputs , while wastewater treatment plant ( wwtp ) effluents made a minor contribution ( < 5% ) . a mass balance of the oder river watershed was assembled to explore the sources of the perfluoroalkyl acids ( pfaas ) in the river inflow . it indicated that wwtp effluents made only a moderate contribution to riverine discharge ( 21% for pfoa , 6% for pfos ) , while atmospheric deposition to the watershed was 12 orders of magnitude greater than wwtp discharges . the input to the baltic sea exceeded the output for all four pfaas , suggesting that inputs were higher during 20052010 than during the previous 20 years despite efforts to reduce emissions of pfaas . one possible explanation is the retention and delayed release of pfaas from atmospheric deposition in the soils and groundwater of the watershed .

Introduction Materials and Methods Results Discussion

, perfluorooctanesulfonic acid ( pfos ) and perfluorooctanoic acid ( pfoa ) have been associated with a wide range of adverse effects on both mammalian and aquatic organisms . the long water residence time of 20 years makes the baltic sea particularly susceptible to pollution , and high levels of a range of persistent organic pollutants such as polychlorinated biphenyls , dibenzo - p - dioxins , and dibenzofurans have been found in baltic sea sediments and food webs . in recent years pfos concentrations in the eggs of baltic sea guillemots are among the highest ever reported in birds , and they are close to the levels that have been shown to cause adverse effects in other bird species . furthermore , temporal trend monitoring in baltic sea herring ( 19802010 ) and sea eagle eggs ( 19662010 ) showed increasing concentrations of pfos and long - chain perfluoroalkylcarboxylic acids ( pfcas , c8c15 ) ( personal communication , anders bignert ) . there is clearly a need to control pfaa levels in the baltic sea environment . in order to develop effective management strategies , the major sources ( both emissions to the environment and transport pathways ) of pfaas to the baltic sea must be identified . to identify the major input pathways of pfaas to the baltic sea , a pfaa mass balance was assembled . available monitoring data were used to estimate pfaa inputs ( from river inflow , atmospheric deposition , wastewater discharges directly to the baltic sea , and inflow from the north sea via the danish straits ) and outputs ( to sediment burial , transformation of the chemical , and outflow via the danish straits to the north sea ) as well as the pfaa inventory in the baltic sea . in addition , a pfaa mass balance was conducted for the oder river watershed that compared the atmospheric deposition to the watershed with the inputs via wwtps throughout the watershed and the output from the watershed via oder river s discharge to the baltic sea . mass balances were conducted for four pfaas : perfluorohexanoic acid ( pfhxa ) , pfoa , perfluorodecanoic acid ( pfda ) and pfos ( see abbreviation list in the supporting information for further frequently used abbreviations and table s1 in supporting information for structures of the target chemicals ) . to set bounds on the uncertainty , two estimates were calculated : high - bound estimate ( hbe ) , for which all of the data points reported as nondetected were set to the mdl ; and low - bound estimate ( lbe ) , for which all of the data points reported as nondetected were set to 0 . two mass balances were conducted , one for the baltic sea and one for the oder river catchment area . the rivers for which pfaa data were available are marked in the figure , and the oder river is highlighted . the properties of the baltic sea of most relevance for the mass balance models are summarized in table 1 . although a one - box model was used for the mass balance of the baltic sea , it was divided into five basins for interpolation of the riverine input ( see figure 1 and table s3 in supporting information ) . each of the basins contained rivers for which pfaa concentration data were available : vindellven and kalixlven in the bothnian bay ( bb ) , kokemen and dallven in the bothnian sea ( bs ) , narva , purtse , and vantaa in the gulf of finland ( gof ) , daugava in the gulf of riga ( gor ) , and vistula , oder , eman , nemunas , norrstrm , and motala in the baltic proper ( bp ) . the processes considered in the mass balances for the baltic sea and the oder river catchment are illustrated in figure 2 . the input pathways ( hereafter also called sources ) treated were river inflow , atmospheric deposition ( which also includes pfaas that were formed from precursors in the atmosphere ) , coastal wastewater discharges , and inflow from the north sea via the danish straits , while the loss processes considered were sediment burial , transformation of the pfaas , and outflow via the danish straits to the north sea . the input processes treated were atmospheric deposition and wastewater discharges , while the outflow from the oder to the baltic sea was the only loss process considered . schematic illustration of the processes included in pfaa mass balances of the baltic sea and the oder river catchment . the riverine input of pfaas , nriver ( kilograms per year ) , was calculated according to1where criver is the pfaa concentration in river water ( kilograms per cubic meter ) and qriver is the river discharge rate ( cubic meters per year ) . the data were scaled up from the river to the basin level by use of the river discharge weighted average concentration ( see section 2.1 in the supporting information for details ) . the input of pfaas via atmospheric deposition , ndeposition ( kilograms per year ) , was calculated according to2where cprecip is the pfaa concentration in precipitation ( kilograms per cubic meter of water ) and qprecip is the amount of precipitation deposited directly on the baltic sea ( cubic meters of water per year ) . cprecip was estimated from the mean pfaa concentrations ( see section 2.2 in the supporting information ) measured in 20 bulk deposition samples that were collected continuously between october 1 , 2007 , and may 8 , 2008 , at a site close to the southwestern corner of the baltic sea . the pfaa input from wwtp discharges directly into the baltic sea , nwwtp ( kilograms per year ) , was estimated according to3where cwwtp is the pfaa concentration in the wwtp effluent ( kilograms per cubic meter ) , qwwtp is the amount of wastewater discharged annually per population equivalent [ cubic meters per year per population equivalent ( pe ) ] , and pequiv is the number of population equivalents connected to wwtps discharging directly into the baltic sea . cwwtp was estimated from recent measurements of pfaa concentrations in effluents from wwtps discharging directly to the baltic sea . the values of qwwtp were taken from the literature , while pequiv was estimated by selecting coastal cities with populations greater than 35 000 inhabitants ( which serve > 80% of the population in the coastal region ) . input of pfaas from the north sea through the danish straits , nns ( kilograms per year ) , was estimated as4where cns is the pfaa concentration in north sea water ( kilograms per cubic meter ) and qns is the annual inflow of water from the north sea through the danish straits ( cubic meters per year ) . hence the transformation half - life t0.5 of pfoa in baltic sea water was assumed to be 25 600 years and the loss due to transformation , ntran ( kilograms per year ) , was calculated according to5where vbaltic is the volume of water in the baltic sea ( cubic kilometers ) and cbaltic is the pfaa concentration in the baltic sea ( kilograms of pfaa per cubic kilometer ) . in the absence of information on the transformation of other pfaas besides pfoa , t0.5 was assumed to be 25 600 years for all four chemicals . outflow of pfaas through the danish straits , nds ( kilograms per year ) , was determined according to6where cbp is the pfaa concentration in water in the baltic proper ( kilograms per cubic meter ) and qds is the annual outflow of water from the baltic sea through the danish straits ( cubic meters per year ) . the rate of loss of pfaa due to sediment burial , nburial ( kilograms per year ) , was calculated according to7where qburial is the rate at which sediment is buried in the baltic sea ( kilograms of sediment dry weight per year ; see table 1 ) and csed is the pfaa concentration in the sediment being buried ( kilograms of pfaa per kilogram of sediment dry weight ) . the pfaa inventory in baltic sea water , mwater ( kilograms ) , was calculated according to8where cwater , basin is the pfaa concentration in water in the basin ( kilograms per cubic meter ) and vbasin is the volume of water in the basin ( cubic meters ) . cwater , basin was estimated from pfaa concentrations measured in 62 water samples collected at depths of 23 and 5 m on two cruises in the baltic sea during the summer of 2008 . the number of samples collected , detection frequency of the pfaas , average pfaa concentrations , and pfaa inventories are given for each basin in tables s16 and s17 ( supporting information ) . the pfaa inventory in baltic sea sediment , msed ( kilograms ) , was calculated according to9where csed , basin is the pfaa concentration in the surface sediment of the basin ( kilograms of pfaa per kilogram of sediment dry weight ) , abasin is the surface area of the basin ( square meters ) , dsed is the average depth of the surface sediment ( meters ) , sed is the porosity of the sediment ( cubic meters of nonsolids per cubic meter of bulk sediment ) , and sed is the density of the dry sediment ( kilograms per cubic meter of solids ) . the mass balance of the oder catchment considered pfaa inputs from wwtps and atmospheric deposition , while riverine discharge was the only output included ( transformation and sedimentation were assumed to be negligible ) . atmospheric deposition was calculated from eq 2 , where qprecip was the amount of precipitation to the oder catchment ( cubic meters per year ) , which was taken from the data supporting the nest decision support system ( erik smedberg , personal communication ) . the riverine discharge was equal to the riverine input of the oder to the baltic sea ( see table s4 in supporting information ) . as with the baltic sea model , this was an input / output mass balance that made no assumptions about how the chemicals behaved within the model domain . the results of the mass balance for the baltic sea are summarized in table 2 . rivers were a major source of the four pfaas to the baltic sea ( table 2 ) . they accounted for 1073% of the total input of pfhxa , 4859% of pfoa , 2867% of pfda , and 77% of pfos ( tables s12 and s13 , supporting information ) . it accounted for 1137% of the total input of pfhxa , 3443% of pfoa , 3172% of pfda , and 2021% of pfos . the north sea made a significant contribution to the lbe scenario for pfhxa ( 49% ) , but otherwise its contribution to the mass balance was insignificant . wwtp discharges directly to the baltic sea made a negligible contribution to the overall mass balance . , 4.97 , 8.98 , and 8.45 ng / l , respectively ) in comparison to the group with lower per capita gdp ( < u.s . in tables s12 and s13 ( supporting information ) , the inputs from rivers , atmospheric deposition , and wwtps are compared on a basin basis . the lbe estimates suggest that wwtps could make a significant contribution to the total input of pfhxa , pfoa , and pfos to the gulf of finland ( 20% , 15% , and 13% , respectively ) . this is particularly true for the riverine inputs of pfhxa and pfda and the north sea inputs of pfda and pfos . however , this uncertainty does not affect the major observation that rivers and atmospheric deposition are much more important sources of pfaas to the baltic sea than wwtps . outflow through the danish straits is the most important output pathway for pfaas in the baltic sea ( table 2 ) . it accounts for 2432% of the total output of pfda and 9% for pfos , while it is an insignificant sink for pfhxa and pfoa ( 1% and 3% , respectively ) . the inventory of most of the pfaas is largely stored in the water column , not in the sediment . water was estimated to contain 96% , 91% , 46% , and 78% of the baltic sea inventory of pfhxa , pfoa , pfda , and pfos , respectively . the results of the pfaa mass balance of the oder catchment are presented in table 3 . a major observation in this study is that wwtps make a minor contribution to the total pfaa input to the baltic sea . the results for the oder catchment mass balance show that this is also true when the indirect inputs of wwtp discharges to rivers flowing into the baltic sea are considered . cohiba , a recent project on the control of hazardous substances in the baltic sea region , estimated the total discharges of pfoa and pfos via wwtps to the baltic sea catchment to be 200 and 100 kg / year , respectively . a second major observation is that atmospheric deposition is a dominant source of pfaas to the baltic sea . this conclusion is reinforced by the indirect contribution of atmospheric deposition through riverine input as indicated by the oder catchment mass balance . for instance , the cohiba project estimated that other sources besides wwtp effluent ( largely the use of firefighting foams on land ) result in emissions of 160 kg / year pfos and 330 kg / year pfoa to the baltic sea watershed . this is < 25% of our estimates of the total input to the baltic sea and < 25% of the atmospheric deposition to the baltic sea catchment ( scaled up from the oder catchment ) . the fact that atmospheric deposition also dominates in the highly populated baltic sea watershed provides stronger evidence that atmospheric deposition is currently a more important source of pfaa contamination of the environment than known anthropogenic emissions to land and water . an explanation for the greater importance of wwtp effluent in the swiss study is the higher population density of 2100 persons / km compared with 49 and 136 persons / km for the baltic sea and oder catchments , respectively . the dominance of atmospheric deposition as a pfaa source to a highly developed region such as the baltic sea has consequences for the environmental risk management of pfaas . focusing management efforts on reducing municipal wwtp emissions of pfaas will not lead to marked reductions of pfaa inputs to the baltic sea . it is currently not clear what the sources of the pfaas in the atmosphere are . one explanation is direct emissions of the chemicals . the understanding of the sources of pfos in the atmosphere is also incomplete , whereby a major contributor is believed to be the transformation of precursor compounds emitted from production facilities and fluorochemical products . a third major observation of the present study was that the input of the pfaas to the baltic sea exceeds the output . this mass balance excess indicates that the concentrations of all chemicals are increasing in the baltic sea . this amounts to 894 years for pfhxa , 1216 years for pfoa , 35 years for pfda , and 4 years for pfos . although the year - to - year variability is considerable , these biomonitoring data suggest doubling times on the order of 1015 years for pfoa , 48 years for pfda , and 510 years for pfos during the first decade of the millennium . a positive mass balance for the baltic sea suggests that inputs during the period when the samples were collected ( 20052011 ) must have been higher than the average input during the previous 20 years ( the water residence time in the baltic sea ) . it is notable that the contribution of direct atmospheric deposition to the total input to the baltic sea was smaller for pfos than for the other substances ; the dominant source was the rivers ( table 2 ) . another factor that could have caused an increase in riverine inputs of pfos and other pfaas during the 2000s is release of residues stored in the watershed . this is more than an order of magnitude greater than the pfos inventory in the baltic sea ( see table 2 ) . this illustrates that soil potentially is a large reservoir of pfos that will buffer pfos inputs from atmospheric deposition , delaying and modulating their transfer via surface runoff and groundwater to surface water . there is clearly a need to reduce the inputs of pfos and other pfaas to the baltic sea . this will require identifying and controlling the sources of pfaas in the atmosphere and obtaining a better understanding of how they travel through the terrestrial environment to surface water .

zinc ( zinc is not commercial ) is a public access database and tool set , initially developed to enable ready access to compounds for virtual screening , that has become ever widely used for virtual screening , ligand discovery , pharamcophore screens , benchmarking , and force field development . increasingly , however , investigators have tried to interrogate it for questions that it was not designed to answer . simple questions , such as how many endogenous human metabolites are there , which of these are purchasable , or what natural product or drug does a compound most closely resemble , were surprisingly difficult to answer . with a target in mind , investigators often wanted a focused library biased toward ligands for that target . with new compounds discovered , they often wanted to find the most similar ligands already known for that target . to optimize that ligand , they might look to the availability of starting products for synthesis , asking , for instance , how many boronic acids that contain an indole ring may be purchased in preparative quantities and how soon will they arrive . for these and many related questions , we wondered whether we could make a system that obviated the need for a computer expert s assistance . here , we describe a new version of zinc designed to address these questions , while retaining the ease of use of the original tool . zinc15 is designed to bring together biology and chemoinformatics with a tool that is easy to use for nonexperts , while remaining fully programmable for chemoinformaticians and computational biologists . 1 ) to integrate and curate biological activity , chemical property , and commercial availability data for small molecules from public sources , supplemented by additional calculated properties into a chemistry - aware relational database . 2 ) to build a general query language and report generator that is web url compatible . 3 ) to design a graphical user interface that requires no programming to interrogate the database using this query language . 4 ) to demonstrate and document the use of this tool to answer previously difficult questions . this effort has resulted in zinc 15 , a new research tool for ligand discovery that connects biological activities by gene product , drugs , and natural products with commercial availability . we describe the system and demonstrate its use to answer questions about biologically active and purchasable chemical space that were previously not easy for nonexperts . previously in zinc , compounds stood on their own and were both the subject of queries to zinc and the answers to such queries . an innovation of this version is to identify those molecules that have known biological effects or are of biological origin , such as drugs and natural products , and to link compounds to the proteins and biological processes that they modulate . correspondingly , one can now interrogate zinc regarding the ligands that bind to a particular protein or regarding the proteins that a particular molecule is known to modulate . extending this , one can also ask what biologically active molecules are most like those that bind a particular protein of interest and what proteins such a compound might be predicted to bind , based on chemical similarity to known ligands . in this way , the mission of zinc is expanded from purely compound - centric to one that links molecules to biological targets , processes , and other bioactive small molecules . the biological annotations the identification of molecules as metabolites , drugs , and natural products and the identification of molecules as ligands for particular proteins and processes all derived from other databases and libraries , such as hmdb , chembl , and drugbank , for which zinc is essentially a client and from whose rapid development in the last several years zinc has benefited . what is new here is that zinc cross - references this information with purchasability of reagents ; this much expands its ability to bring readily available reagents to biological questions . doing so has demanded optimization of the mechanics of zinc and its interface . some of these have obvious impacts on the user and the questions they can ask , for instance , expanding the number of molecules they can address by 2 orders of magnitude . for convenience , we divide the results into descriptions of the new associations with which purchasable molecules are freighted and examples of their use . zinc15 draws upon third party databases such as chembl , hmdb , drugbank , and https://clinicaltrials.gov to annotate high information compounds that are active in , or created by , nature . these include biogenic molecules , such as natural products , metabolites , and fda approved drugs , among others ( figure 1a ) . what zinc brings is not only lists of these molecules , which after all are derived from other sources , but their purchasability and , as we will see , their predicted as well as known associations . for instance , there are 70539 metabolites that have been annotated , 15006 of which may be purchased from vendors . restricting this to endogenous human metabolites , there are 46941 molecules , of which 8271 are for sale . properties of catalogs and , in turn , the molecules they contains are ranked in three independent series , thus : biogenic : endogenous human metabolites > nonhuman metabolites > natural products > unknown biogenicity . drug - series : fda approved > world drugs > investigational > in man > in vivo > in cells > in vitro > unknown bioactivity . purchasability : in - stock > via agent > on - demand > boutique > annotated ( not for sale ) . within each series , we classify each compound by the highest level it attains by catalog membership . molecules found in catalogs categorized as containing only endogenous human metabolites such as oxedrine ( zinc733 ) , for instance , inherit that property . compounds are also annotated with molecular properties that speak to their potential behavior and mechanism . for instance , 16485 compounds have appeared in the literature and have been shown to aggregate , a mechanism that is the origin of the greatest number of artifacts in early drug discovery . of these , 15072 are purchasable . interestingly , 53 metabolites have been observed to aggregate as have 23 world drugs , i.e. drugs approved by major national regulatory agencies such as the fda . the zinc15 web interface . a : selected zinc substance subsets showing the number of purchasable bioactive and biogenic subsets . click on the subset name ( 2 ) to browse or download a subset . ( 3 ) estimates of the number of each subset . ( 4 ) inset of dropdown menu providing access to other resources . the page navigation tool ( 1 ) , the get total tool ( 2 ) , the breadcrumbs ( 3 ) , the selection tool ( 4 ) , and the download tool ( 5 ) . click on any molecule s number ( 6 ) to view its detail page . for instance , to answer the following question : how many endogenous human metabolites are there . the user would browse to http://zinc15.docking.org , click on substances in the navigation bar ( top ) , click on subsets ( top , left ) , and select endogenous from the list ( bottom ) ( figure 1a ) . the endogenous human metabolites are displayed , as tiles , but often there are too many to count immediately ( figure 1b ) . to count the number if it is not displayed , the user clicks on the get total button ( top , left ) ( 47319 ) . the user selects now from the selection tool dropdown menu ( top , right ) and clicks on the count button again ( now 7190 ) . to download these , the user would click on the download button ( top , right ) . the same procedure is applicable for drugs , investigational compounds , and biogenic compounds , among others . thus , the user may ask for drugs that are also metabolites or natural products that have been investigated in clinical trials . having drawn on databases such as chembl and drugbank to associate compounds with their individual targets for instance , in zinc15 there are 2737 eukaryotic proteins that have compounds binding at 10 m or better annotated to them . over 100,000 distinct compounds hit at least one eukaryotic target at 10 nm or better , rising to over 1/3 of a million at 10 m . intriguingly , only 361 bacterial proteins have molecules annotated to them amounting to only 4283 compounds at 1 m or better ; the numbers for archael and viral targets are , notwithstanding intense interest , lower still . zinc may be used to acquire focused libraries of ligands annotated to a particular gene . for instance , a user seeking new ligands for the ionotropic glutamate receptor grin1 might want the 59 known ligands as controls in 3d sdf format files of the usual relevant forms expected at physiological ph . in the previous example orthologous genes were combined into gene symbols , but some questions require a particular species be specified . thus , 2025 compounds bind the human beta-2 adrenergic receptor at 10 m or better , while 2050 distinct compounds bind any of its orthologs , which includes the 2025 above plus 25 additional compounds . eight distinct uniprot annotations are available for this gene , and 2021 distinct ligands bind either the rat or human form at 1 m or better . the user may look for molecules either one at a time or in bulk . for a single molecule , the chemical drawing may be seeded with a drug or chemical name , smiles string , smarts pattern , inchi , inchikey , zinc i d , or even original catalog ids such as chembl ids ( figure 2a ) . four buttons below the chemical drawing tool allow for exact , substructure , and two kinds of similarity searches , using either tanimoto or dice coefficients , each based on 512 bit ecfp4 fingerprints . to look up many compounds at once , the user may use the resolver ( figure 2b ) , specifying one molecule per line , again using smiles , name , and i d but not smarts . ( 1 ) the user may seed the drawing with chemical or drug names , zinc ids , inchi , inchikey , or original catalog numbers . the search options may be edited prior to search ( 2 ) , which is run using one of four buttons below drawing ( 3 ) : exact match , substructure , and two kinds of similarity . specify one molecule per line in a file ( 4 ) or by pasting ( 5 ) . additional search options ( 6 ) . format of results may be specified ( 7 , inset 8) prior to running the resolver . buttons to browse subsets or view subsets ( 9 ) as well as general free text search . there is no set limit on the number of molecules that may be returned in a single similarity search calculation . using the api , it should be possible to download 1 million or more compounds , in any format , based on similarity and/or substructure . however , these queries may take a long time and would likely be run in batch mode . zinc is a public resource , and occasionally the most pragmatic solution may be to download a large portion of zinc and run a chemical search locally . we look forward to discovering the practical limits of this new technology . in previous versions of zinc , we supported similarity searches for multiple molecules , apparently in parallel . internally , they were run serially , and the results concatenated . currently , there is no mechanism to run queries with multiple query molecules . 2 ) use the resolver , which is limited to a minimum similarity of 0.7 ( ecfp4 ) . when a hit is found in a screening campaign , a common next step is to identify , possibly model , and then purchase analogs , often called sar - by - catalog . how well explored is the annotated or purchasable chemical space around a compound ? to investigate analogs of olaparib ( zinc 40430143 ) , a recently approved parp inhibitor , the user clicks on substance in the navigation bar and types olaparib in the input line above the drawing tool ( figure 2a ) . at time of writing there were 38885 analogs within a tanimoto of 50% ( ecfp4 ) , 297 of which were in stock for immediate delivery . zinc can answer questions about novelty of a newly discovered ligand for a particular target . for instance , the drug cariprazine is known to hit drd2 , but what are the most similar ligands that hit drd3 or drd4 ? to investigate this , the user would click on substances in the navigation bar and enter cariprazine in the draw / search structure field above the drawing tool ( the molecule appears ) ( figure 2a ) . on the results page , the user selects the relations selector ( the chain link icon ) , selects gene from the popup , types drd3 as the resource name , and clicks on the blue chain link icon to apply this constraint . zinc supports full smarts using rdkit , enabling complex chemical patterns to be matched . the same search tool used for similarity search may be used , in conjunction with the substructure button . smarts pattern searching can be slow , and thus many of these queries will probably end up being run in batch mode . for instance , to find benzylamines , the user would click on substances in the navigation bar , enter the smarts c1ccccc1cn in the draw / search structure bar above the drawing tool , and click on the substructure button below the drawing tool . to select only compounds available in preparative quantities , the users would click on the subsets popup ( the label icon ) and click on bb ( building blocks ) . to only select compounds available for immediate delivery , the user would select now from the same popup . if the user needs to look up more that a few molecules , a bulk facility can simplify this task . to do this , the user selects substances from the navigation bar and using the resolver ( figure 2b ) either selects a file containing molecules to look up or specifies them in the paste smiles field . in either case , there should be one molecule per line , which may be smiles , cas number , name , zinc i d , or an original catalog i d , such as chembl i d . options include allowing close matches , looking for analogs , and whether a single or multiple matches should be returned per input line . the output may be to a web page or a downloadable file in 11 supported formats ( figure 3a ) . when ready , the user clicks resolve file to start the process . if more than 300 molecules are specified , the job is automatically run in batch mode . a. zinc results may be accessed in 11 formats plus the web pages . three line - oriented formats are easy to parse for both people and computers . three machine readable formats provide for rich and flexible data interchange between programs . b. compounds are classified by how they may be purchased based on their current catalog membership . there are five primary levels and three derived levels . c. we classify substances into six levels by the most reactive group they possess , based on smarts patterns.d . 3d representations are associated progressively with the ph range at which they become relevant for docking . these patterns enable new features , such as computing a reactivity attribute for each molecule , accelerating substructure search , and providing a basis for new features . we have calculated patterns for 480 pains patterns using the rdkit version of the guha translation of the original sln format smarts . we also include 40 filtering patterns used in the previous version of zinc for backward compatibility . we have calculated statistics on the prevalence of these functional groups allowing the most popular and the least popular to be easily identified . we compute a reactivity score ( figure 3c ) , which classifies each molecule by the worst functionality it contains , enabling queries as well as subsets that follow community opinion in the tranche browser . for instance , which pains patterns are most common among drugs ? to answer this , the user would select patterns from the navigation bar , click browse , and then click on the drugs column heading twice to sort it in descending order . to find purchasable sulfonyl halides , the user would select patterns from the more menu in the navigation bar and then click browse . in the lookup field , the user would type sulfonyl halide and click on the blue go button . the user can see that there are 85487 sulfonyl halides for sale and clicks on the number to view the substances . the user may further specify building block or now subsets to further narrow the query . the statistics of occurrence documents the popularity of rings by subset . to browse these , the user would select rings from the navigation bar followed by browse . rings may be ordered by their frequency of occurrence in drugs , natural products , or purchasable subsets by clicking on the column heading . the interface allows the user to , for instance , rapidly identify all compounds containing indole rings available in preparative quantities , all investigational compounds containing quinazoline rings , or all compounds containing both pyrimidine and morpholine rings . the latter is interpreted as all substances containing a morpholine ring , and among these , those where any ring in the compound is pyrimidine . interesting questions may be answered from the molecule detail page alone ( figure 4 ) . to look up an individual drug by name or zinc i d , click on substances in the navigation bar and enter its name or code into the text input field above the molecular drawing tool ( we will use the example for isoniazid , zinc1590 ) and click exact ( below , left ) . the molecule detail page contains purchasing information , annotated catalog membership ( figure 4a ) , biological activity data derived from chembl , biological activity predictions from sea and chembl , similar interesting molecules ( figure 4b ) , publications from chembl , chemical patterns , rings , publications from chembl , clinical trial information , and more . molecules may be downloaded in either 2d or 3d in 11 formats ( figure 3a ) . large subsets and slow - to - download ones will be queued and run in batch mode when resources permit . a special class of downloads lead - like and fragment - like subsets , for which we recommend the tranche browser , accessed from the tranches button in the navigation bar ( figure 5 ) . the tranche browser divides physical property space into 121 tranches based on two properties in 11 bins each : the horizontal axis is size ( molecular weight ) and the vertical axis is polarity ( logp ) . the tranche browser allows the user to select the characteristics of the database subset required and then download it , in 2d ( smiles ) for chemoinformatics or 3d formats for docking . the user may select or deselect individual tranches by clicking on them or use the presets selector ( top right ) to choose a popular subset . for 2d databases , the user may also specify purchasability ( figure 3b ) and reactivity ( figure 3c ) and two downloading options , format ( figure 3a ) and download method . in addition to purchasability and reactivity , 3d users may specify restrictions on net molecular charge and ph range ( figure 5 ) . a. showing ( 1 ) zinc i d , name if known , subset membership , ( 2 ) properties and 2d depiction , ( 3 ) 3d representations if available , ( 4 ) purchasing information , and ( 5 ) annotated catalog membership , ( 6 ) breadcrumbs indicating current location , ( 7 ) selection tool for refinement of query , and ( 8) download tool . b. interesting bioactive and biogenic analogs section of molecule detail page : ( 1 ) similar biogenic compounds , ( 2 ) similar bioactive compounds , ( 3 ) compounds with a shared scaffold , ( 4 ) similar aggregators , and ( 5 ) similar purchasable compounds currently too slow to calculate . physical - chemical space has been divided into 11 bins of polarity - hydrophobicity ( calculated logp , vertical ) ( 1 ) and size ( molecular weight , horizontal ) ( 2 ) . subsets of this space may be selected in 2d ( smiles ) or 3d ( figure 3a ) ( 3 ) . purchasability level ( figure 3b ) ( 4 ) and reactivity ( figure 3c ) ( 5 ) may also be specified . for 3d only , net molecular charge ( 6 ) and ph range ( figure 3d ) ( 7 ) may also be specified . presets ( 8) correspond to community practice ( e.g. , lead - like , fragment - like ) , and download ( 9 ) provides for five methods to access the selected molecules . knowing that a molecule resembles a drug or natural product can help generate hypotheses of mechanism of action , while the absence of similar annotated compounds might suggest biological novelty . each substance detail page includes an analogs section , in which the nearest endogenous human metabolite , any metabolite , natural product , drug , in man compounds , and bioactives are shown , if there is one within a tanimoto index of 0.6 ( 512 bit ecfp4 fingerprints ) . a find more button may be used to find more . we have already seen earlier how the most similar compounds annotated for an individual gene target , its major target or subclass , or from a particular catalog may also be found . downloading a 3d screening library for docking subset for docking , the user selects tranches from the navigation bar and then clicks on 3d ( top left ) to switch to the 3d downloading tool ( figure 5 ) . from the preset popup ( top right ) the user chooses lead - like , which sets the molecular weight and logp range of tranches . individual tranches may be selected or deselected by clicking on them . by default , the purchasability selector ( figure 3b ) is set to wait - ok , which includes both in stock and on demand compounds . the reactivity selector ( figure 3c ) is set to mild by default , which includes pains and weakly reactive compounds . to download a script to download the database , the user clicks on the download icon ( top right ) , where two further choices are possible . the user downloads the script , which may then be run to download the database tranches . target classes such as membrane receptors , ion channels , transporters , and enzymes group proteins by function . we have adapted the top two classification tiers in chembl to assign one of 15 major and 42 target subclasses to all genes ( table 2 ) . zinc may be used to select compounds that are known or predicted to hit particular classes of targets . thus , for instance , there are 35080 commercially available ligands for class a gcprs ( purchasable ) , and for epigenetic regulator targets , there are 1286 purchasable annotated ligands . the number of genes total as well as the number of genes for which one or more compounds active at 10 m or better was for sale at the time of publication . the number of distinct compounds that are for sale and not for sale for each target class . zinc encompasses more of the purchasable and annotated chemical space as the catalogs it includes grow in size and number , currently adding around 50 new vendor and annotated catalogs each year . the date each catalog was last updated is available in tabular form and on the detail page of each catalog . two - dimensional smiles have been decoupled from 3d models , allowing us to load molecules for which we do not build noncovalent 3d docking models such as boronic acids , tin , and silicon containing compounds . zinc now includes molecules with molecular weight of up to 1000 da , offering more complete coverage of commercially available chemical space , currently with 220 million molecules , over half of which are for sale . zinc is now a good way to find molecules even if they are too big to be practical for docking , as long as they are available for purchase . building blocks available in preparative quantities are now included and are easier to find . we now use chemaxon s jchem to protonate and prepare biologically relevant tautomers , resulting in an average of 2.2 biologically relevant forms per molecule at physiological ph . zinc now uses the latest version of omega ( openeye scientific software , http://eyesopen.com ) for improved 3d conformations for docking . we have added pdbqt to sdf , mol2 , and our own flexibase formats , for both dock37 and previous versions . zinc15 will now generate dude - style decoys for any molecule directly from the database . zinc now provides iupac inchi and inchikeys for every molecule to allow better interoperability with other resources such as wikipedia , chembl , pubchem , chemspider , and unichem . the first part of an inchikey , which specifies the framework without stereochemistry or protonation , offers a reliable way to find stereoisomers ( e.g. , for praziquantel ) . the canonicalization required by inchis has reduced molecule duplication in zinc so that search results are better estimates of their true values . the anatomical therapeutic chemical classification system , curated by the world health organization , organizes drugs by their therapeutic , pharmacological , and chemical properties . zinc acquires atc codes of drugs via chembl20 and drugbank , allowing drugs to be selected by anatomical , therapeutic , and chemical classes . for instance , the user may find all purchasable drugs for cancer , all dermatologicals of biological origin , and opioid anesthetics such as fentanyl . integration of atc codes also allows for more consistent identification of who - assigned names for substances . we load clinical trials information from https://clinicaltrials.gov , enabling zinc to answer questions about these important compounds . for instance , to see the current clinical trials , the user would click on clinical trials in the navigation bar and select current from the selection tool ( top right ) . it is possible to ask to see which compounds are in clinical trials that hit a eukaryotic target at 10 nm or better and also to ask to see compounds in clinical trials for cancer that are sold by cayman chemicals . publications information from chembl enables the literature to be browsed , active compounds for any paper to be displayed , papers to be found based on which compounds they report on , and many other questions . the user need only enter the pmid of a paper to retrieve all the active structures it reports . suppose the user would like the active compounds from a paper in 2d or 3d form . 2014 , 57 , 9 , the user would click on publication in the navigation bar and enter either the pmid ( here 24684293 ) or select the journal , year , volume , and page number from the selectors . the user clicks search to arrive at the page where the two genes and the first five compounds that bind them at 10 m or better as described in that paper are shown . to download these compounds , use the download selector ( top , center ) . to find out whether any of the compounds reported in this paper can be purchased , the user would click on purchasable in the selection tool ( top , right ) . for example , it turns out that the subnanomolar ligand for protein kinase c , zinc4096162 , is available , in both screening and preparative quantities . this compound , in turn , is reported in 15 papers , which are summarized at the bottom of its detail page or listed at the references relation to zinc4096162 . a new api that is almost identical to the web page url structure allows zinc to be scripted and integrated into third - party applications . documentation for both the web page and the api is available using the help and examples endpoints , and the api ( url ) syntax is described on our wiki . machine - readable formats such as json , xml , sdf , csv , and txt may be read directly into third party client programs such as knime , pipelinepilot , cytoscape , datawarrior , instantjchem , and ipython notebook via pandas . in many cases , the content of the help pages each resource supports up to ten endpoints , including the relation endpoint , which intersects one relation with another . the subsets endpoint allows the curators to define popular subsets of the resource , which can help simplify query syntax . the supported subsets for each resource are available at the respective subsets endpoint . three themes emerge from this work . first , a new research tool zinc15 is now available . it enables chemists and biologists to answer questions that before would have required the assistance of a chemoinformatician . second , zinc has also been improved for experts , enabling them to integrate its features into their applications using the new api . third , zinc has undergone a wide variety of improvements for its original constituency , molecular docking . this required the design of a new database , the use of new software such as rdkit , a new url - compatible query language and report generator , and new web pages designed to simplify complex tasks . the data are structured in 20 resources , which are further divided into subsets . questions may now be asked not only about molecules but also genes and their target classes , catalogs , chemical patterns and rings , publications , and clinical trials as well as individual activity data points . orthologous targets from chembl are now grouped by gene symbol and organized by major and subclasses . the system offers focused libraries of known compounds , purchasable or otherwise , organized by gene , subclass , or major target class . zinc answers questions about chemical novelty and similarity to known drugs , bioactives , and natural products . chemoinformaticsts may now embed zinc and its tools into their own applications . for instance , zinc has been integrated into cytoscape ( zincytoscape ) and r ( spelter ) . the new api offers a modular interface using industry - standard formats like xml and json , and reports are flexible in format and content . resources and their attributes are fully documented and may also be retrieved in a machine - readable format allowing the creation of rich and dynamic tools . the interface accepts molecular queries represented not only as smiles and smarts but also inchi , inchikey , and even binary fingerprints . finally , the virtual screening community can benefit from many innovations and improvements here . among these are new vendors , new annotated catalogs , improved 3d representations , tranches for more efficient physical property subsets , less duplication , faster and more comprehensive searches by similarity and substructure , annotations grouped by gene and organism class , and search results that may be hundreds or thousands of times larger than before . zinc provides a view of biologically precedented and commercially available chemical space organized by genes and the major and subclasses to which they belong . for over one - third of genes that have ligands reported in the literature not a single one of them is for sale , underscoring an urgent need for synthesis to fill gaps in screening libraries . it inherits errors and ambiguity from the catalogs it incorporates , including stereochemical ambiguity , an ongoing challenge with few solutions that are not labor intensive . whereas our goal is to make the interface capable of creating every query without programming expertise , the zinc query and report language ( api ) allow many options for which we have not yet been able to build a point - and - click interface . due to its size and to the generality of questions supported , some queries will take a long time and must be run in batch . batch mode , meant to handle long - running queries , will not be released until december 2015 . , zinc allows compounds to be annotated for bioactivity and biogenicity , adding value to their library . synthetic organic chemists may use zinc to identify neglected metabolites or drugs for synthesis or other bioactives that are not currently purchasable , as well as the building blocks with which to make them . curators of annotated libraries such as chembl and drugbank may use zinc to enhance their offerings with supporting information such as purchasability and biogenicity information . dockers and chemoinformaticians may download commercially available libraries for screening , in 2d or 3d , as well as sets of known actives as controls . medicinal chemists may use zinc to compare their discoveries to what is known publically and then to find purchasable analogs or building blocks to make new libraries . we expect the zinc tools and libraries to have broad utility in the community . new software was written for loading , curating , and querying the database in python using the chemoinformatics software system rdkit 2014_09_01 , the python structured query language toolkit and object relational mapper sqlalchemy version 0.9.8 , and the python web framework flask version 0.10.1 . some sources such as hmdb and drugbank were loaded as several distinct catalogs in zinc allowing us to leverage the curation of metabolite origin such as plant metabolites in hmdb or drug status such as investigational drugs in drugbank . all catalogs in zinc are categorized by their biogenic and bioactivity status , if any . for instance , the approved subset of drugbank was categorized as world drugs since it contains over 100 drugs approved in other countries but not by fda , and the endogenous these values are computed and stored and are accessible in the interface as molecular features . there are four biogenic catalog levels : 1 ) endogenous human metabolites , i.e. compounds that are synthesized in man . interestingly , this may include compounds produced by our bacterial flora ; 2 ) metabolites of any species , i.e. small molecules that are involved in metabolism , development , and reproduction but not metabolites of xenobiotics ; 3 ) biogenic compounds , often called natural products ; 4 ) unknown biogenic status . 1 ) fda approved ; 2 ) world drugs ; 3 ) investigational , compounds reported to be used in clinical trials ; 4 ) in man , which including nutraceuticals , for instance ; 5 ) in vivo , which includes drugbank experimental compounds that have been in animals ; 6 ) in cells , which includes compounds reported active in cell based assays ; 7 ) in vitro , compounds active or assumed active at 10 m or better in a direct binding assay . the categories are ordered to be progressively inclusive within each series , thus all fda approved drugs are also world drugs and all compounds active in cells are also active in vitro . we annotate as building blocks those catalogs of compounds available in preparative quantities , typically 250 mg or more . commercial vendors are categorized by the speed and cost of compound acquisition , allowing the best purchasability of every compound to be computed based on its current catalog membership . catalog categorizations are refined continually by purchasing experience in our lab and reports from colleagues , as follows : 1 ) in stock , delivery in under 2 weeks , 95% typical acquisition success rate ; 2 ) procurement agent , in stock , delivery in 2 weeks , 95% typical acquisition success rate ; 3 ) make - on - demand , delivery typically within 8 to 10 weeks , 70% typical acquisition success rate ; 4 ) boutique , where the cost may be high but still likely cheaper than making it yourself , 70% typical acquisition success rate . source catalogs are processed and loaded into the database ( 2d only ) as follows . name and cas numbers are loaded into a synonyms table , while selected bioactivity and other selected data are stored in a provided_values table . we convert sdf to smiles using rdkit and take the largest organic part of the compound ( desalting ) , enumerating up to four stereoisomers from stereochemically ambiguous smiles using oechem tk version 1.7 ( openeye scientific software , santa fe , nm ) . because of the combinatorial problem of ambiguous stereocenters in sterols , we used smarts filters to prioritize the most probable implied stereoisomers based on biosynthetic pathways ( prof . the smiles are neutralized with mitools ( http://molinspiration.com ) , which also filters out incorrectly coded molecules well . molecules are loaded using python / rdkit scripts by attempting to map them to existing zinc ids or creating new zinc substances as necessary , as well as any additional required datastructures . inchi and inchikeys are calculated on loading , and the inchikey is used as a unique constraint in the database . 512 bit morgan fingerprints with radius 2 ( effectively ecfp4 ) are calculated for each molecule using rdkit . the 3d molecule processing pipeline is now disconnected from the 2d loading process , above . we now use chemaxon s package and the command line tool cxcalc to calculate protonation states and tautomers at or near physiologically relevant ph in three ph tranches . these are physiological , covering roughly ph 6.4 to 8.4 , high , roughly ph 8.4 to 9.0 , and low , roughly ph 5.8 to 6.4 . each protomer is rendered into 3d using jchem s molconvert ( chemaxon , budapest , hungary ) and conformationally sampled using omega ( openeye scientific software , santa fe nm ) . atomic charges and desolvation penalties are calculated using amsol 7.1 and our previously published protocol . files are formatted for docking as flexibase files , mol2 , sdf , and pdbqt . we normalized pki , ic50 , ec50 , ac50 , and pic50 to a single standard pki value , which we rounded to two decimal places . we filtered out data flagged with the data_validity_comment field indicating possible problems in the source document . we associate compounds annotated for protein complexes to each of the genes involved in that complex . two common areas of biology where multigene complexes are observed is for the cell surface receptor integrins and the ligand - gated ion channels such as the nicotinic acetylcholine receptor . for instance , integrin vla-1 is an alpha-1/beta-1 heterodimer of two genes , itga1 and itgb1 , respectively . likewise , nachrs such as ( alpha-3)2(beta-4)3 is a heteropentamer of two genes chrna3 and chrnb4 , respectively . in such cases , for single proteins , we used uniprot gene symbols based on the uniprot accession codes in chembl . orthologs in the trembl part of uniprot often did not have assigned gene symbols , in which case we used the uniprot accession code as a provisional gene name . we assigned target classes and subclasses based on the first two subfields of the protein_class field of the protein_classification table of chembl . in this version of zinc there are 42 subclasses grouped into 15 major target classes : membrane receptor , ion channel , transporter , transcription factor , enzyme , epigenetic , and 9 other catch - all classes for the few cases when none of these fit . we computed 512 bit fingerprints using the morgan algorithm with radius 2 as implemented in rdkit . stereoisomers and some very near neighbors have identical fingerprints , resulting in approximately 50% fewer fingerprints than substances , on average . we grouped fingerprints into three classes , interesting , current , and benched for faster searching . queries that limit their results to annotated compounds need only search fingerprints in the interesting subsets , while benched fingerprints , corresponding to compounds not in any current catalog , are never searched . we have implemented a very general chemical search api that automatically parallelizes chemical search queries using python green threads to search in parallel increments of 1 million molecules at a time . executed on a 64-core computer , we often see full database smarts searches completing in 30 s or less , although smarts can be of almost unlimited complexity , and some queries will certainly take far longer . similarity searches features label molecules with computed properties often derived from catalog membership that would be prohibitive to calculate interactively . there are four biogenic class annotations : biogenic ( natural products ) , metabolites , endogenous human metabolites , and unknown . there are eight bioactivity classes , which includes drugs : fda approved , world drugs , investigational , in man , in vivo , in cells , in vitro , and unknown . there has been considerable interest in pan - assay interference ( pains ) smarts patterns recently . we used the rdkit version of the guha translation of the original 480 pains expressed in sybyl line notation ( sln ) . all molecules in zinc have been annotated and are searchable by pains and other smarts patterns . the reactivity categories are a ) anodyne ; b ) clean ( pains - ok ) ; c ) mild ( weakly reactive , typically as a nucleophile or electrophile ) ; d ) reactive ; e ) unstable or irrelevant for screening . for e , we do not build molecular models for noncovalent docking ( e.g. , boronic acids ) . the web site and api were coded in python using rdkit , sqlalchemy , and flask . a curator s tool ( zincmanage ) is used to load , update , and curate the database . a command line interface ( zinccli ) provides a unix - shell like interface for additional testing and curation . the stereochemical ambiguity problem is particularly acute in sterol rings , but since these are almost always biological in origin and are derived from the sterol biosynthetic pathway , sensible guesses of stereochemistry are reasonable . we have therefore created a special sterol processing pipeline for loading molecules into zinc . leslie kuhn for drawing our attention to this and for providing smarts patterns and advice . we used mitools ( http://molinspiration.com ) to extract ring systems from every zinc molecule and loaded them into the database . we calculate static counts of the number of molecules that have biogenic or bioactivity annotations , allowing rapid reports of approximate counts of numbers of qualifying compounds per ring . we built an interface to the docs table in chembl20 and integrated it into the docs resource on the molecule detail , gene detail , and target detail pages . we attempt to identify names for substances in zinc from who - assigned names via the atc , chembl molecule names , and synonyms extracted from hmdb , drugbank , ttd , and other catalogs . all drug or dietary supplement interventions are then queried using a free text search against substance names to map the indications to the corresponding substances in zinc . there are five classes of endpoints , ten static and an almost unlimited number of dynamic ones . the five endpoint classes are list , detail , relation , subsets , and special . thus , for example , the substances help endpoint provides guidance on how to find substances of interest and a table provides a list of available catalogs in zinc and the time of their last update and shows the available subsets of genes in zinc per chembl20 . the major classes home endpoint provides an overview of target classes in zinc and gives examples of how to query and select individual observations of compound - target affinities from chembl , with or without additional purchasability constraints .

many questions about the biological activity and availability of small molecules remain inaccessible to investigators who could most benefit from their answers . to narrow the gap between chemoinformatics and biology , we have developed a suite of ligand annotation , purchasability , target , and biology association tools , incorporated into zinc and meant for investigators who are not computer specialists . the new version contains over 120 million purchasable drug - like compounds effectively all organic molecules that are for sale a quarter of which are available for immediate delivery . zinc connects purchasable compounds to high - value ones such as metabolites , drugs , natural products , and annotated compounds from the literature . compounds may be accessed by the genes for which they are annotated as well as the major and minor target classes to which those genes belong . it offers new analysis tools that are easy for nonspecialists yet with few limitations for experts . zinc retains its original 3d roots all molecules are available in biologically relevant , ready - to - dock formats . zinc is freely available at http://zinc15.docking.org .

Introduction Results Discussion Methods

zinc ( zinc is not commercial ) is a public access database and tool set , initially developed to enable ready access to compounds for virtual screening , that has become ever widely used for virtual screening , ligand discovery , pharamcophore screens , benchmarking , and force field development . to optimize that ligand , they might look to the availability of starting products for synthesis , asking , for instance , how many boronic acids that contain an indole ring may be purchased in preparative quantities and how soon will they arrive . for these and many related questions , we wondered whether we could make a system that obviated the need for a computer expert s assistance . here , we describe a new version of zinc designed to address these questions , while retaining the ease of use of the original tool . 1 ) to integrate and curate biological activity , chemical property , and commercial availability data for small molecules from public sources , supplemented by additional calculated properties into a chemistry - aware relational database . this effort has resulted in zinc 15 , a new research tool for ligand discovery that connects biological activities by gene product , drugs , and natural products with commercial availability . we describe the system and demonstrate its use to answer questions about biologically active and purchasable chemical space that were previously not easy for nonexperts . previously in zinc , compounds stood on their own and were both the subject of queries to zinc and the answers to such queries . an innovation of this version is to identify those molecules that have known biological effects or are of biological origin , such as drugs and natural products , and to link compounds to the proteins and biological processes that they modulate . in this way , the mission of zinc is expanded from purely compound - centric to one that links molecules to biological targets , processes , and other bioactive small molecules . the biological annotations the identification of molecules as metabolites , drugs , and natural products and the identification of molecules as ligands for particular proteins and processes all derived from other databases and libraries , such as hmdb , chembl , and drugbank , for which zinc is essentially a client and from whose rapid development in the last several years zinc has benefited . for convenience , we divide the results into descriptions of the new associations with which purchasable molecules are freighted and examples of their use . zinc15 draws upon third party databases such as chembl , hmdb , drugbank , and https://clinicaltrials.gov to annotate high information compounds that are active in , or created by , nature . these include biogenic molecules , such as natural products , metabolites , and fda approved drugs , among others ( figure 1a ) . what zinc brings is not only lists of these molecules , which after all are derived from other sources , but their purchasability and , as we will see , their predicted as well as known associations . for instance , there are 70539 metabolites that have been annotated , 15006 of which may be purchased from vendors . restricting this to endogenous human metabolites , there are 46941 molecules , of which 8271 are for sale . within each series , we classify each compound by the highest level it attains by catalog membership . for instance , 16485 compounds have appeared in the literature and have been shown to aggregate , a mechanism that is the origin of the greatest number of artifacts in early drug discovery . interestingly , 53 metabolites have been observed to aggregate as have 23 world drugs , i.e. drugs approved by major national regulatory agencies such as the fda . the user would browse to http://zinc15.docking.org , click on substances in the navigation bar ( top ) , click on subsets ( top , left ) , and select endogenous from the list ( bottom ) ( figure 1a ) . the same procedure is applicable for drugs , investigational compounds , and biogenic compounds , among others . thus , the user may ask for drugs that are also metabolites or natural products that have been investigated in clinical trials . eight distinct uniprot annotations are available for this gene , and 2021 distinct ligands bind either the rat or human form at 1 m or better . for a single molecule , the chemical drawing may be seeded with a drug or chemical name , smiles string , smarts pattern , inchi , inchikey , zinc i d , or even original catalog ids such as chembl ids ( figure 2a ) . four buttons below the chemical drawing tool allow for exact , substructure , and two kinds of similarity searches , using either tanimoto or dice coefficients , each based on 512 bit ecfp4 fingerprints . the search options may be edited prior to search ( 2 ) , which is run using one of four buttons below drawing ( 3 ) : exact match , substructure , and two kinds of similarity . format of results may be specified ( 7 , inset 8) prior to running the resolver . buttons to browse subsets or view subsets ( 9 ) as well as general free text search . there is no set limit on the number of molecules that may be returned in a single similarity search calculation . zinc is a public resource , and occasionally the most pragmatic solution may be to download a large portion of zinc and run a chemical search locally . in previous versions of zinc , we supported similarity searches for multiple molecules , apparently in parallel . at time of writing there were 38885 analogs within a tanimoto of 50% ( ecfp4 ) , 297 of which were in stock for immediate delivery . on the results page , the user selects the relations selector ( the chain link icon ) , selects gene from the popup , types drd3 as the resource name , and clicks on the blue chain link icon to apply this constraint . smarts pattern searching can be slow , and thus many of these queries will probably end up being run in batch mode . for instance , to find benzylamines , the user would click on substances in the navigation bar , enter the smarts c1ccccc1cn in the draw / search structure bar above the drawing tool , and click on the substructure button below the drawing tool . to only select compounds available for immediate delivery , the user would select now from the same popup . in either case , there should be one molecule per line , which may be smiles , cas number , name , zinc i d , or an original catalog i d , such as chembl i d . a. zinc results may be accessed in 11 formats plus the web pages . three line - oriented formats are easy to parse for both people and computers . b. compounds are classified by how they may be purchased based on their current catalog membership . these patterns enable new features , such as computing a reactivity attribute for each molecule , accelerating substructure search , and providing a basis for new features . we have calculated statistics on the prevalence of these functional groups allowing the most popular and the least popular to be easily identified . we compute a reactivity score ( figure 3c ) , which classifies each molecule by the worst functionality it contains , enabling queries as well as subsets that follow community opinion in the tranche browser . to answer this , the user would select patterns from the navigation bar , click browse , and then click on the drugs column heading twice to sort it in descending order . rings may be ordered by their frequency of occurrence in drugs , natural products , or purchasable subsets by clicking on the column heading . interesting questions may be answered from the molecule detail page alone ( figure 4 ) . the molecule detail page contains purchasing information , annotated catalog membership ( figure 4a ) , biological activity data derived from chembl , biological activity predictions from sea and chembl , similar interesting molecules ( figure 4b ) , publications from chembl , chemical patterns , rings , publications from chembl , clinical trial information , and more . large subsets and slow - to - download ones will be queued and run in batch mode when resources permit . a special class of downloads lead - like and fragment - like subsets , for which we recommend the tranche browser , accessed from the tranches button in the navigation bar ( figure 5 ) . b. interesting bioactive and biogenic analogs section of molecule detail page : ( 1 ) similar biogenic compounds , ( 2 ) similar bioactive compounds , ( 3 ) compounds with a shared scaffold , ( 4 ) similar aggregators , and ( 5 ) similar purchasable compounds currently too slow to calculate . , lead - like , fragment - like ) , and download ( 9 ) provides for five methods to access the selected molecules . each substance detail page includes an analogs section , in which the nearest endogenous human metabolite , any metabolite , natural product , drug , in man compounds , and bioactives are shown , if there is one within a tanimoto index of 0.6 ( 512 bit ecfp4 fingerprints ) . we have already seen earlier how the most similar compounds annotated for an individual gene target , its major target or subclass , or from a particular catalog may also be found . from the preset popup ( top right ) the user chooses lead - like , which sets the molecular weight and logp range of tranches . target classes such as membrane receptors , ion channels , transporters , and enzymes group proteins by function . we have adapted the top two classification tiers in chembl to assign one of 15 major and 42 target subclasses to all genes ( table 2 ) . zinc may be used to select compounds that are known or predicted to hit particular classes of targets . the number of genes total as well as the number of genes for which one or more compounds active at 10 m or better was for sale at the time of publication . the number of distinct compounds that are for sale and not for sale for each target class . zinc encompasses more of the purchasable and annotated chemical space as the catalogs it includes grow in size and number , currently adding around 50 new vendor and annotated catalogs each year . two - dimensional smiles have been decoupled from 3d models , allowing us to load molecules for which we do not build noncovalent 3d docking models such as boronic acids , tin , and silicon containing compounds . zinc now includes molecules with molecular weight of up to 1000 da , offering more complete coverage of commercially available chemical space , currently with 220 million molecules , over half of which are for sale . zinc is now a good way to find molecules even if they are too big to be practical for docking , as long as they are available for purchase . we have added pdbqt to sdf , mol2 , and our own flexibase formats , for both dock37 and previous versions . zinc now provides iupac inchi and inchikeys for every molecule to allow better interoperability with other resources such as wikipedia , chembl , pubchem , chemspider , and unichem . the anatomical therapeutic chemical classification system , curated by the world health organization , organizes drugs by their therapeutic , pharmacological , and chemical properties . for instance , the user may find all purchasable drugs for cancer , all dermatologicals of biological origin , and opioid anesthetics such as fentanyl . we load clinical trials information from https://clinicaltrials.gov , enabling zinc to answer questions about these important compounds . publications information from chembl enables the literature to be browsed , active compounds for any paper to be displayed , papers to be found based on which compounds they report on , and many other questions . suppose the user would like the active compounds from a paper in 2d or 3d form . 2014 , 57 , 9 , the user would click on publication in the navigation bar and enter either the pmid ( here 24684293 ) or select the journal , year , volume , and page number from the selectors . this compound , in turn , is reported in 15 papers , which are summarized at the bottom of its detail page or listed at the references relation to zinc4096162 . machine - readable formats such as json , xml , sdf , csv , and txt may be read directly into third party client programs such as knime , pipelinepilot , cytoscape , datawarrior , instantjchem , and ipython notebook via pandas . the supported subsets for each resource are available at the respective subsets endpoint . second , zinc has also been improved for experts , enabling them to integrate its features into their applications using the new api . this required the design of a new database , the use of new software such as rdkit , a new url - compatible query language and report generator , and new web pages designed to simplify complex tasks . questions may now be asked not only about molecules but also genes and their target classes , catalogs , chemical patterns and rings , publications , and clinical trials as well as individual activity data points . zinc answers questions about chemical novelty and similarity to known drugs , bioactives , and natural products . chemoinformaticsts may now embed zinc and its tools into their own applications . the new api offers a modular interface using industry - standard formats like xml and json , and reports are flexible in format and content . the interface accepts molecular queries represented not only as smiles and smarts but also inchi , inchikey , and even binary fingerprints . among these are new vendors , new annotated catalogs , improved 3d representations , tranches for more efficient physical property subsets , less duplication , faster and more comprehensive searches by similarity and substructure , annotations grouped by gene and organism class , and search results that may be hundreds or thousands of times larger than before . zinc provides a view of biologically precedented and commercially available chemical space organized by genes and the major and subclasses to which they belong . for over one - third of genes that have ligands reported in the literature not a single one of them is for sale , underscoring an urgent need for synthesis to fill gaps in screening libraries . it inherits errors and ambiguity from the catalogs it incorporates , including stereochemical ambiguity , an ongoing challenge with few solutions that are not labor intensive . whereas our goal is to make the interface capable of creating every query without programming expertise , the zinc query and report language ( api ) allow many options for which we have not yet been able to build a point - and - click interface . , zinc allows compounds to be annotated for bioactivity and biogenicity , adding value to their library . synthetic organic chemists may use zinc to identify neglected metabolites or drugs for synthesis or other bioactives that are not currently purchasable , as well as the building blocks with which to make them . dockers and chemoinformaticians may download commercially available libraries for screening , in 2d or 3d , as well as sets of known actives as controls . for instance , the approved subset of drugbank was categorized as world drugs since it contains over 100 drugs approved in other countries but not by fda , and the endogenous these values are computed and stored and are accessible in the interface as molecular features . small molecules that are involved in metabolism , development , and reproduction but not metabolites of xenobiotics ; 3 ) biogenic compounds , often called natural products ; 4 ) unknown biogenic status . molecules are loaded using python / rdkit scripts by attempting to map them to existing zinc ids or creating new zinc substances as necessary , as well as any additional required datastructures . inchi and inchikeys are calculated on loading , and the inchikey is used as a unique constraint in the database . files are formatted for docking as flexibase files , mol2 , sdf , and pdbqt . we normalized pki , ic50 , ec50 , ac50 , and pic50 to a single standard pki value , which we rounded to two decimal places . two common areas of biology where multigene complexes are observed is for the cell surface receptor integrins and the ligand - gated ion channels such as the nicotinic acetylcholine receptor . in such cases , for single proteins , we used uniprot gene symbols based on the uniprot accession codes in chembl . in this version of zinc there are 42 subclasses grouped into 15 major target classes : membrane receptor , ion channel , transporter , transcription factor , enzyme , epigenetic , and 9 other catch - all classes for the few cases when none of these fit . we grouped fingerprints into three classes , interesting , current , and benched for faster searching . executed on a 64-core computer , we often see full database smarts searches completing in 30 s or less , although smarts can be of almost unlimited complexity , and some queries will certainly take far longer . there are four biogenic class annotations : biogenic ( natural products ) , metabolites , endogenous human metabolites , and unknown . there are eight bioactivity classes , which includes drugs : fda approved , world drugs , investigational , in man , in vivo , in cells , in vitro , and unknown . the web site and api were coded in python using rdkit , sqlalchemy , and flask . we have therefore created a special sterol processing pipeline for loading molecules into zinc . we calculate static counts of the number of molecules that have biogenic or bioactivity annotations , allowing rapid reports of approximate counts of numbers of qualifying compounds per ring . the major classes home endpoint provides an overview of target classes in zinc and gives examples of how to query and select individual observations of compound - target affinities from chembl , with or without additional purchasability constraints .

nucleophilic additions to conformationally biased cyclohexanones can provide two stereoisomeric alcohol products via reaction at the axial or the equatorial face of the carbonyl ( figure 1 ) . the factors controlling selectivities have been studied and debated for roughly three - quarters of a century . the facial selectivity of addition is influenced by the size of the nucleophilic reagent : small nucleophiles tend to add to the axial face , whereas bulky nucleophiles preferentially attack the equatorial face . for example , nabh4 and lialh4 both deliver hydride to the axial face of 4-tert - butylcyclohexanone ( 1 ) , giving an equatorial alcohol as the major product , whereas bulky hydride reagents such as lihbbu3 ( l - selectride ) preferentially yield the axial alcohol via equatorial hydride delivery ( figure 1 ) . the observed preferential addition of bulky reagents to the equatorial face is generally attributed to steric factors : the axial face is more hindered than equatorial due to 1,3-diaxial interactions with the incoming nucleophile . historically , conversely , dauben attributed the axial preference observed with smaller reagents to product development control , reflecting the greater stability of the resulting equatorial alcohol . a more widely accepted model based on torsional strain was first proposed by felkin and later supported computationally by anh and eisenstein . anh model posits that the transition state ( ts ) for addition to the equatorial face of a cyclohexanone chair involves torsional strain greater than that of axial attack ; that is , there are more eclipsing interactions during equatorial attack compared to axial ( figure 2 ) . the stereoelectronic basis of these facial preferences has been studied extensively through computational methods by houk and others using small hydride reagents ( lih , nabh4 , lialh4 ) as computationally affordable systems . although other models have been proposed , including cieplak s model that emphasizes overlap between an antiperiplanar orbital and the developing * orbital , theoretical studies using small hydride reagents overall support the torsional strain model . increased eclipsed interactions ( torsional strain ) in equatorial face attack of hydrides to carbonyl compounds . this general trend in facial selectivity does not hold , however , for some six - membered cyclic ketones . cis-2,6-disubstituted n - acylpiperidones ( e.g. , compounds 2 and 3 in scheme 1 ) have been studied extensively by the comins group and others . for 2 and 3 , hydride addition from the equatorial face is favored even when a small hydride reagent ( nabh4 ) is used ( scheme 1 , eqs 1 and 2 ) . indeed , the axial face of this class of substrates should be particularly hindered due to the axial 2,6-substituents , forced into this conformation by a strain with the n - substituent . surprisingly , however , the more conformationally restricted bridged analogues ( tropinones , e.g. , compound 4 ) undergo favored axial attack by small hydride reagents despite the steric hindrance imposed by the bridge ( eqs 3 and 4 ) . low energy twist - boat conformations of piperidones have been observed in solution by nmr spectroscopy and in the solid state by x - ray diffraction . we wondered if these twist - boat conformers might be relevant to the transition states of reactions of these compounds . to study this possibility experimentally , we compared the reactivity of piperidone 3 and its tropinone analogue 5 toward k - selectride . notably , although the former substrate can access a twist - boat conformation , the latter can not due to geometrical constraints . based on the results of a competition experiment ( scheme 2 ) , the rate of reduction of tropinone 5 with k - selectride is about 3-fold slower than that of piperidone 3 ; however , both processes are completely selective toward formation of the axial alcohol ( equatorial attack of hydride ) . this result , in combination with the contrasting stereoselectivity of these two classes of substrates with nonbulky hydrides ( scheme 1 , eqs 3 and 4 ) , encouraged us to computationally explore the hypothesis that a twist - boat transition state could in fact be contributing significantly to the reaction of cis-2,6-disubstituted n - acylpiperidones . we have undertaken a computational study of the factors influencing the differences in reactivity and facial selectivity of reduction of these two structurally similar piperidone and tropinone substrate classes . we have analyzed the roles of the six - membered ring conformation and the size of the nucleophile on stereoselectivities . the contributions of both steric repulsion and torsional strain to the activation energy upon hydride addition , factors that have been found to be particularly relevant in cyclic ketones , were analyzed . our calculations indicate that , although a chair conformation experiences greater torsional strain during attack at the equatorial face by a small hydride , attack at the two faces of a twist - boat do not necessarily follow the same trend . additionally , with very bulky hydride reagents , the difference in torsional strain during attack at the axial vs equatorial face of a chair is nearly negligible due to a late transition state . figure 3 depicts the substrates and hydride reagents used in our computational studies . while 4-tert - butylcyclohexanone ( 1 ) is a well - studied conformationally biased cyclic ketone , chosen as a baseline , n - methoxycarbonyl - cis-2,6-dimethylpiperidone ( 6 ) and n - methoxycarbonyltropinone ( 7 ) were selected as piperidone and tropinone model substrates . lithium aluminum hydride ( lah ) is the small hydride model . for a bulky hydride , we mimicked the reactivity of l - selectride ( lithium tri - sec - butylborohydride ) using a computationally affordable surrogate ( l - selectride is conformationally and stereochemically very complex , with thousands of low energy conformers ) . we initially tested libhme3 as a bulky hydride model but were unable to reproduce the experimental selectivities using this relatively simple trialkylborohydride . however , a more sterically demanding hydride , lithium triisopropylborohydride ( ltbh ) was found to satisfactorily reproduce the experimental selectivities of l - selectride , albeit at greater computational cost than libhme3 due to the large conformational space of ltbh . figure 4 depicts the calculated geometries of the most stable chair ( 1ch , 6ch , and 7ch ) and twist - boat ( 1 tb and 6 tb ) conformations of the cyclic ketone substrates . newman projections sighting down the ring carbon carbonyl carbon bond are also provided . because of an internal plane of symmetry in the chair conformations , the two possible newman projections are enantiomeric ( e.g. , sighting down the c2c1 bond of 1ch provides the mirror image of sighting down c6c1 ) . conversely , two different newman projections are given for 1 tb and 6 tb , as the twist - boat conformations of these substrates lack a plane or axis of symmetry . in the ground state , 1 adopts a chair conformation ( 1ch ) that is more stable than the minimum energy twist - boat conformation ( 1 tb , in which c1 and c4 are at the bow and stern ) by 3.2 kcal mol ( figure 4a ) . conformations of cyclic ketone substrates calculated at the smd(thf)/b3lyp/6 - 311+g(2d , p)//b3lyp/6 - 31g(d , p ) level . gibbs free energies ( g ) are referenced to the lowest energy conformer , when applicable , and are given in kcal mol ; angles are given in degrees . both chair and twist boat conformations were also located for piperidone 6 ( figure 4b ) . previous studies on cis-2,6-disubstituted n - acylpiperidones have indicated that the chair conformation with equatorial 2- and 6-substituents is unstable . the resulting a - strain between the 2,6-diequatorial and the n - substituents disfavors such a conformation , and instead these substituents exist in an axial ( or pseudoaxial ) orientation in conformation 6ch . indeed , our calculations show that the energy penalty for placing these two groups equatorial is 6.1 kcal mol relative to 6ch ( see supporting information ) . despite the greater intrinsic stability of chair conformations , 6ch has a disadvantageous 1,3-diaxial interaction between the two axial methyl substituents , which is alleviated in 6 tb ( the distances between the proximal hydrogens of the two methyl groups in 6ch and 6 tb are 2.19 and 2.76 , respectively ) . the calculated geometry of the most stable 6 tb very closely matches that predicted by the nmr studies of venkatraj et al . , in which c6 ( the ring carbon syn to the carbonyl of the n - acyl group ) and c3 are at the bow and stern positions . the twist - boat conformation of 6 is predicted to be only 0.5 kcal mol higher in energy than 6ch , suggesting the coexistence of both isomers in solution . as described above , previous experimental and computational studies have indicated that similar piperidones ( albeit bearing bulkier 2- and 6-substituents ) exist predominantly in a twist - boat conformation in solution and in the solid state . the six - membered ring of tropinone 7 is locked into a chair conformation ( figure 4c ) . the bridging ch2ch2 cinches the chair together on one side , making the nitrogen flap more folded in 7ch , with a flap angle ( out - of - plane dihedral angle ) of 115 compared to 138 in 6ch ( , figure 4d ) . conversely , the carbonyl flap on the opposite side of 7ch is more flattened , with a flap angle of 146 ( , figure 4d ) , compared to 131 in 6ch and 137 in 1ch . the lowest energy reactant species for the reduction of cyclic ketones 1 , 6 , and 7 by lah are prereaction coordination complexes that are stabilized by 10.9 to 12.3 kcal mol with respect to the separated reactants ( see , for example , figure 5a ) . substrate geometries are not significantly distorted by formation of the coordination complexes , and the energy differences between chair and twist - boat conformations ( for 1 and 6 ) are very nearly conserved ( figure 5b ) . thus , all of the calculated activation barriers reported herein are measured from the lowest - energy prereaction coordination complexes . although the most realistic representation of the lithium counterion would likely include coordinated solvent ( thf ) molecules , we were unable to obtain optimized geometries of all the transition states needed to account for stereoselectivity using explicit solvent . however , significant computational precedent exists for successfully reproducing experimental selectivities of hydride reductions involving nonexplicitly solvated lithium species . ( a ) example of formation of the reactant complex with lah , shown for 1ch . ( b ) comparison of the gibbs free energies ( g ) of chair and twist - boat conformations of free ketones 1 , 6 , and 7 and their prereaction complexes with lah . optimized structures were calculated at the smd(thf)/b3lyp/6 - 311+g(2d , p)//b3lyp/6 - 31g(d , p ) level . gibbs free energies ( g ) are referenced to the lowest energy free ketone conformation and are given in kcal mol . because neither face of a twist - boat experiences nucleophilic attack via a true axial or equatorial trajectory , attack on the twist - boat face that corresponds to the axial face of the analogous chair will be herein be referred to as pro - axial attack or attack at the pro - axial face ( the term pro - equatorial will also be used ) . these terms refer to the orientation of the added nucleophile in the product ( i.e. , the axial or equatorial orientation of the hydride ) and not of the hydroxyl . the torsional strain associated with the transition states herein is represented graphically by newman projections and described numerically by the parameter , which corresponds to the average deviation from 60 of the 12 dihedral angles of both newman projections involving the carbonyl carbon ( figure 6 ) . it should be noted that due to the intrinsic conformational differences between chair and twist - boat conformations , there is a certain amount of eclipsing already associated with the twist - boats . hence , the ranges of values for these two ring conformers are different , and these parameters should only be compared between attack at the axial and equatorial faces of the same ring conformation , not across different conformations . it should be also noted that , depending on the position of the ts in the reaction coordinate , the range of values for can change significantly due to the greater sp character of the carbonyl carbon in late and more distorted ts . measurement of torsional strain ( ) in the ts of hydride addition to cyclic ketones . the transition structures obtained for addition of lah to both faces of the different conformations of ketones 1 , 6 , and 7 are provided in figures 79 . consistent with experimental selectivity trends,1ch was found to favor attack at the axial face by lah , rather than the equatorial face , by 1.1 kcal mol ( corresponding to an 86:14 ratio of axial : equatorial addition products at 25 c ) . the analysis of the geometries of the transition states for attack at the axial and equatorial faces ( 1ch - ts - lahax and 1ch - ts - laheq , figure 7 ) revealed features consistent with the previously described models in which attack on the equatorial face experiences greater torsional strain compared to axial face attack . although the corresponding equatorial and axial transition states are located at similar points on the reaction coordinate ( the forming c h and breaking al h bond lengths are 1.66 and 1.71 , respectively , for both transition structures ) , addition to the equatorial face of 1ch involves slightly greater eclipsing interactions ( = 12 for axial face attack vs 14 for equatorial face attack ) . moreover , addition to the chair equatorial face requires greater distortion of the ring dihedral angle relative to the geometry of the reactant 1ch ( dihedral angle c c c c = 40 and 60 for 1ch - ts - lahax and 1ch - ts - laheq , respectively , compared to 48 for 1ch ) . these results illustrate how changes in geometry occurring in the ts region can sometimes have opposing effects : the expansion of a single dihedral angle to optimal values of a c(sp)c(sp ) bond can be detrimental if it implies a great distortion from the reactant structure . the delicate balance between these stabilizing / destabilizing geometric features ultimately results in the overall relative energies of competing pathways . lowest energy transition structures for the addition of lah to ( a ) the axial face of chair 1ch , ( b ) the equatorial face of chair 1ch , ( c ) the pro - axial face of twist - boat 1 tb , and ( d ) the pro - equatorial face of twist - boat 1 tb . optimized structures were calculated at the smd(thf)/b3lyp/6 - 311+g(2d , p)//b3lyp/6 - 31g(d , p ) level . activation gibbs free energies ( g ) are referenced to the lowest energy prereaction coordination complex and are given in kcal mol ; distances are given in angstroms and angles in degrees . the twist - boat transition states for 1 ( figure 7c and 7d ) are slightly later ( the forming c h bond length is 1.60 for 1tb - ts - lah and 1.66 for 1ch - ts - lah ) and , consequently , higher in energy than the chair transition states for both pro - axial and pro - equatorial hydride addition . this difference in position on the reaction coordinate contributes to amplifying the intrinsic preference for the chair conformation in the transition state ( gtwist - chair = 6.2 and 4.7 kcal mol for pro - axial and pro - equatorial addition , respectively ) with respect to the initial reactant ( gtwist - chair = 2.8 kcal mol ) . the increased destabilization of the twist - boat relative to the chair can also be attributed , at least in part , to a greater difference in torsional strain between the two transition states . h = 8 ) to transition state , especially for axial attack ( o c c h of 1ch - ts - lahax = 45 ) , albeit with an 8 compression in the c c c c angle . in contrast , the twist - boat transition states maintain an eclipsed arrangement ( the smaller o c c h angle = 4 in 1 tb , compared to 611 in 1tb - ts - lah ) . notably , neither face of the twist boat is strongly preferred for hydride addition by lah due to similar torsional strain occurring in both approaches , as represented by very similar values . in fact , pro - equatorial attack on the twist boat is slightly favored over pro - axial attack by 0.4 kcal mol ( figure 7c and 7d ) . this weak preference for pro - equatorial attack in the twist boat is opposite to the preference for axial attack on 1ch and alludes to the possibility that stable twist - boat conformations can alter the usual stereoselectivity . consistent with literature reports , the equatorial face of piperidone 6 is predicted to be more reactive than the axial face toward lah by at least 1 kcal mol ( figure 8) . the predicted stereoselectivity for this reaction , considering all feasible pathways , is a 89:11 ratio of axial : equatorial alcohols at 25 c ( or 95:5 at 78 c ) . this equatorial preference is predicted for both chair and twist - boat transition structures ( geq - ax = 0.8 and 2.0 kcal mol for 6ch - ts - lah and 6tb - ts - lah , respectively ) . in the chair conformation , the destabilization of the ts for axial face attack is likely caused by the steric hindrance with the 2,6-dimethyl substituents , which translates into a longer forming c h bond distance ( 1.75 vs 1.66 in the cyclohexanone ts ) . attack at the equatorial face of the chair conformation also benefits from a slight mitigation of the 1,3-diaxial interactions between the two methyl groups in the transition state ( dh h = 2.22 ) , while axial attack does not provide any such relief ( dh h = 2.18 , compared to 2.19 in the reactant ) . these steric factors override the intrinsically greater torsional strain generated in attack on the equatorial face . lowest energy transition structures for the addition of lah to ( a ) the axial face of chair 6ch , ( b ) the equatorial face of chair 6ch , ( c ) the pro - axial face of twist - boat 6 tb , and ( d ) the pro - equatorial face of twist - boat 6 tb . optimized structures were calculated at the smd(thf)/b3lyp/6 - 311+g(2d , p)//b3lyp/6 - 31g(d , p ) level . activation gibbs free energies ( g ) are referenced to the lowest energy prereaction coordination complex and are given in kcal mol ; distances are given in angstroms and angles in degrees . consistent with our hypothesis that twist - boat transition states may be involved , pro - equatorial attack on the twist - boat conformation of 6 is not only unusually stable but is even slightly favored ( by 0.3 kcal mol ) over attack on the chair conformation . in fact , 6tb - ts - laheq was calculated to be the lowest energy pathway for the addition of lah to 6 . the trajectory of hydride addition to the pro - axial face of 6 tb is remote from the 2,6-dimethyl substituents ( dh h = 3.85 in 6tb - ts - lahax ) . nevertheless , pro - equatorial attack on the twist - boat is preferred , and is 5 kcal mol lower than that of the twist - boat transition state of cyclohexanone 1 . consistent with its experimentally observed reactivity , and contrary to its nonbridged analogue 6 , tropinone 7 is predicted to undergo preferential hydride attack by lah at the axial face ( typical felkin anh selectivity ) , although with a lower stereoselectivity ( geq - ax = + 0.7 kcal mol , leading to a 85:15 ratio of equatorial : axial alcohols at 78 c ) . steric hindrance in the axial addition trajectory of 7ch - ts - lahax is slightly less important , since the alh4 approaches somewhat further away from the ethylene bridge in 7 than from the dimethyls of piperidone 6 . also , the advantage of alleviating the 1,3-diaxial interactions described for the equatorial attack in 6ch and both approaches in 6 tb does not apply to 7ch , in which these substituents are bridged . the geometric constraints imposed by the bicyclic structure of 7ch preclude relaxation of the torsional strain generated in the transition states , as reflected by the activation barriers that are calculated to be 24 kcal mol higher for 7 than for 6 and 1 and by the 712 increase in the values for 7 with respect to 6 and 1 . in view of the different stereochemical outcomes observed for the addition of lah to piperidone 6 and tropinone 7 , it can be concluded that relaxation of 1,3-diaxial interactions , either by accessing twist - boat conformations or by favoring equatorial addition trajectories , is the key factor determining facial stereoselectivity in the reduction of this type of system with small hydrides , overriding the contribution of other steric factors . lowest energy transition structures for the addition of lah to ( a ) the axial face of 7ch , and ( b ) the equatorial face of 7ch . optimized structures were calculated at the smd(thf)/b3lyp/6 - 311+g(2d , p)//b3lyp/6 - 31g(d , p ) level . activation gibbs free energies ( g ) are referenced to the lowest energy prereaction coordination complex and are given in kcal mol ; distances are given in angstroms and angles in degrees . as observed with lah , all three substrates studied form prereaction coordination complexes with lithium triisopropylborohydride ( ltbh ) that are lower in energy than the separated reactants ( see , for example , figure 10a ) . the activation energies reported here for reduction with ltbh are also measured from the lowest - energy prereaction coordination complexes . for 1 , the difference in energy between chair and twist - boat conformations is essentially conserved upon formation of the prereaction complex ( figure 10b ) . however , for piperidone 6 , the twist - boat conformation is destabilized relative to the chair upon formation of the prereaction complex ( gtwist - chair = 1.8 kcal mol for the prereaction complexes vs 0.5 kcal mol for the uncomplexed ketone conformations , respectively ) . ( a ) example of formation of the reactant complex with ltbh , shown for 1ch . ( b ) comparison of the gibbs free energies ( g ) of chair and twist - boat conformations of free ketones 1 , 6 , and 7 and their prereaction complexes with ltbh . optimized structures were calculated at the smd(thf)/b3lyp/6 - 311+g(2d , p)//b3lyp/6 - 31g(d , p ) level . gibbs free energies ( g ) are referenced to the lowest energy free ketone conformation and are given in kcal mol . the bulky hydride reagent is less reactive than the sterically less - demanding reagent lah , as reflected by higher overall activation barriers calculated for ltbh . the reactivity trend observed for the three cyclic ketones toward lah is maintained for reduction with ltbh , although the differences in the calculated activation energies are smaller ( g = 24.7 , 24.9 , and 25.3 kcal mol for 1 , 6 , and 7 , respectively ; figures 1113 ) . both the lithium and the potassium salts of selectride are commonly used experimentally as a bulky hydride reagents . therefore , we also investigated the reactivity of piperidone 6 and tropinone 7 toward potassium triisopropylborohydride ( ktbh ) , in addition to ltbh . the corresponding calculated activation energies indicate a somewhat greater reactivity of ktbh with respect to ltbh ( g = 22.9 and 25.1 kcal mol for the reaction of ktbh with 6 and 7 , respectively ) . this may be due to a weaker interaction between k and the incoming hydride in the transition structure , as suggested by the computed transition state geometries . the kinetic preference for 6 vs 7 predicted by our calculations is consistent with the results of the competition experiment described in scheme 2 . due to the very similar chair vs twist - boat selectivity trends calculated for ltbh and ktbh ( gtwist - chair = 2.8 and 2.6 kcal mol for the reaction of 6 with ltbh and ktbh , respectively ) , and to facilitate a more direct comparison to lah a more detailed description of the reactivity of ktbh with 6 and 7 can be found in the supporting information . in accordance with experimental results , the three substrates studied are consistently predicted to prefer attack at the equatorial face by the bulky hydride reagent ltbh . despite the large number of conformers accessible for each productive reaction pathway ( e.g. , 108 conformers representing approach of ltbh to just one face of 6 ) , in all cases reaction with ltbh proceeds through significantly later transition states ( forming dc h = 1.381.45 with ltbh vs 1.661.75 with lah , figure 1113 ) . ltbh favors addition to the equatorial face of 1ch by 3.7 kcal mol ( figure 11a and 11b ) . thus , the surrounding dihedral angles in the chair ts are more staggered than in the transition structures with lah . this feature is reflected in the slightly smaller values of for the chair ts with ltbh ( = 1011 ) vs with lah ( = 1214 ) . furthermore , the difference in torsional strain between attack at the axial and equatorial faces ( range of values ) with ltbh is smaller than with lah , rendering torsional strain less influential on facial selectivity of these chair ts . only a small energy difference is calculated between the transition states for reaction at the two faces of the twist boat with ltbh ( geq - ax = + 0.6 kcal mol ) . addition to either face of the twist boat is prohibitively high in energy relative to the favored addition to the equatorial face of 1ch ( gtwist - chair 6 kcal mol for pro - equatorial addition , figure 11d vs 11b ) . lowest energy transition structures for the addition of ltbh to ( a ) the axial face of chair 1ch , ( b ) the equatorial face of chair 1ch , ( c ) the pro - axial face of twist - boat 1 tb , and ( d ) the pro - equatorial face of twist - boat 1 tb . optimized structures were calculated at the smd(thf)/b3lyp/6 - 311+g(2d , p)//b3lyp/6 - 31g(d , p ) level . activation gibbs free energies ( g ) are referenced to the lowest energy prereaction coordination complex and are given in kcal mol ; distances are given in angstroms and angles in degrees . due to exceedingly large steric repulsions between ltbh and the substrate 2,6-substituents , only equatorial - face addition transition structures could be located for the reaction of piperidone 6 ( figure 12 ) and tropinone 7 ( figure 13 ) with this bulky hydride reagent . the energetic degeneracy predicted for the pro - equatorial addition of lah to both the chair and twist - boat conformations of 6 is not conserved with ltbh , for which the twist - boat transition state is disfavored by 3 kcal mol due to a simultaneous reduction in the torsional strain of 6ch - ts - ltbheq ( = 10 ) and increase in torsional strain of 6tb - ts - ltbheq ( = 33 ) . taken together with the results using lah , these studies show that twist - boat conformations can be relevant for both reactivity and selectivity of cis-2,6-disubstituted piperidones for reduction by small hydride reagents but not with ltbh or , presumably , other bulky nucleophiles . lowest energy transition structures for the addition of ltbh to ( a ) the equatorial face of chair 6ch and ( b ) the pro - equatorial face of twist - boat 6 tb . optimized structures were calculated at the smd(thf)/b3lyp/6 - 311+g(2d , p)//b3lyp/6 - 31g(d , p ) level . activation gibbs free energies ( g ) are referenced to the lowest energy prereaction coordination complex and are given in kcal mol ; distances are given in angstroms and angles in degrees . lowest energy transition structure for the addition of ltbh to the equatorial face of 7ch . optimized structures were calculated at the smd(thf)/b3lyp/6 - 311+g(2d , p)//b3lyp/6 - 31g(d , p ) level . activation gibbs free energy ( g ) is referenced to the lowest energy prereaction coordination complex and is given in kcal mol ; distances are given in angstroms and angles in degrees . the computational results described in this paper are consistent with the felkin anh model for predicting the facial selectivity of the reaction of tert - butylcyclohexanone 1 and tropinone 7 with a small hydride reagent : lah preferentially adds to the axial face of both 1 and 7 . an exception to this common trend is found in piperidone 6 , for which a twist - boat conformation is calculated to be relevant to the transition state for addition of a small hydride reagent , and pro - equatorial attack by lah is overall preferred . our results indicate that pro - equatorial attack on a twist - boat ( i.e. , attack at the face that would lead to an equatorial nucleophile in the chair conformation of the product ) with a small nucleophile does not necessarily incur more torsional strain than pro - axial attack . additionally , our calculations show that the torsional strain developed during both attack on the axial and equatorial faces of a chair depends also on the nature of the incoming nucleophile . with a bulky hydride , the degree of torsional strain experienced in the transition states for attack at the equatorial and axial faces are similar , and selectivity is dominated by steric effects . all synthetic reactions described in this paper were performed using oven - dried glassware under an argon or dry nitrogen atmosphere . thf , toluene , and diethyl ether were dried by distillation from sodium / benzophenone . other reagents and solvents were stored over molecular sieves under argon and used directly . radial plc was performed using a model 7924 t chromatotron using thin layers of silica gel the mass analyzer type used for the hrms measurements was tof with electrospray as the ionization method . chemical shifts are in units ( ppm ) with tms ( 0.0 ppm ) used as an internal standard for h nmr spectra and the cdcl3 absorption at 77.23 ppm for c nmr . to cubrsme2 ( 177 mg , 0.86 mmol ) in dms ( 4 ml ) at 78 c the reaction mixture was allowed to warm to 30 c over 30 min and then cooled to 78 c . a solution of n-(phenoxycarbonyl)-2-methyl-2,3-dihydropyridone ( 100 mg , 0.43 mmol ) in 0.5 ml of dms was added via syringe . the mixture was stirred at 78 c for 3 h and then at 42 c for 30 min . the cooling bath was removed and saturated aqueous nh4cl ( 0.5 ml ) was added followed by anhydrous na2so4 ( 8 g ) . after stirring for 2 h , purification by radial plc ( sio2 , 1020% etoac / hexanes ) afforded 91 mg ( 85% ) of 3 as a clear oil ( product contains 7% of the trans isomer ) . ir ( neat ) 2974 , 1710 , 1336 , 1204 cm ; h nmr ( 300 mhz , cdcl3 ) 7.36 ( m , 2h ) , 7.2 ( m , 1h ) , 7.11 ( d , 2h , j = 8.4 hz ) , 4.90 ( m , 2h ) , 2.79 ( dd , 2h , j = 7.6 , 15.1 hz ) , 2.38 ( dd 2h , j = 2.1 , 15.0 hz ) , 1.38 ( d , 6h , j = 7.0 hz ) ; c nmr ( 75 mhz , cdcl3 ) 207.9 , 154.0 , 151.4 , 129.6 , 125.7 , 121.9 , 49.5 , 45.5 , 23.2 ; hrms calcd for c14h17no3 [ ( m + h ) ] 248.1281 , found 248.1275 . to a solution of piperidone 3 in thf ( 2 ml ) at 78 c was added k - selectride ( 1 m / thf , 0.33 ml , 0.33 mmol ) , and the mixture was stirred at 78 c for 1 h. anhydrous acetone ( 0.3 ml ) was added , and stirring was continued for 5 min . the cooling bath was removed , saturated nh4cl ( 0.5 ml ) added , and the mixture stirred at rt for 1 h. etoac ( 15 ml ) and dry na2so4 ( 3 g ) were added . after stirring for 1 h , filtration and concentration gave the crude product . purification by radial plc ( sio2 , etoac / hexanes ) afforded 63 mg ( 84% ) of alcohol 3a as a white solid , mp 127128 c ( 10% etoac / hexanes ) . ir ( neat ) 3466 , 2967 , 1710 , 1688 ; h nmr ( 300 mhz , cdcl3 ) 7.33 ( t , 2h , j = 8.4 hz ) , 7.19 ( t , 1h , j = 8.4 hz ) , 7.1 ( d , 2h , j = 8.4 hz ) , 4.46 ( m , 2h ) , 4.0 ( m , 1h ) , 2.28 ( s , 1h ) , 2.06 ( m , 2h ) , 1.62 ( m , 2h ) , 1.46 ( d , 6h , j = 6.6 hz ) ; c nmr ( 75 mhz , cdcl3 ) 154.3 , 151.6 , 129.5 , 125.4 , 122.0 , 65.1 , 46.8 , 37.1 , 24.0 ; hrms calcd for c14h19no3 [ ( m + h ) ] 250.1438 , found 250.1433 . to a 50/50 mixture of piperidones 3 ( 0.12 mmol ) and 5 ( 0.12 mmol ) in thf ( 3 ml ) at 74 c was added k - selectide ( 1 m / thf , 0.12 ml , 0.12 mmol ) dropwise . anhydrous acetone ( 0.2 ml ) was added , and stirring was continued for 5 min . the cooling bath was removed , saturated aqueous nh4cl ( 0.5 ml ) added , and the mixture stirred for 1 h at room temperature . etoac ( 10 ml ) and anhydrous na2so4 ( 4 g ) were added . after stirring for 1 h analysis by hplc and nmr showed that the ketones 3 and 5 were reduced in a ratio of 75:25 ( see supporting information ) . all geometry optimizations were carried out with the b3lyp hybrid functional and 6 - 31g(d , p ) basis set . single - point energy calculations were performed on the optimized geometries using the 6 - 311+g(2d , p ) basis set . the meta - hybrid m06 - 2x functional was also tested for both geometry optimization and single - point energy calculations , using the same basis sets described above . similar results were obtained with both methods , although the b3lyp functional showed a better agreement with experimental results . the theoretical ratio of reaction products was obtained through the gibbs free energy of the different transition states ( g ) using a maxwell boltzmann distribution at the appropriate temperature . the nature of the stationary points was determined in each case according to the appropriate number of negative eigenvalues of the hessian matrix from the frequency calculations . scaled frequencies were not considered , because significant errors in the calculated thermodynamic properties are not found at this theoretical level . mass - weighted intrinsic reaction coordinate ( irc ) calculations were carried out using the gonzalez and schlegel scheme to ensure that the tss indeed connect the appropriate reactants and products . bulk solvent effects were considered implicitly by performing single - point energy calculations on the gas - phase optimized geometries , through the smd polarizable continuum model of cramer and truhlar as implemented in gaussian 09 . the parameters for tetrahydrofuran were used to calculate solvation free energies ( gsolv ) . cartesian coordinates , electronic energies , entropies , enthalpies , gibbs free energies , and lowest frequencies of the different conformations of all structures are available as supporting information .

the role of twist - boat conformers of cyclohexanones in hydride reductions was explored . the hydride reductions of a cis-2,6-disubstituted n - acylpiperidone , an n - acyltropinone , and tert - butylcyclohexanone by lithium aluminum hydride and by a bulky borohydride reagent were investigated computationally and compared to experiment . our results indicate that in certain cases , factors such as substrate conformation , nucleophile bulkiness , and remote steric features can affect stereoselectivity in ways that are difficult to predict by the general felkin anh model . in particular , we have calculated that a twist - boat conformation is relevant to the reactivity and facial selectivity of hydride reduction of cis-2,6-disubstituted n - acylpiperidones with a small hydride reagent ( lialh4 ) but not with a bulky hydride ( lithium triisopropylborohydride ) .

Introduction Results and Discussion Conclusions Experimental Section

the facial selectivity of addition is influenced by the size of the nucleophilic reagent : small nucleophiles tend to add to the axial face , whereas bulky nucleophiles preferentially attack the equatorial face . for example , nabh4 and lialh4 both deliver hydride to the axial face of 4-tert - butylcyclohexanone ( 1 ) , giving an equatorial alcohol as the major product , whereas bulky hydride reagents such as lihbbu3 ( l - selectride ) preferentially yield the axial alcohol via equatorial hydride delivery ( figure 1 ) . anh model posits that the transition state ( ts ) for addition to the equatorial face of a cyclohexanone chair involves torsional strain greater than that of axial attack ; that is , there are more eclipsing interactions during equatorial attack compared to axial ( figure 2 ) . cis-2,6-disubstituted n - acylpiperidones ( e.g. for 2 and 3 , hydride addition from the equatorial face is favored even when a small hydride reagent ( nabh4 ) is used ( scheme 1 , eqs 1 and 2 ) . indeed , the axial face of this class of substrates should be particularly hindered due to the axial 2,6-substituents , forced into this conformation by a strain with the n - substituent . , compound 4 ) undergo favored axial attack by small hydride reagents despite the steric hindrance imposed by the bridge ( eqs 3 and 4 ) . low energy twist - boat conformations of piperidones have been observed in solution by nmr spectroscopy and in the solid state by x - ray diffraction . we wondered if these twist - boat conformers might be relevant to the transition states of reactions of these compounds . to study this possibility experimentally , we compared the reactivity of piperidone 3 and its tropinone analogue 5 toward k - selectride . notably , although the former substrate can access a twist - boat conformation , the latter can not due to geometrical constraints . based on the results of a competition experiment ( scheme 2 ) , the rate of reduction of tropinone 5 with k - selectride is about 3-fold slower than that of piperidone 3 ; however , both processes are completely selective toward formation of the axial alcohol ( equatorial attack of hydride ) . this result , in combination with the contrasting stereoselectivity of these two classes of substrates with nonbulky hydrides ( scheme 1 , eqs 3 and 4 ) , encouraged us to computationally explore the hypothesis that a twist - boat transition state could in fact be contributing significantly to the reaction of cis-2,6-disubstituted n - acylpiperidones . we have undertaken a computational study of the factors influencing the differences in reactivity and facial selectivity of reduction of these two structurally similar piperidone and tropinone substrate classes . the contributions of both steric repulsion and torsional strain to the activation energy upon hydride addition , factors that have been found to be particularly relevant in cyclic ketones , were analyzed . our calculations indicate that , although a chair conformation experiences greater torsional strain during attack at the equatorial face by a small hydride , attack at the two faces of a twist - boat do not necessarily follow the same trend . additionally , with very bulky hydride reagents , the difference in torsional strain during attack at the axial vs equatorial face of a chair is nearly negligible due to a late transition state . while 4-tert - butylcyclohexanone ( 1 ) is a well - studied conformationally biased cyclic ketone , chosen as a baseline , n - methoxycarbonyl - cis-2,6-dimethylpiperidone ( 6 ) and n - methoxycarbonyltropinone ( 7 ) were selected as piperidone and tropinone model substrates . lithium aluminum hydride ( lah ) is the small hydride model . for a bulky hydride , we mimicked the reactivity of l - selectride ( lithium tri - sec - butylborohydride ) using a computationally affordable surrogate ( l - selectride is conformationally and stereochemically very complex , with thousands of low energy conformers ) . figure 4 depicts the calculated geometries of the most stable chair ( 1ch , 6ch , and 7ch ) and twist - boat ( 1 tb and 6 tb ) conformations of the cyclic ketone substrates . conversely , two different newman projections are given for 1 tb and 6 tb , as the twist - boat conformations of these substrates lack a plane or axis of symmetry . in the ground state , 1 adopts a chair conformation ( 1ch ) that is more stable than the minimum energy twist - boat conformation ( 1 tb , in which c1 and c4 are at the bow and stern ) by 3.2 kcal mol ( figure 4a ) . gibbs free energies ( g ) are referenced to the lowest energy conformer , when applicable , and are given in kcal mol ; angles are given in degrees . previous studies on cis-2,6-disubstituted n - acylpiperidones have indicated that the chair conformation with equatorial 2- and 6-substituents is unstable . the resulting a - strain between the 2,6-diequatorial and the n - substituents disfavors such a conformation , and instead these substituents exist in an axial ( or pseudoaxial ) orientation in conformation 6ch . , in which c6 ( the ring carbon syn to the carbonyl of the n - acyl group ) and c3 are at the bow and stern positions . the twist - boat conformation of 6 is predicted to be only 0.5 kcal mol higher in energy than 6ch , suggesting the coexistence of both isomers in solution . as described above , previous experimental and computational studies have indicated that similar piperidones ( albeit bearing bulkier 2- and 6-substituents ) exist predominantly in a twist - boat conformation in solution and in the solid state . the bridging ch2ch2 cinches the chair together on one side , making the nitrogen flap more folded in 7ch , with a flap angle ( out - of - plane dihedral angle ) of 115 compared to 138 in 6ch ( , figure 4d ) . conversely , the carbonyl flap on the opposite side of 7ch is more flattened , with a flap angle of 146 ( , figure 4d ) , compared to 131 in 6ch and 137 in 1ch . the lowest energy reactant species for the reduction of cyclic ketones 1 , 6 , and 7 by lah are prereaction coordination complexes that are stabilized by 10.9 to 12.3 kcal mol with respect to the separated reactants ( see , for example , figure 5a ) . substrate geometries are not significantly distorted by formation of the coordination complexes , and the energy differences between chair and twist - boat conformations ( for 1 and 6 ) are very nearly conserved ( figure 5b ) . however , significant computational precedent exists for successfully reproducing experimental selectivities of hydride reductions involving nonexplicitly solvated lithium species . ( b ) comparison of the gibbs free energies ( g ) of chair and twist - boat conformations of free ketones 1 , 6 , and 7 and their prereaction complexes with lah . because neither face of a twist - boat experiences nucleophilic attack via a true axial or equatorial trajectory , attack on the twist - boat face that corresponds to the axial face of the analogous chair will be herein be referred to as pro - axial attack or attack at the pro - axial face ( the term pro - equatorial will also be used ) . the torsional strain associated with the transition states herein is represented graphically by newman projections and described numerically by the parameter , which corresponds to the average deviation from 60 of the 12 dihedral angles of both newman projections involving the carbonyl carbon ( figure 6 ) . it should be noted that due to the intrinsic conformational differences between chair and twist - boat conformations , there is a certain amount of eclipsing already associated with the twist - boats . moreover , addition to the chair equatorial face requires greater distortion of the ring dihedral angle relative to the geometry of the reactant 1ch ( dihedral angle c c c c = 40 and 60 for 1ch - ts - lahax and 1ch - ts - laheq , respectively , compared to 48 for 1ch ) . lowest energy transition structures for the addition of lah to ( a ) the axial face of chair 1ch , ( b ) the equatorial face of chair 1ch , ( c ) the pro - axial face of twist - boat 1 tb , and ( d ) the pro - equatorial face of twist - boat 1 tb . the twist - boat transition states for 1 ( figure 7c and 7d ) are slightly later ( the forming c h bond length is 1.60 for 1tb - ts - lah and 1.66 for 1ch - ts - lah ) and , consequently , higher in energy than the chair transition states for both pro - axial and pro - equatorial hydride addition . the increased destabilization of the twist - boat relative to the chair can also be attributed , at least in part , to a greater difference in torsional strain between the two transition states . in contrast , the twist - boat transition states maintain an eclipsed arrangement ( the smaller o c c h angle = 4 in 1 tb , compared to 611 in 1tb - ts - lah ) . this weak preference for pro - equatorial attack in the twist boat is opposite to the preference for axial attack on 1ch and alludes to the possibility that stable twist - boat conformations can alter the usual stereoselectivity . this equatorial preference is predicted for both chair and twist - boat transition structures ( geq - ax = 0.8 and 2.0 kcal mol for 6ch - ts - lah and 6tb - ts - lah , respectively ) . in the chair conformation , the destabilization of the ts for axial face attack is likely caused by the steric hindrance with the 2,6-dimethyl substituents , which translates into a longer forming c h bond distance ( 1.75 vs 1.66 in the cyclohexanone ts ) . lowest energy transition structures for the addition of lah to ( a ) the axial face of chair 6ch , ( b ) the equatorial face of chair 6ch , ( c ) the pro - axial face of twist - boat 6 tb , and ( d ) the pro - equatorial face of twist - boat 6 tb . consistent with our hypothesis that twist - boat transition states may be involved , pro - equatorial attack on the twist - boat conformation of 6 is not only unusually stable but is even slightly favored ( by 0.3 kcal mol ) over attack on the chair conformation . nevertheless , pro - equatorial attack on the twist - boat is preferred , and is 5 kcal mol lower than that of the twist - boat transition state of cyclohexanone 1 . consistent with its experimentally observed reactivity , and contrary to its nonbridged analogue 6 , tropinone 7 is predicted to undergo preferential hydride attack by lah at the axial face ( typical felkin anh selectivity ) , although with a lower stereoselectivity ( geq - ax = + 0.7 kcal mol , leading to a 85:15 ratio of equatorial : axial alcohols at 78 c ) . the geometric constraints imposed by the bicyclic structure of 7ch preclude relaxation of the torsional strain generated in the transition states , as reflected by the activation barriers that are calculated to be 24 kcal mol higher for 7 than for 6 and 1 and by the 712 increase in the values for 7 with respect to 6 and 1 . in view of the different stereochemical outcomes observed for the addition of lah to piperidone 6 and tropinone 7 , it can be concluded that relaxation of 1,3-diaxial interactions , either by accessing twist - boat conformations or by favoring equatorial addition trajectories , is the key factor determining facial stereoselectivity in the reduction of this type of system with small hydrides , overriding the contribution of other steric factors . as observed with lah , all three substrates studied form prereaction coordination complexes with lithium triisopropylborohydride ( ltbh ) that are lower in energy than the separated reactants ( see , for example , figure 10a ) . however , for piperidone 6 , the twist - boat conformation is destabilized relative to the chair upon formation of the prereaction complex ( gtwist - chair = 1.8 kcal mol for the prereaction complexes vs 0.5 kcal mol for the uncomplexed ketone conformations , respectively ) . ( b ) comparison of the gibbs free energies ( g ) of chair and twist - boat conformations of free ketones 1 , 6 , and 7 and their prereaction complexes with ltbh . the bulky hydride reagent is less reactive than the sterically less - demanding reagent lah , as reflected by higher overall activation barriers calculated for ltbh . the reactivity trend observed for the three cyclic ketones toward lah is maintained for reduction with ltbh , although the differences in the calculated activation energies are smaller ( g = 24.7 , 24.9 , and 25.3 kcal mol for 1 , 6 , and 7 , respectively ; figures 1113 ) . both the lithium and the potassium salts of selectride are commonly used experimentally as a bulky hydride reagents . due to the very similar chair vs twist - boat selectivity trends calculated for ltbh and ktbh ( gtwist - chair = 2.8 and 2.6 kcal mol for the reaction of 6 with ltbh and ktbh , respectively ) , and to facilitate a more direct comparison to lah a more detailed description of the reactivity of ktbh with 6 and 7 can be found in the supporting information . in accordance with experimental results , the three substrates studied are consistently predicted to prefer attack at the equatorial face by the bulky hydride reagent ltbh . furthermore , the difference in torsional strain between attack at the axial and equatorial faces ( range of values ) with ltbh is smaller than with lah , rendering torsional strain less influential on facial selectivity of these chair ts . lowest energy transition structures for the addition of ltbh to ( a ) the axial face of chair 1ch , ( b ) the equatorial face of chair 1ch , ( c ) the pro - axial face of twist - boat 1 tb , and ( d ) the pro - equatorial face of twist - boat 1 tb . due to exceedingly large steric repulsions between ltbh and the substrate 2,6-substituents , only equatorial - face addition transition structures could be located for the reaction of piperidone 6 ( figure 12 ) and tropinone 7 ( figure 13 ) with this bulky hydride reagent . the energetic degeneracy predicted for the pro - equatorial addition of lah to both the chair and twist - boat conformations of 6 is not conserved with ltbh , for which the twist - boat transition state is disfavored by 3 kcal mol due to a simultaneous reduction in the torsional strain of 6ch - ts - ltbheq ( = 10 ) and increase in torsional strain of 6tb - ts - ltbheq ( = 33 ) . taken together with the results using lah , these studies show that twist - boat conformations can be relevant for both reactivity and selectivity of cis-2,6-disubstituted piperidones for reduction by small hydride reagents but not with ltbh or , presumably , other bulky nucleophiles . lowest energy transition structures for the addition of ltbh to ( a ) the equatorial face of chair 6ch and ( b ) the pro - equatorial face of twist - boat 6 tb . the computational results described in this paper are consistent with the felkin anh model for predicting the facial selectivity of the reaction of tert - butylcyclohexanone 1 and tropinone 7 with a small hydride reagent : lah preferentially adds to the axial face of both 1 and 7 . an exception to this common trend is found in piperidone 6 , for which a twist - boat conformation is calculated to be relevant to the transition state for addition of a small hydride reagent , and pro - equatorial attack by lah is overall preferred . our results indicate that pro - equatorial attack on a twist - boat ( i.e. , attack at the face that would lead to an equatorial nucleophile in the chair conformation of the product ) with a small nucleophile does not necessarily incur more torsional strain than pro - axial attack . with a bulky hydride , the degree of torsional strain experienced in the transition states for attack at the equatorial and axial faces are similar , and selectivity is dominated by steric effects .

the kaiser permanente southern california ( kpsc ) medical care program is a large , prepaid , group - practice , managed health care organization with > 3.3 million members in 2010 . members receive their health care in kpsc - owned facilities throughout the seven - county region . the study population consisted of women who had a live singleton birth at 20 weeks ' gestation at the kpsc bellflower medical center between 1 october 2005 and 31 march 2010 , who underwent a prenatal 2-h 75-g ogtt with no prior 50-g oral glucose challenge test and had prepregnancy and delivery anthropometric data ( n = 9,199 ) . in this medical center , routine clinical practice involved treating women whose prenatal 75-g ogtt results met at least two thresholds : fpg 100 mg / dl , 1-h glucose 195 mg / dl , and 2-h glucose 160 mg / dl with diet and exercise therapy . of these , women whose fpg was consistently 105 mg / dl or 1-h postprandial glucose was 140 mg / dl were treated with insulin or glyburide in addition to diet and exercise . this practice is based on the analyses of ogtt results and pregnancy outcomes published by sacks et al . ( 11 ) . after excluding women who received any form of treatment during pregnancy ( n = 488 ) , we used ogtt results to identify women with gdm based on iadpsg guidelines ( 10 ) . for untreated women with more than one ogtt during pregnancy , outcomes are reported based on the test result within or nearest to 2428 weeks ' gestation . for women with more than one birth during the study period , only data from the first pregnancy were analyzed . maternal age at delivery , race / ethnicity ( hispanic , non - hispanic white , non - hispanic black , asian / pacific islander , and other / multiple races ) , and parity ( 0 , 1 , and 2 ) were obtained from infant birth certificates . information on prenatal smoking was obtained from the electronic health record ( ehr ) during prenatal encounters . infant sex , gestational age at birth , birth weight , and birth length were obtained from the birth certificate or ehr . based on the results of the 2-h 75-g ogtt , we categorized women into five mutually exclusive categories : no glucose impairment ( no gdm ) , single isolated impaired glucose tolerance ( i - igt1 ) if either 1-h glucose 180 mg / dl or 2-h glucose 153 mg / dl and fpg < 92 mg / dl , isolated impaired fasting glucose ( i - ifg ) if fpg 92 mg / dl and both 1-h glucose < 180 mg / dl and 2-h glucose < 153 mg / dl , double - isolated impaired glucose tolerance ( i - igt2 ) if both 1-h glucose 180 mg / dl and 2-h glucose 153 mg / dl but fpg < 92 mg / dl , and combined ifg and igt ( ifg+igt ) if fpg 92 mg / dl and either 1-h glucose 180 mg / dl and/or 2-h glucose 153 mg / dl . prepregnancy bmi and weight gain during pregnancy both have been shown to be associated with the development of gdm ( 1315 ) as well as adverse outcomes independent of gdm ( 1619 ) . maternal prepregnancy weight and height were obtained from the ehr ( n = 7,894 [ 90.6% ] ) or from the infant 's birth certificate if not available from the ehr ( n = 817 [ 9.4% ] ) . among those with data available from the ehr , identification of measured prepregnancy weight was contingent upon the timing of the clinical visit closest to last menstrual period ( lmp ) . to establish the lmp date , infant gestational age at delivery ( in days ) height and prepregnancy weight were selected hierarchically , if available , 03 months prior to lmp ( n = 3,046 [ 38.6% ] ) , 03 months after lmp ( n = 4,690 [ 59.4% ] ) , or 36 months prior to lmp ( n = 158 [ 2.0% ] ) . delivery weight was obtained within 30 days prior to delivery ; 96.1% were measured within 14 days of delivery . prepregnancy bmi was classified as normal ( bmi < 25 kg / m ) , overweight ( 25 bmi < 30 kg / m ) , or obese ( bmi 30 kg / m ) ( 20 ) . the 2009 institute of medicine ( iom ) guidelines were used to classify excessive weight gain during pregnancy based on categories of prepregnancy bmi ( normal : > 35 lb , overweight : > 25 lb , or obese : > 20 lb ) ( 21 ) . large - for - gestational - age ( lga ) infants were defined as sex- , race- , and gestational age consistent with the methods used by the hapo study group ( 11 ) , percentiles for birth weight were determined by quantile regression stratified by sex and race / ethnicity , with adjustment for gestational age and maternal parity . an infant was considered to have a birth weight > 90th percentile if the birth weight was greater than the estimated 90th percentile for the infant 's sex , gestational age , race / ethnicity , and maternal parity . delivery by primary cesarean section was obtained from infant birth certificates ; data for women who had a previous cesarean section ( n = 963 ) were excluded from analysis of this outcome . shoulder dystocia / birth injury was defined by icd-9 codes 653.4 , 653.5 , 660.4 , 767.0767.9 , or 959.0959.9 at delivery . we identified women with gestational hypertensive disorders by icd-9 codes 642.3642.6 and/or 642.9 during pregnancy . in analyses of gestational hypertension , we excluded women with pregestational hypertension ( icd-9 codes 401405.9 , 642.0642.2 , and/or 642.7 ; n = 323 ) . we examined the associations between maternal demographic , clinical , and anthropometric characteristics ; adverse clinical outcomes ; and gdm subtypes ( i - igt1 , i - ifg , i - igt2 , and ifg+igt ) . associations between categorical variables and gdm subtype were assessed using tests ; differences in mean continuous variables by subtype were evaluated using anova with tukey honestly - significant - difference adjustment for multiple comparisons . multiple logistic regression models were used to calculate adjusted odds ratios ( aors ) and corresponding 95% cis for a 1-sd increase in continuous fasting and 1-h and 2-h glucose levels associated with adverse outcomes , after controlling for maternal age , race / ethnicity , parity , prepregnancy bmi , and gestational weight gain . all analyses were performed with sas version 9.1 ( sas institute , cary , nc ) . the kaiser permanente southern california ( kpsc ) medical care program is a large , prepaid , group - practice , managed health care organization with > 3.3 million members in 2010 . members receive their health care in kpsc - owned facilities throughout the seven - county region . the study population consisted of women who had a live singleton birth at 20 weeks ' gestation at the kpsc bellflower medical center between 1 october 2005 and 31 march 2010 , who underwent a prenatal 2-h 75-g ogtt with no prior 50-g oral glucose challenge test and had prepregnancy and delivery anthropometric data ( n = 9,199 ) . in this medical center , routine clinical practice involved treating women whose prenatal 75-g ogtt results met at least two thresholds : fpg 100 mg / dl , 1-h glucose 195 mg / dl , and 2-h glucose 160 mg / dl with diet and exercise therapy . of these , women whose fpg was consistently 105 mg / dl or 1-h postprandial glucose was 140 mg / dl were treated with insulin or glyburide in addition to diet and exercise . this practice is based on the analyses of ogtt results and pregnancy outcomes published by sacks et al . ( 11 ) . after excluding women who received any form of treatment during pregnancy ( n = 488 ) , we used ogtt results to identify women with gdm based on iadpsg guidelines ( 10 ) . for untreated women with more than one ogtt during pregnancy , outcomes are reported based on the test result within or nearest to 2428 weeks ' gestation . for women with more than one birth during the study period , only data from the first pregnancy were analyzed . maternal age at delivery , race / ethnicity ( hispanic , non - hispanic white , non - hispanic black , asian / pacific islander , and other / multiple races ) , and parity ( 0 , 1 , and 2 ) were obtained from infant birth certificates . information on prenatal smoking was obtained from the electronic health record ( ehr ) during prenatal encounters . infant sex , gestational age at birth , birth weight , and birth length were obtained from the birth certificate or ehr . based on the results of the 2-h 75-g ogtt , we categorized women into five mutually exclusive categories : no glucose impairment ( no gdm ) , single isolated impaired glucose tolerance ( i - igt1 ) if either 1-h glucose 180 mg / dl or 2-h glucose 153 mg / dl and fpg < 92 mg / dl , isolated impaired fasting glucose ( i - ifg ) if fpg 92 mg / dl and both 1-h glucose < 180 mg / dl and 2-h glucose < 153 mg / dl , double - isolated impaired glucose tolerance ( i - igt2 ) if both 1-h glucose 180 mg / dl and 2-h glucose 153 mg / dl but fpg < 92 mg / dl , and combined ifg and igt ( ifg+igt ) if fpg 92 mg / dl and either 1-h glucose 180 mg / dl and/or 2-h glucose 153 mg / dl . prepregnancy bmi and weight gain during pregnancy both have been shown to be associated with the development of gdm ( 1315 ) as well as adverse outcomes independent of gdm ( 1619 ) . maternal prepregnancy weight and height were obtained from the ehr ( n = 7,894 [ 90.6% ] ) or from the infant 's birth certificate if not available from the ehr ( n = 817 [ 9.4% ] ) . among those with data available from the ehr , identification of measured prepregnancy weight was contingent upon the timing of the clinical visit closest to last menstrual period ( lmp ) . to establish the lmp date , infant gestational age at delivery ( in days ) height and prepregnancy weight were selected hierarchically , if available , 03 months prior to lmp ( n = 3,046 [ 38.6% ] ) , 03 months after lmp ( n = 4,690 [ 59.4% ] ) , or 36 months prior to lmp ( n = 158 [ 2.0% ] ) . delivery weight was obtained within 30 days prior to delivery ; 96.1% were measured within 14 days of delivery . kg / m ) , overweight ( 25 bmi < 30 kg / m ) , or obese ( bmi 30 kg / m ) ( 20 ) . the 2009 institute of medicine ( iom ) guidelines were used to classify excessive weight gain during pregnancy based on categories of prepregnancy bmi ( normal : > 35 lb , overweight : > 25 lb , or obese : > 20 lb ) ( 21 ) . large - for - gestational - age ( lga ) infants were defined as sex- , race- , and gestational age specific birth weight > 90th percentile . consistent with the methods used by the hapo study group ( 11 ) , percentiles for birth weight were determined by quantile regression stratified by sex and race / ethnicity , with adjustment for gestational age and maternal parity . an infant was considered to have a birth weight > 90th percentile if the birth weight was greater than the estimated 90th percentile for the infant 's sex , gestational age , race / ethnicity , and maternal parity . delivery by primary cesarean section was obtained from infant birth certificates ; data for women who had a previous cesarean section ( n = 963 ) were excluded from analysis of this outcome . shoulder dystocia / birth injury was defined by icd-9 codes 653.4 , 653.5 , 660.4 , 767.0767.9 , or 959.0959.9 at delivery . we identified women with gestational hypertensive disorders by icd-9 codes 642.3642.6 and/or 642.9 during pregnancy . in analyses of gestational hypertension , we excluded women with pregestational hypertension ( icd-9 codes 401405.9 , 642.0642.2 , and/or 642.7 ; n = 323 ) . we examined the associations between maternal demographic , clinical , and anthropometric characteristics ; adverse clinical outcomes ; and gdm subtypes ( i - igt1 , i - ifg , i - igt2 , and ifg+igt ) . associations between categorical variables and gdm subtype were assessed using tests ; differences in mean continuous variables by subtype were evaluated using anova with tukey honestly - significant - difference adjustment for multiple comparisons . multiple logistic regression models were used to calculate adjusted odds ratios ( aors ) and corresponding 95% cis for a 1-sd increase in continuous fasting and 1-h and 2-h glucose levels associated with adverse outcomes , after controlling for maternal age , race / ethnicity , parity , prepregnancy bmi , and gestational weight gain . all analyses were performed with sas version 9.1 ( sas institute , cary , nc ) . the study population consisted of 9,199 women , of which 2,179 ( 23.7% ) met the iadpsg criteria for gdm ( 10 ) . after excluding 488 women who received any form of treatment ( 5.3% of the population ; 22.4% of all women with gdm ) , the remaining sample for this analysis was comprised of 8,711 untreated women with a mean age of 29.1 5.9 years , the majority of whom were hispanic ( table 1 ) . among all women , the mean prepregnancy bmi was 27.5 6.1 kg / m , which exceeds the threshold for overweight , and the mean weeks of gestation at which ogtt was performed was 26.7 2.9 . mean fasting and 1-h and 2-h glucose levels were 83.4 7.9 , 131.8 30.7 , and 107.6 23.0 mg / dl , respectively . correlations among these three glucose measures were 0.39 for fpg and 1-h glucose , 0.30 for fpg and 2-h glucose , and 0.62 for 1-h and 2-h glucose ( p < 0.0001 for each comparison ) . maternal characteristics and frequency of maternal and infant outcomes by gdm subtype * p values based on test , with fisher exact test used for variables with any cell count < 10 . anova adjusted with tukey honestly significant difference for continuous variables , p < 0.05 : significantly different than i - igt1 ; significantly different than i - ifg ; significantly different than i - igt2 ; significantly different than ifg+igt ; significantly different than no gdm . # the distribution of each variable within the population is shown as a column percent . * * ponderal index was available for 5,893 infants . data for women who had pre - gestational hypertension were excluded . of these women , 1,691 ( 19.4% ) had iadpsg - defined gdm . compared with women without gdm , those with gdm tended to be slightly older , more parous , have higher mean prepregnancy bmi , and have relatively similar mean gestational weight gain ( table 1 ) . among women with gdm , 52% were based on i - ifg , 23% on i - igt1 , 5% on i - igt2 , and 20% on ifg+igt . of 391 women with i - igt1 , 131 ( 33.5% ) had an abnormal 2-h glucose value only ( 7.7% of all women with gdm ) . of those with ifg+igt ( n = 331 ) , only 55 had three abnormal ogtt results ( 0.6% of gdm women ) , reflecting the fact that women with the most abnormal ogtt results were treated and therefore excluded from these analyses . as shown in table 1 , women in the i - igt1 group had significantly lower prepregnancy bmi than those with i - ifg or ifg+igt ; bmi for these women was similar to those with i - igt2 . on average , weight gain for the i - igt1 group was similar to women of all other gdm subtypes . women with i - ifg had significantly higher prepregnancy bmi than those with i - igt1 or i - igt2 ; bmi was similar to those with ifg+igt . mean weight gain for these women was similar to those with i - igt1 , i - igt2 , or ifg+igt . among all gdm subtypes , prepregnancy bmi and gestational weight gain were highest for women with i - ifg and ifg+igt . associations between adverse outcomes and continuous fasting and 1-h and 2-h glucose values , as well as glucose dichotomized at iadpsg cut points , are shown in table 2 . odds ratios for each continuous glucose measure are expressed as risk per 1-sd increase above the mean . odds ratios presented are adjusted for maternal age , race / ethnicity , parity , prepregnancy bmi , gestational weight gain , infant sex , and gestational age at ogtt . when modeled separately , we found a significant association between each continuous ogtt value and lga , primary cesarean , preterm delivery , and gestational hypertension . we additionally observed significant associations between the continuous 1-h and 2-h postglucose challenge values and shoulder dystocia / birth injury , as well as hyperbilirubinemia . odds ratios for significant risk of adverse outcomes categorized by iadpsg cut points for each glucose measure were relatively consistent with those of the continuous measures , except that those with 1-h glucose 180 mg / dl were not at significantly increased risk for primary cesarean section or hyperbilirubinemia , compared with women with 1-h glucose < 180 mg / dl . similarly , women with 2-h glucose 153 mg / dl were not at significantly increased risk for lga , shoulder dystocia / birth injury , or hyperbilirubinemia , compared with those with 2-h glucose < 153 mg / dl . women with gdm , as defined by iadpsg guidelines , were at significantly increased risk for all outcomes except infant hyperbilirubinemia . adjusted odds ratios * and 95% cis for the association of adverse outcomes with ogtt values and gdm * odds ratios for ogtt measures are for an increase in glucose level of 1 sd ( 7.99 mg / dl for fpg , 30.84 mg / dl for 1-h glucose , and 23.08 mg / dl for 2-h glucose ) and are adjusted for maternal age , race / ethnicity , parity , prepregnancy bmi , gestational weight gain , infant sex , and gestational age at ogtt . associations between gdm subtype ( i - igt1 , i - ifg , i - igt2 , and ifg+igt ) and each adverse outcome , adjusting for maternal age , race / ethnicity , parity , prepregnancy bmi , and gestational weight gain are presented in table 3 . we did not observe statistically significant associations between i - igt1 and lga , primary cesarean , and shoulder dystocia / birth injury . women classified as having gdm based on a single abnormal postload glucose value were at significantly increased risk for preterm delivery , gestational hypertension , and infant hyperbilirubinemia . conversely , women with i - ifg were twice as likely to have an lga infant ( 95% ci 1.622.45 ) and 45% more likely to have shoulder dystocia / birth injury at delivery ( 1.052.00 ) than those with no glucose impairment . women with i - ifg were also 29% more likely to have gestational hypertension ( 1.041.72 ) compared with women without gdm . women with the i - igt2 gdm subtype were 2.33 times as likely to develop gestational hypertension ( 1.204.51 ) than women without gdm , and infants born to these women were 2.85 times as likely to be delivered preterm ( 1.595.10 ) than infants of mothers without gdm . women with ifg+igt were more than twice as likely to have gestational hypertension ( 1.422.84 ) , 87% more likely to have shoulder dystocia / birth injury during delivery ( 1.182.96 ) , and 2.32 times as likely to have an lga infant ( 1.723.13 ) than women without gdm . adjusted odds ratios * with 95% cis for the association between adverse outcomes and gdm subtype * odds ratios are adjusted for maternal age , race / ethnicity , parity , prepregnancy bmi , gestational weight gain , infant sex , and gestational age at ogtt . lga was more strongly associated with gdm subtypes defined by an abnormal fasting glucose ( i - ifg and ifg+igt ) than those based on abnormal postload glucose values only . additionally , while risk for shoulder dystocia / birth injury was elevated in all four gdm subtype groups , the outcome was only significantly associated with i - ifg and ifg+igt . in contrast , preterm delivery , gestational hypertension , and hyperbilirubinemia appeared to be more strongly associated with categories of gdm involving elevated postload glucose ( i - igt1 and/or i - igt2 ) . in fact , women with i - igt2 had the highest risk for preterm delivery and gestational hypertension among all gdm subtypes . associations between adverse outcomes and continuous fasting and 1-h and 2-h glucose values , as well as glucose dichotomized at iadpsg cut points , are shown in table 2 . odds ratios for each continuous glucose measure are expressed as risk per 1-sd increase above the mean . odds ratios presented are adjusted for maternal age , race / ethnicity , parity , prepregnancy bmi , gestational weight gain , infant sex , and gestational age at ogtt . results were similar after additional adjustment for maternal prenatal smoking . when modeled separately , we found a significant association between each continuous ogtt value and lga , primary cesarean , preterm delivery , and gestational hypertension . we additionally observed significant associations between the continuous 1-h and 2-h postglucose challenge values and shoulder dystocia / birth injury , as well as hyperbilirubinemia . odds ratios for significant risk of adverse outcomes categorized by iadpsg cut points for each glucose measure were relatively consistent with those of the continuous measures , except that those with 1-h glucose 180 mg / dl were not at significantly increased risk for primary cesarean section or hyperbilirubinemia , compared with women with 1-h glucose < 180 mg / dl . similarly , women with 2-h glucose 153 mg / dl were not at significantly increased risk for lga , shoulder dystocia / birth injury , or hyperbilirubinemia , compared with those with 2-h glucose < 153 mg / dl . women with gdm , as defined by iadpsg guidelines , were at significantly increased risk for all outcomes except infant hyperbilirubinemia . adjusted odds ratios * and 95% cis for the association of adverse outcomes with ogtt values and gdm * odds ratios for ogtt measures are for an increase in glucose level of 1 sd ( 7.99 mg / dl for fpg , 30.84 mg / dl for 1-h glucose , and 23.08 mg / dl for 2-h glucose ) and are adjusted for maternal age , race / ethnicity , parity , prepregnancy bmi , gestational weight gain , infant sex , and gestational age at ogtt . associations between gdm subtype ( i - igt1 , i - ifg , i - igt2 , and ifg+igt ) and each adverse outcome , adjusting for maternal age , race / ethnicity , parity , prepregnancy bmi , and gestational weight gain are presented in table 3 . we did not observe statistically significant associations between i - igt1 and lga , primary cesarean , and shoulder dystocia / birth injury . women classified as having gdm based on a single abnormal postload glucose value were at significantly increased risk for preterm delivery , gestational hypertension , and infant hyperbilirubinemia . conversely , women with i - ifg were twice as likely to have an lga infant ( 95% ci 1.622.45 ) and 45% more likely to have shoulder dystocia / birth injury at delivery ( 1.052.00 ) than those with no glucose impairment . women with i - ifg were also 29% more likely to have gestational hypertension ( 1.041.72 ) compared with women without gdm . women with the i - igt2 gdm subtype were 2.33 times as likely to develop gestational hypertension ( 1.204.51 ) than women without gdm , and infants born to these women were 2.85 times as likely to be delivered preterm ( 1.595.10 ) than infants of mothers without gdm . women with ifg+igt were more than twice as likely to have gestational hypertension ( 1.422.84 ) , 87% more likely to have shoulder dystocia / birth injury during delivery ( 1.182.96 ) , and 2.32 times as likely to have an lga infant ( 1.723.13 ) than women without gdm . adjusted odds ratios * with 95% cis for the association between adverse outcomes and gdm subtype * odds ratios are adjusted for maternal age , race / ethnicity , parity , prepregnancy bmi , gestational weight gain , infant sex , and gestational age at ogtt . lga was more strongly associated with gdm subtypes defined by an abnormal fasting glucose ( i - ifg and ifg+igt ) than those based on abnormal postload glucose values only . additionally , while risk for shoulder dystocia / birth injury was elevated in all four gdm subtype groups , the outcome was only significantly associated with i - ifg and ifg+igt . in contrast , preterm delivery , gestational hypertension , and hyperbilirubinemia appeared to be more strongly associated with categories of gdm involving elevated postload glucose ( i - igt1 and/or i - igt2 ) . in fact , women with i - igt2 had the highest risk for preterm delivery and gestational hypertension among all gdm subtypes . in this sample of 8,711 untreated women , 19.4% of whom had gdm by iadpsg criteria , we found a significant association between adverse maternal and perinatal outcomes and increasing fasting and 1- and 2-h ogtt glucose values . the magnitude and significance of these associations were consistent with those reported by the hapo study group ( 8) . as iadpsg guidelines for diagnosis of gdm were determined by the average glucose values in the hapo study , at which the adjusted risk for selected adverse outcomes increased by 75% compared with the risk at mean glucose levels overall , it was not surprising that we additionally observed significant association between gdm status and all adverse outcomes , even after adjusting for confounders such as prepregnancy bmi and gestational weight gain , which were not included in the analyses conducted by the hapo study group . however , the formulation of the new guidelines , whereby a single abnormal fasting and 1-h or 2-h value is sufficient to diagnose gdm , implies that each glucose value contributes a significant independent effect on adverse outcomes and that such effects are equally important for outcome development . models that examine only the associations between a single glucose measure and outcome , with or without adjustment for multiple confounders , do not account for the underlying correlation between fasting and 1-h and 2-h ogtt values . we observed significant correlations of moderate magnitude among the three glucose measures ( range 0.300.62 ) that are consistent with those reported by hapo investigators ( 8) , suggesting that a woman 's response to glucose is at least partially associated with her fpg levels . therefore , we examined various combinations of abnormal values , categorized women according to one of four combinations that result in a diagnosis of gdm under iadpsg guidelines ( i - igt1 , i - ifg , i - igt2 , and ifg+igt ) and investigated group - specific risk for adverse outcomes compared with women without gdm . our results suggest that women with i - igt1 may have modestly elevated risk for primary cesarean delivery , shoulder dystocia / birth injury , or having an lga infant , compared with women without gdm , but these risks were not statistically significant . most importantly , women diagnosed with gdm based on i - ifg may have different risks for specific outcomes , compared with women with gdm based on abnormal 1-h and 2-h ogtt results . for example , lga and shoulder dystocia appear to be more strongly associated with categories of gdm based on abnormal fasting values ; the risk for lga among those with elevated fasting glucose may be further compounded by abnormal postload glucose . in contrast , preterm delivery , gestational hypertension , and hyperbilirubinemia appear to be more closely related to elevated postload glucose than abnormal fasting values , with the highest risks observed among women with normal fasting and two elevated postload glucose values . first , we observed considerable variation in sample sizes for specific gdm subgroups , which is largely attributed to the fact that women with the most abnormal results were likely to be treated and thus excluded from our analyses . the smaller numbers of women in i - igt1 and i - igt2 categories reduced our power to detect statistically significant differences for some outcomes in these groups . additionally , given that data used for these analyses were collected during the course of clinical care , there was some variability in the timing of ascertainment of prepregnancy and delivery weight . among women with prepregnancy weight extracted from the ehr , there was little variability in the mean prepregnancy bmi by timing of measurement ( mean bmi sd : 03 months prior to lmp 27.69 6.20 kg / m ; and 36 months before lmp 27.03 5.90 kg / m ) . additionally , delivery weight was ascertained 24 weeks prior to delivery for 3.9% of the women with measurements in the ehr ( 31.36 5.88 kg / m ) and is likely an underestimate of their true weight at delivery ( for women measured within 2 weeks of delivery : 32.72 5.84 kg / m ) , as is delivery weight by self - report ( 31.74 5.72 kg / m ) . moreover , we abstracted gestational age from the ehr or infant birth certificate , which is largely based on date of lmp rather than estimates from first or midtrimester ultrasound dates . this may have resulted in some misclassification of preterm delivery , which in turn may have influenced our estimates of preterm delivery risk for the various gdm subtypes . lack of ultrasound data , as well as the lack of high - quality information on maternal behaviors and family history , precluded us from controlling for additional confounders which may have affected some outcomes . the utilization of clinical and administrative information allowed for the identification and characterization of women with gdm based on standard laboratory test ( 75-g 2-h ogtt ) results , which allowed us to disaggregate potential subgroups of gdm using ogtt values . in addition , we were able to exclude women who were treated with diet , exercise , and/or pharmacotherapy for gdm during pregnancy , which provided an opportunity to examine outcomes for untreated women with modestly elevated glycemia . this afforded us an opportunity to examine the relationship between glucose measures in untreated women , including those who meet contemporary criteria for gdm , and adverse clinical outcomes . as more women with mild hyperglycemia will be diagnosed with gdm under iadpsg criteria , examining this cohort allowed us to assess outcomes for these women if left untreated . our data suggest that the risks for different adverse maternal and perinatal outcomes vary depending on which single or combined iadpsg - defined ogtt thresholds are equaled or exceeded . prospective studies are needed to determine whether changing pre- and postprandial glucose targets will more uniformly reduce adverse outcomes for women whose pregnancies are complicated by mild gdm .

objectiveto examine the association between levels of hyperglycemia , determined by each prenatal oral glucose tolerance test ( ogtt ) value ( fasting , 1 and 2 h ) , and maternal and perinatal outcomes and to determine whether the risk for these outcomes differs for women whose value(s ) equaled or exceeded the thresholds for gestational diabetes mellitus ( gdm ) established by the international association of diabetes in pregnancy study groups ( iadpsg).research design and methodsthis article discusses a retrospective study of 8,711 women , delivering at 20 weeks ' gestation , who had a prenatal 2-h 75-g ogtt without a prior 50-g challenge and were not treated with insulin , glyburide , diet , and/or exercise during pregnancy . associations between adverse outcomes and elevated ogtt values are reported.resultsafter excluding treated women , 19.4% of the remaining women had iadpsg - defined gdm . continuous fasting , 1- and 2-h ogtt measures , and gdm ( yes / no ) were significantly associated with most adverse outcomes . however , the magnitude and significance of risk for these outcomes differed by various combinations of abnormal glucose values . women with normal fasting and elevated postload values were at higher risk for preterm delivery , gestational hypertension , and having an infant with hyperbilirubinema , whereas women with elevated fasting and normal postload values were at higher risk of having a large - for - gestational - age infant , compared with women without gdm.conclusionsrisks for different adverse outcomes vary depending on which single or combined iadpsg - defined ogtt thresholds are equaled or exceeded . prospective studies are needed to determine whether changing pre- and postprandial glucose targets during pregnancy will more uniformly reduce adverse outcomes .

RESEARCH DESIGN AND METHODS Population and data sources Categorization of OGTT results Measures of prepregnancy BMI and gestational weight gain Maternal and infant outcomes Statistical analyses RESULTS Glucose values and adverse outcomes GDM subtypes and adverse outcomes CONCLUSIONS

the study population consisted of women who had a live singleton birth at 20 weeks ' gestation at the kpsc bellflower medical center between 1 october 2005 and 31 march 2010 , who underwent a prenatal 2-h 75-g ogtt with no prior 50-g oral glucose challenge test and had prepregnancy and delivery anthropometric data ( n = 9,199 ) . in this medical center , routine clinical practice involved treating women whose prenatal 75-g ogtt results met at least two thresholds : fpg 100 mg / dl , 1-h glucose 195 mg / dl , and 2-h glucose 160 mg / dl with diet and exercise therapy . of these , women whose fpg was consistently 105 mg / dl or 1-h postprandial glucose was 140 mg / dl were treated with insulin or glyburide in addition to diet and exercise . for untreated women with more than one ogtt during pregnancy , outcomes are reported based on the test result within or nearest to 2428 weeks ' gestation . maternal age at delivery , race / ethnicity ( hispanic , non - hispanic white , non - hispanic black , asian / pacific islander , and other / multiple races ) , and parity ( 0 , 1 , and 2 ) were obtained from infant birth certificates . based on the results of the 2-h 75-g ogtt , we categorized women into five mutually exclusive categories : no glucose impairment ( no gdm ) , single isolated impaired glucose tolerance ( i - igt1 ) if either 1-h glucose 180 mg / dl or 2-h glucose 153 mg / dl and fpg < 92 mg / dl , isolated impaired fasting glucose ( i - ifg ) if fpg 92 mg / dl and both 1-h glucose < 180 mg / dl and 2-h glucose < 153 mg / dl , double - isolated impaired glucose tolerance ( i - igt2 ) if both 1-h glucose 180 mg / dl and 2-h glucose 153 mg / dl but fpg < 92 mg / dl , and combined ifg and igt ( ifg+igt ) if fpg 92 mg / dl and either 1-h glucose 180 mg / dl and/or 2-h glucose 153 mg / dl . prepregnancy bmi and weight gain during pregnancy both have been shown to be associated with the development of gdm ( 1315 ) as well as adverse outcomes independent of gdm ( 1619 ) . large - for - gestational - age ( lga ) infants were defined as sex- , race- , and gestational age consistent with the methods used by the hapo study group ( 11 ) , percentiles for birth weight were determined by quantile regression stratified by sex and race / ethnicity , with adjustment for gestational age and maternal parity . an infant was considered to have a birth weight > 90th percentile if the birth weight was greater than the estimated 90th percentile for the infant 's sex , gestational age , race / ethnicity , and maternal parity . delivery by primary cesarean section was obtained from infant birth certificates ; data for women who had a previous cesarean section ( n = 963 ) were excluded from analysis of this outcome . in analyses of gestational hypertension , we excluded women with pregestational hypertension ( icd-9 codes 401405.9 , 642.0642.2 , and/or 642.7 ; n = 323 ) . multiple logistic regression models were used to calculate adjusted odds ratios ( aors ) and corresponding 95% cis for a 1-sd increase in continuous fasting and 1-h and 2-h glucose levels associated with adverse outcomes , after controlling for maternal age , race / ethnicity , parity , prepregnancy bmi , and gestational weight gain . the study population consisted of women who had a live singleton birth at 20 weeks ' gestation at the kpsc bellflower medical center between 1 october 2005 and 31 march 2010 , who underwent a prenatal 2-h 75-g ogtt with no prior 50-g oral glucose challenge test and had prepregnancy and delivery anthropometric data ( n = 9,199 ) . in this medical center , routine clinical practice involved treating women whose prenatal 75-g ogtt results met at least two thresholds : fpg 100 mg / dl , 1-h glucose 195 mg / dl , and 2-h glucose 160 mg / dl with diet and exercise therapy . of these , women whose fpg was consistently 105 mg / dl or 1-h postprandial glucose was 140 mg / dl were treated with insulin or glyburide in addition to diet and exercise . after excluding women who received any form of treatment during pregnancy ( n = 488 ) , we used ogtt results to identify women with gdm based on iadpsg guidelines ( 10 ) . for untreated women with more than one ogtt during pregnancy , outcomes are reported based on the test result within or nearest to 2428 weeks ' gestation . maternal age at delivery , race / ethnicity ( hispanic , non - hispanic white , non - hispanic black , asian / pacific islander , and other / multiple races ) , and parity ( 0 , 1 , and 2 ) were obtained from infant birth certificates . based on the results of the 2-h 75-g ogtt , we categorized women into five mutually exclusive categories : no glucose impairment ( no gdm ) , single isolated impaired glucose tolerance ( i - igt1 ) if either 1-h glucose 180 mg / dl or 2-h glucose 153 mg / dl and fpg < 92 mg / dl , isolated impaired fasting glucose ( i - ifg ) if fpg 92 mg / dl and both 1-h glucose < 180 mg / dl and 2-h glucose < 153 mg / dl , double - isolated impaired glucose tolerance ( i - igt2 ) if both 1-h glucose 180 mg / dl and 2-h glucose 153 mg / dl but fpg < 92 mg / dl , and combined ifg and igt ( ifg+igt ) if fpg 92 mg / dl and either 1-h glucose 180 mg / dl and/or 2-h glucose 153 mg / dl . prepregnancy bmi and weight gain during pregnancy both have been shown to be associated with the development of gdm ( 1315 ) as well as adverse outcomes independent of gdm ( 1619 ) . large - for - gestational - age ( lga ) infants were defined as sex- , race- , and gestational age specific birth weight > 90th percentile . consistent with the methods used by the hapo study group ( 11 ) , percentiles for birth weight were determined by quantile regression stratified by sex and race / ethnicity , with adjustment for gestational age and maternal parity . an infant was considered to have a birth weight > 90th percentile if the birth weight was greater than the estimated 90th percentile for the infant 's sex , gestational age , race / ethnicity , and maternal parity . delivery by primary cesarean section was obtained from infant birth certificates ; data for women who had a previous cesarean section ( n = 963 ) were excluded from analysis of this outcome . in analyses of gestational hypertension , we excluded women with pregestational hypertension ( icd-9 codes 401405.9 , 642.0642.2 , and/or 642.7 ; n = 323 ) . multiple logistic regression models were used to calculate adjusted odds ratios ( aors ) and corresponding 95% cis for a 1-sd increase in continuous fasting and 1-h and 2-h glucose levels associated with adverse outcomes , after controlling for maternal age , race / ethnicity , parity , prepregnancy bmi , and gestational weight gain . after excluding 488 women who received any form of treatment ( 5.3% of the population ; 22.4% of all women with gdm ) , the remaining sample for this analysis was comprised of 8,711 untreated women with a mean age of 29.1 5.9 years , the majority of whom were hispanic ( table 1 ) . data for women who had pre - gestational hypertension were excluded . of these women , 1,691 ( 19.4% ) had iadpsg - defined gdm . compared with women without gdm , those with gdm tended to be slightly older , more parous , have higher mean prepregnancy bmi , and have relatively similar mean gestational weight gain ( table 1 ) . associations between adverse outcomes and continuous fasting and 1-h and 2-h glucose values , as well as glucose dichotomized at iadpsg cut points , are shown in table 2 . when modeled separately , we found a significant association between each continuous ogtt value and lga , primary cesarean , preterm delivery , and gestational hypertension . odds ratios for significant risk of adverse outcomes categorized by iadpsg cut points for each glucose measure were relatively consistent with those of the continuous measures , except that those with 1-h glucose 180 mg / dl were not at significantly increased risk for primary cesarean section or hyperbilirubinemia , compared with women with 1-h glucose < 180 mg / dl . similarly , women with 2-h glucose 153 mg / dl were not at significantly increased risk for lga , shoulder dystocia / birth injury , or hyperbilirubinemia , compared with those with 2-h glucose < 153 mg / dl . adjusted odds ratios * and 95% cis for the association of adverse outcomes with ogtt values and gdm * odds ratios for ogtt measures are for an increase in glucose level of 1 sd ( 7.99 mg / dl for fpg , 30.84 mg / dl for 1-h glucose , and 23.08 mg / dl for 2-h glucose ) and are adjusted for maternal age , race / ethnicity , parity , prepregnancy bmi , gestational weight gain , infant sex , and gestational age at ogtt . women classified as having gdm based on a single abnormal postload glucose value were at significantly increased risk for preterm delivery , gestational hypertension , and infant hyperbilirubinemia . women with i - ifg were also 29% more likely to have gestational hypertension ( 1.041.72 ) compared with women without gdm . women with the i - igt2 gdm subtype were 2.33 times as likely to develop gestational hypertension ( 1.204.51 ) than women without gdm , and infants born to these women were 2.85 times as likely to be delivered preterm ( 1.595.10 ) than infants of mothers without gdm . women with ifg+igt were more than twice as likely to have gestational hypertension ( 1.422.84 ) , 87% more likely to have shoulder dystocia / birth injury during delivery ( 1.182.96 ) , and 2.32 times as likely to have an lga infant ( 1.723.13 ) than women without gdm . adjusted odds ratios * with 95% cis for the association between adverse outcomes and gdm subtype * odds ratios are adjusted for maternal age , race / ethnicity , parity , prepregnancy bmi , gestational weight gain , infant sex , and gestational age at ogtt . additionally , while risk for shoulder dystocia / birth injury was elevated in all four gdm subtype groups , the outcome was only significantly associated with i - ifg and ifg+igt . in contrast , preterm delivery , gestational hypertension , and hyperbilirubinemia appeared to be more strongly associated with categories of gdm involving elevated postload glucose ( i - igt1 and/or i - igt2 ) . in fact , women with i - igt2 had the highest risk for preterm delivery and gestational hypertension among all gdm subtypes . associations between adverse outcomes and continuous fasting and 1-h and 2-h glucose values , as well as glucose dichotomized at iadpsg cut points , are shown in table 2 . when modeled separately , we found a significant association between each continuous ogtt value and lga , primary cesarean , preterm delivery , and gestational hypertension . odds ratios for significant risk of adverse outcomes categorized by iadpsg cut points for each glucose measure were relatively consistent with those of the continuous measures , except that those with 1-h glucose 180 mg / dl were not at significantly increased risk for primary cesarean section or hyperbilirubinemia , compared with women with 1-h glucose < 180 mg / dl . similarly , women with 2-h glucose 153 mg / dl were not at significantly increased risk for lga , shoulder dystocia / birth injury , or hyperbilirubinemia , compared with those with 2-h glucose < 153 mg / dl . women with gdm , as defined by iadpsg guidelines , were at significantly increased risk for all outcomes except infant hyperbilirubinemia . adjusted odds ratios * and 95% cis for the association of adverse outcomes with ogtt values and gdm * odds ratios for ogtt measures are for an increase in glucose level of 1 sd ( 7.99 mg / dl for fpg , 30.84 mg / dl for 1-h glucose , and 23.08 mg / dl for 2-h glucose ) and are adjusted for maternal age , race / ethnicity , parity , prepregnancy bmi , gestational weight gain , infant sex , and gestational age at ogtt . women classified as having gdm based on a single abnormal postload glucose value were at significantly increased risk for preterm delivery , gestational hypertension , and infant hyperbilirubinemia . women with i - ifg were also 29% more likely to have gestational hypertension ( 1.041.72 ) compared with women without gdm . women with the i - igt2 gdm subtype were 2.33 times as likely to develop gestational hypertension ( 1.204.51 ) than women without gdm , and infants born to these women were 2.85 times as likely to be delivered preterm ( 1.595.10 ) than infants of mothers without gdm . women with ifg+igt were more than twice as likely to have gestational hypertension ( 1.422.84 ) , 87% more likely to have shoulder dystocia / birth injury during delivery ( 1.182.96 ) , and 2.32 times as likely to have an lga infant ( 1.723.13 ) than women without gdm . adjusted odds ratios * with 95% cis for the association between adverse outcomes and gdm subtype * odds ratios are adjusted for maternal age , race / ethnicity , parity , prepregnancy bmi , gestational weight gain , infant sex , and gestational age at ogtt . additionally , while risk for shoulder dystocia / birth injury was elevated in all four gdm subtype groups , the outcome was only significantly associated with i - ifg and ifg+igt . in contrast , preterm delivery , gestational hypertension , and hyperbilirubinemia appeared to be more strongly associated with categories of gdm involving elevated postload glucose ( i - igt1 and/or i - igt2 ) . in fact , women with i - igt2 had the highest risk for preterm delivery and gestational hypertension among all gdm subtypes . in this sample of 8,711 untreated women , 19.4% of whom had gdm by iadpsg criteria , we found a significant association between adverse maternal and perinatal outcomes and increasing fasting and 1- and 2-h ogtt glucose values . the magnitude and significance of these associations were consistent with those reported by the hapo study group ( 8) . as iadpsg guidelines for diagnosis of gdm were determined by the average glucose values in the hapo study , at which the adjusted risk for selected adverse outcomes increased by 75% compared with the risk at mean glucose levels overall , it was not surprising that we additionally observed significant association between gdm status and all adverse outcomes , even after adjusting for confounders such as prepregnancy bmi and gestational weight gain , which were not included in the analyses conducted by the hapo study group . however , the formulation of the new guidelines , whereby a single abnormal fasting and 1-h or 2-h value is sufficient to diagnose gdm , implies that each glucose value contributes a significant independent effect on adverse outcomes and that such effects are equally important for outcome development . models that examine only the associations between a single glucose measure and outcome , with or without adjustment for multiple confounders , do not account for the underlying correlation between fasting and 1-h and 2-h ogtt values . therefore , we examined various combinations of abnormal values , categorized women according to one of four combinations that result in a diagnosis of gdm under iadpsg guidelines ( i - igt1 , i - ifg , i - igt2 , and ifg+igt ) and investigated group - specific risk for adverse outcomes compared with women without gdm . our results suggest that women with i - igt1 may have modestly elevated risk for primary cesarean delivery , shoulder dystocia / birth injury , or having an lga infant , compared with women without gdm , but these risks were not statistically significant . most importantly , women diagnosed with gdm based on i - ifg may have different risks for specific outcomes , compared with women with gdm based on abnormal 1-h and 2-h ogtt results . for example , lga and shoulder dystocia appear to be more strongly associated with categories of gdm based on abnormal fasting values ; the risk for lga among those with elevated fasting glucose may be further compounded by abnormal postload glucose . in contrast , preterm delivery , gestational hypertension , and hyperbilirubinemia appear to be more closely related to elevated postload glucose than abnormal fasting values , with the highest risks observed among women with normal fasting and two elevated postload glucose values . additionally , delivery weight was ascertained 24 weeks prior to delivery for 3.9% of the women with measurements in the ehr ( 31.36 5.88 kg / m ) and is likely an underestimate of their true weight at delivery ( for women measured within 2 weeks of delivery : 32.72 5.84 kg / m ) , as is delivery weight by self - report ( 31.74 5.72 kg / m ) . this may have resulted in some misclassification of preterm delivery , which in turn may have influenced our estimates of preterm delivery risk for the various gdm subtypes . the utilization of clinical and administrative information allowed for the identification and characterization of women with gdm based on standard laboratory test ( 75-g 2-h ogtt ) results , which allowed us to disaggregate potential subgroups of gdm using ogtt values . in addition , we were able to exclude women who were treated with diet , exercise , and/or pharmacotherapy for gdm during pregnancy , which provided an opportunity to examine outcomes for untreated women with modestly elevated glycemia . our data suggest that the risks for different adverse maternal and perinatal outcomes vary depending on which single or combined iadpsg - defined ogtt thresholds are equaled or exceeded . prospective studies are needed to determine whether changing pre- and postprandial glucose targets will more uniformly reduce adverse outcomes for women whose pregnancies are complicated by mild gdm .

it is no surprise that alcohol , as a topic , is inherently interesting to high school students ; it is one of the most commonly used drugs by this population . according to the center for disease control s youth risk behavior surveillance system , 71% of high school students have consumed alcohol on at least one day in their life . it is even more alarming that 57% of 12th graders have been drunk at least once . clearly , there is a need to educate students on the dangers of drinking alcohol , especially at a young age . typically , substance abuse topics are covered in health education courses not courses in chemistry or biology . yet , basic principles of chemistry and biology are crucial in understanding our bodies , health , and disease . educators can take advantage of the fact that the topic of alcohol interests high school students . promoting interest has been shown to be an important factor in helping motivate students to learn and increase achievement . moreover , linnenbrink - garcia , et al . have shown that using topics that connect to real life in the classroom is one way to increase interest and motivation in science learning . in fact , high school students indicate that the topics of most interest to them in their science classes include drugs , disease , and the environment . moreover , students with relatively low expectations for success in science display more interest and perform better in science when they are asked to connect the relevance of their science topics in class to their lives . there have been relatively few studies that assess how specific topics affect student achievement in chemistry and biology . one study that used the chemistry in the community curriculum ( chemcomm ) , which focuses on areas such as the environment , industry , food , and health , demonstrated increased achievement in a small group of high school chemistry students . in several larger studies , we have shown that curricula focused on pharmacology topics such as drugs of abuse can markedly improve high school student achievement in chemistry and biology . the very nature of pharmacology , which integrates basic principles of chemistry and biology to uncover the mechanisms by which drugs and chemicals affect organisms , lends itself to a useful approach for providing relevance and context especially for teenagers . in our previous studies , teachers used a series of modules in their classrooms as part of our program called the pharmacology education partnership ( pep ) . the pep modules covered various drug - related topics with catchy titles such as acids , bases , and cocaine addicts and steroids and athletes genes work overtime . the pep studies provided the basis for the current study , which brings the subject of alcohol pharmacology to high school teaching . the teachers who participated in pep suggested that the subject of alcohol was of great interest to their students and thought that a similar program devoted to alcohol as a context for learning chemistry and biology would be well received . moreover , teachers mentioned that there is considerable misconception among their students about the chemistry and biology of alcohol . for example , many adolescents believe that drinking alcohol once an hour can avoid legal intoxication , yet a basic understanding of chemistry and biology can help to correct this misconception . in addition , teachers reported that students have misconceptions of basic chemical and biological principles as well , such as the meaning of equilibrium and the relationship between genes and proteins . research in areas of cognitive psychology reveals that students prior knowledge ( including misconceptions ) and their prior experiences are crucial elements in formulating their own understanding of phenomena ( constructivism theory ) . thus , a program that can challenge prior knowledge and correct misconceptions about chemistry and biology that relate to a topic such as alcohol may help students construct meaning from their everyday life and better their learning in these sciences . to implement such a program , professional development at the teacher level is just as important . effective professional development aims to improve teacher knowledge , teaching practices , and ultimately student achievement . as compiled by supovitz and turner , high - quality professional development should include at least the following:inquiry - based approaches and active learning.an intensive and sustained program.a mechanism to engage teachers in concrete teaching tasks that they can use in their own classrooms.a focus on subject - matter knowledge and content skills . inquiry - based approaches and active learning . an intensive and sustained program . a mechanism to engage teachers in concrete teaching tasks that they can use in their own classrooms . we addressed each of these features in our three previous pep studies , which delivered professional development at ( i ) a five - day residential workshop , ( ii ) a one - day workshop at a national meeting or ( iii ) a six - hour workshop via distance learning ( two hours per week ) . regardless of the workshop format , teacher content knowledge in chemistry and biology increased and was maintained for at least a year . additionally , in each case , there was greater student achievement compared to students who did not have the pep modules in their classes . thus , our findings support research indicating that rich content , rather than the duration of professional development , is associated with student learning . for this study , we recruited a new set of teachers from the united states to participate in the alcohol pharmacology education partnership ( apep ) . we used the more cost - effective forms of professional development , a full - day workshop onsite at an annual conference and an interactive three - session workshop delivered by distance learning technology . each of the apep modules that we developed addresses several basic principles of chemistry and biology . additionally , the modules capitalize on the natural curiosity that teens typically have concerning the subject of alcohol . as we demonstrated previously using pep , students scored higher when tested for knowledge of chemistry and biology concepts after using the apep modules than students who did not use the modules . high school chemistry and biology teachers were recruited from across the united states by placing announcements in the national science teachers association ( nsta ) newspaper , nsta reports . ultimately , 156 teachers participated in the study . details of the teacher demographics are provided in the supporting information , table s1 . in brief , 50% of teachers were of biology or chemistry , 88% were from public schools , 30% were teaching in schools with at least a 40% population of minorities , and 67% were from urban or suburban schools . teachers selected to attend six hours of apep professional development at either a full - day conference - based workshop conducted concurrently with the nsta or north carolina science teachers association ( ncsta ) annual meetings or a series of distance learning ( dl ) workshops equal in length to the residential sessions . of the 156 participating teachers , 100 attended the conference - based workshops and 56 attended the dl workshops . teachers students ( n = 14 014 ) who participated in the study were from chemistry and biology classes in grades 912 representing schools from urban , suburban , and rural districts . the demographics of students participating in the study are shown in table 1 . during the first year of the study , all students served as the control group ( before teachers attended any apep professional development ) ; the following year , a second set of students served as the experimental group . at the start of the second year , teachers attended the professional development workshop and then field - tested the apep curriculum in their classrooms over the ensuing school year . there was no indication that demographics of the school student populations differed systematically from year to year . all teachers enrolled in the study received professional development provided by three of us ( r.d.s .- b . see supporting information table s2 for the regional demographics of broadcast sites for the dl workshops . the dl workshops were conducted using two - way live audio and video plus high - speed data to allow interaction between instructors and teachers as we reported previously . this two - way system allowed connections to four sites simultaneously and was broadcast from the north carolina school for science and mathematics in durham , nc . ( an example of the dl broadcast is available . ) the formats for both the conference - based and dl workshops were based on our previous pep studies . central chemistry concepts ( e.g. , dynamic equilibrium , solubility , molecule polarity , enzymes , catalysts ) and biology concepts ( e.g. , cell structure and function , transport of molecules across membranes , protein synthesis ) were presented within the context of four apep modules developed by the authors ( see below ) to be used in field - testing in the teachers classes the following year . thus , during the workshops , teachers not only were able to review each of these concepts and learn how to incorporate them within the apep modules but also tackled one of the apep modules themselves , working in groups , just as their students would . they read the opening story , researched answers to the inquiries built into the module , discussed their findings with their partners , and then presented their answers to all of the teachers . the teachers also participated in one of the hands - on laboratory exercises contained in the apep curriculum . teachers prepared a chemical breathalyzer test that can be adapted for standard or advanced placement ( ap ) courses . teachers in the dl course also participated in the laboratory exercise during the live broadcast with materials provided to them ahead of time . the team of authors created four apep modules to be field - tested in classrooms during the study . the four modules integrate chemistry and biology concepts within the context of alcohol - related pharmacology topics that have relevance to high school students . each module contains the following elements:an overview containing a description of the module with the associated education standards.a set of learning objectives.a student handout that contains the story followed by a set of questions that students must answer ( similar to a problem in problem - based learning).the content the bulk of the module that contains all the answers to the student questions , including animations.a student quiz with 46 questions about the chemistry and biology concepts relevant to the module ( and answers provided).activities ( integrating math calculations or lab activities with the module content).resources ( a list of references and url links ) . . a student handout that contains the story followed by a set of questions that students must answer ( similar to a problem in problem - based learning ) . the content the bulk of the module that contains all the answers to the student questions , including animations . a student quiz with 46 questions about the chemistry and biology concepts relevant to the module ( and answers provided ) . activities ( integrating math calculations or lab activities with the module content ) . resources ( a list of references and url links ) . in addition to these module - specific items , there are several other resources that address all the modules . these include:biology and chemistry connections a table showing the specific chemistry and biology concepts that are covered in each module and links to a review of specific chemistry and biology concepts normally covered in high school courses.explore more , a set of pages ( also linked within the modules ) that cover topics related to the modules such as alcoholism and neuroscience , and technologies such as magnetic resonance imaging .. a glossary.list of the next generation science standards and former national science education standards . a table showing the specific chemistry and biology concepts that are covered in each module and links to a review of specific chemistry and biology concepts normally covered in high school courses . explore more , a set of pages ( also linked within the modules ) that cover topics related to the modules such as alcoholism and neuroscience , and technologies such as magnetic resonance imaging .. list of the next generation science standards and former national science education standards . the module content is aligned with the former national science education standards and with the new next generation science standards ( ngss ) ( see table 2 ) . in addition , the apep module content addresses typical teen misconceptions and myths related to alcohol consumption . these include misconceptions such as ( i ) drinking one drink an hour will not get you drunk , ( ii ) you can get rid of alcohol by urinating it out , and ( iii ) beer and wine are less intoxicating than liquor . the modules were developed to foster student engagement in the form of a story , similar to a problem - based or case - based learning approach . working in groups , students read the story contained in the student handout , which is followed by a series of questions relating to the story . it is up to the teacher as to whether the students can use web - based literature to find the answers , or use the apep module content pages to find the answers . a student self - assessment quiz is included within each module with an explanation of the correct or incorrect answers . in addition , the modules contain activities , many of which include mathematical calculations . the general structure of the apep modules is shown in figure 1 and the story for module 4 is provided in the supporting information . the apep modules may be accessed online ( both a teacher and student version ) . teachers were instructed to field test the four apep modules in their classrooms over the year . we did not prescribe a specific approach for teachers to use the apep modules ; instead , we asked teachers to report how they incorporated the content into their courses . previously , we showed that the more pep modules used , the greater the beneficial effect on student scores in both basic and advanced science knowledge . first , we conducted a summative evaluation to determine the attitudes of teachers about the quality of the workshop . there were three strands within the survey : content , teaching approaches , and format of the workshop . items were assessed using a five - point likert - type scale , followed by several open - ended questions pertaining to what teachers liked most and least about the workshop . the evaluation items can be found in the supporting information , table s3 . to determine the effectiveness of the workshop on teachers knowledge gain and retention after one year , we administered a short test consisting of 20 multiple - choice questions that addressed the chemistry and biology content listed in table 2 . the test was administered to the teachers at the beginning of the workshop ( pretest ) , at the end of the workshop ( post - test ) , and again at the end of the year ( one - year follow - up ) without prior notification . sample items are included in the supporting information . at the end of the school year after field - testing the apep modules , we sent the teachers a multiple - choice content test similar to that used in our previous pep studies to give to their students ; the tests were unannounced . the tests were constructed by the authors , with input from high school chemistry and biology teachers at the north carolina school of science and math , where one of us ( m.j.h . ) teaches . the test comprised two parts , a basic knowledge and an advanced knowledge section . the basic test consisted of 20 questions ( 11 chemistry and 9 biology ) similar to those found in first - year chemistry and biology textbooks ( see the supporting online material for examples ) . the multiple choice questions assessed student knowledge of concepts in chemistry and biology according to the framework provided by the 1996 national assessment of educational progress science test . following the 20 basic knowledge questions , there were 10 questions that were specific for the new knowledge about alcohol in the context of chemistry and biology ( advanced knowledge ) . these questions assessed concepts not normally taught in the standard curriculum ( see the supporting information for examples ) . to establish reliability of the assessment instrument , we conducted a test retest with 58 high school students in chemistry and biology classes ( unrelated to the study ) . an intraclass correlation of scores between the two tests generated a reliability coefficient of 0.85 ( 95% confidence interval 0.730.92 ) . reliability coefficients 0.811.0 are considered substantial . to establish validity of the assessment instrument , we asked a team of nine high school chemistry and biology teachers ( not related to any of the authors ) to rate each of the questions as to whether it was relevant and appropriate to their courses . for the biology and chemistry basic questions , eight of nine teachers rated at least 80% and 90% , respectively , of the questions as relevant and appropriate . for the advanced questions , seven of nine teachers rated at least 80% of the biology questions as relevant and appropriate , and all of the teachers rated at least 80% of the chemistry questions as relevant and appropriate . we posited that students with different backgrounds could score differently on average on the apep tests . therefore , we obtained demographic information from the students regarding these demographic parameters : gender , race and ethnicity , year in high school ( i.e. , 9th12th grade ) , course type ( i.e. , chemistry or biology ) , and course level ( i.e. , first - year , second - year , or ap ) . the demographic representation of students within classes of teachers who administered the apep tests is presented in table 1 . we compiled the percent correct scores of the 14 014 students on both the basic and advanced tests . to estimate the effects of the modules , we used logistic regression models with random effects for the teachers ( which is a type of multilevel model ) , adjusting for demographic characteristics ( see table 1 ) , and number of modules as a series of indicator variables . the outcome variables are the number of correct answers out of 20 questions on the basic test and the number of correct answers out of 10 questions on the advanced test . for simplicity student - level random effects for students taught by the same teacher are centered around their teacher - specific mean , which enables us to account for nesting of students within teachers . thus , the random effects account for the correlations among outcomes of students taught by the same teacher . this research received an irb approval of exemption from human research subjects according to federal rules ( 45 cfr 46.101(b)(1 ) ) and is not subject to the privacy rule ( hippa ) ( 45 cfr 164.500(a ) ) . to assess the effect of providing the pharmacology - based professional development workshop on teacher knowledge of basic chemistry and biology concepts , teachers were assessed for content knowledge at the beginning of the workshop ( pretest ) , at the end of the workshop ( post - test ) , and one year following the workshop ( one - year follow - up ) . based on separate repeated measures anovas for each workshop , teacher scores differed significantly among the three tests for the dl workshop , f ( 1.852 , 53.70 ) = 11.79 and p < 0.0001 , and for the conference workshop , f ( 1.78 , 144.3 ) = 42.33 and p < 0.0001 . all score distributions were approximately distributed as gaussian , with a geisser greenhouse epsilon of 0.93 and 0.89 , respectively . comparisons between the pre- and post - tests revealed that average scores on both the post - test and the one - year follow - up test were significantly higher than the pretest averages ( tukey s multiple comparison test ) . there was no difference between the scores in the post - test and the one - year follow - up test for both workshops , indicating persistence of the knowledge gain . the effect sizes ( cohen s d ) for knowledge gains were in the moderate to large range . data are from teachers who participated in all three testing modalities : pretest , post - test , and one - year follow up . there was no significant difference between the post - test and one - year follow - up test for either workshop . we also assessed whether teachers knowledge gain was similar on the biology and chemistry questions depending on whether the teacher taught biology or chemistry . there were no statistically significant differences in knowledge gains over the year for any comparison with the exception of chemistry teachers who attended the conference workshop . their average gain in scores on the biology questions over the year was considerably larger than their gain in scores on the chemistry questions ( mean gain in scores sd was 31 32 versus 11 26 percentage points , respectively ( n = 31 ) ; p < 0.05 , paired student s t test ) . at the conclusion of the workshop , teachers provided an evaluation of the workshop approach and content delivery a summary of their evaluation scores is provided in the supporting information , table s3 . teachers participating in the workshop were instructed to use as many apep modules as possible ( i.e. , four ) in their classes throughout the year . although there was no set prescription for implementing the modules , we did collect information on how the teachers used the modules in their classes ( table 4 ) . of the 426 classes in which modules were used , the most common format was to cover the module content over several class periods ( 49% of classes ) . teachers administered the apep tests , unannounced , at the end of their courses . the results revealed that the use of the apep modules was a significant predictor of greater student achievement on both the basic and advanced knowledge tests , as shown in figure 2 and in the supporting information . using more modules resulted in progressively higher scores on both tests . the average scores in the control teachers classrooms using zero modules did not differ practically or significantly from the average scores in classrooms of teachers in the experimental group who did not use any apep modules ; hence , we used a common indicator variable for zero apep modules for these two sets of students . the regression results suggested that usage of modules is associated with higher test scores . for example , a typical student in a class that used all four modules had 1.21 times the odds of answering basic knowledge questions correctly compared to a typical student in a class that used no modules . not surprisingly , the strongest predictor of higher basic and advanced scores was being in an advanced biology or chemistry course . a typical biology 2 student had 1.58 times the odds of answering a basic knowledge question correctly compared to a typical biology 1 student , and a typical chemistry 2 student had 1.84 times the odds of answering correctly compared to a typical biology 1 student . for the basic knowledge questions , students in chemistry 1 had a small increase in odds of answering correctly compared to biology 1 students . because most chemistry students had already had biology , this might be expected . several student demographic characteristics were also statistically significant predictors in both models , though with smaller coefficients than the class type . all results are in the regression tables in the supporting information , tables s4 and s5 . student knowledge assessed at the end of the course ( chemistry or biology ) in which apep was administered . student basic and advanced knowledge scores were higher as more modules were used in their classroom . data represent the mean sem scores ( unadjusted ) from students in classes of teachers enrolled in the apep study . the zero module data includes students from the control year and teachers who taught zero modules in the experimental year . use of modules was a significant predictor of higher scores ( logistic regression ; n = 14 014 students ; see the supporting information , tables s4 and s5 ) . students in the apep study were enrolled in either chemistry or biology classes . we also analyzed subsets of questions and students separately ( e.g. , the performance of biology students on chemistry questions , the performance of chemistry students on chemistry questions , etc . ) . in general , using more modules was associated with higher test scores , preserving the dose - response relationship we see in the overall analysis ( table 5 ) . however , the higher chemistry student scores on biology and chemistry questions remained relatively flat when one , two , and three modules were used compared to no modules . thus , the steady increase in scores from biology students when using one , two , and three modules may have accounted for most of the higher scores as reported in the overall analysis ( figure 2 ) . interestingly , students in biology class whose teachers used three or four apep modules achieved chemistry scores similar to the baseline scores of students in chemistry ( see table 5 ) . use of modules was a significant predictor of higher scores ( logistic regression ; n = 14 014 students ) . students in biology class whose teachers used three or four apep modules achieved chemistry scores similar to the baseline scores of students in chemistry . in this study , we demonstrate that implementing pharmacology - based science education ( using alcohol as the context ) in high school classrooms increases student and teacher achievement in chemistry and biology . these findings reproduce the findings obtained in our three previous pep studies that focused on drugs . in the present study , we found up to 10 percentage - point increases in achievement ( compared to no module use ) when students were administered unannounced tests . even greater increases can be achieved when using up to six modules , as we showed in a previous pep study that focused on drugs . in the apep study , all of the modules focused on one drug ( alcohol ) , so it is possible that limiting the subject matter may have contributed somewhat to a smaller effect compared to our previous studies that incorporated a variety of drug topics . the format of the apep curriculum is similar to the pep curriculum , so it would be easy to combine the two , thereby increasing the number of modules ( to 10 ) that could be used to teach chemistry and biology concepts . the content within apep was designed to address the 1996 national science education standards . since then , the standards have been revised , recommending an integrated , research - based curriculum . the integration of chemistry , biology , and math concepts in apep addresses several components of the new ngss , including scientific practices and the integration of disciplinary core ideas . we surmise that integration of chemistry and biology may have been successful in our study because students in biology , who typically take chemistry a year after biology , were able to increase their chemistry scores to the same level as that achieved by control chemistry students just by using at least three apep modules . the apep curriculum has several features that may have contributed to the positive outcome , including the following:the relevance of the various alcohol - focused stories ( and the content ) to the high school population.an inquiry - based approach to learn the content.content that addresses myths and misconceptions about alcohol.repetition of core concepts among the modules . the relevance of the various alcohol - focused stories ( and the content ) to the high school population . each of these features are important elements in teaching strategies for effective learning . additional studies would be required to isolate these variables to determine which is the greatest predictor of higher scores . as mentioned above , we have shown in a previous pep study that use of six modules predicts even higher scores in chemistry and biology than reported here , supporting the importance of dose . however , we suggest that it is the combination of these features that makes the apep curriculum complete , as it includes elements of best practices in curriculum design and teaching . in terms of professional development , both on - site conference - based workshops and the distance learning workshops helped teachers improve their knowledge in chemistry and biology and maintain that improvement for at least a year . however , merely attending the professional development workshop was not sufficient for increased student achievement . student scores of the control teachers did not differ from the experimental teachers who attended the workshop but did not use the apep modules . this is important because there is a plethora of professional development activities for teachers many providing continuing education credit that assume the experience will improve teaching . our findings suggest that teachers must use what they have learned in the workshop along with the curriculum to help students learn science better . although the results presented here replicate what we ve found in our previous studies , there are several limitations . first , although teachers used the modules in a variety of ways , we do not know which of the implementation methods was a better predictor for the stepwise increase in scores . because several teachers used multiple implementation methods in their classes at different times , it was not realistic to determine whether any method was more effective than another . however , presenting the module material over several classes was the most prevalent implementation format ( 50% of classes used this format ) , so it is reasonable to conclude that at least this particular method may have contributed significantly to the increasing scores . in contrast , only 6% of classes worked on the apep modules online , so it is unlikely that this method alone contributed significantly to the positive findings . in addition , we do not know whether any specific module was important in explaining the increasing scores . as there was considerable overlap of concepts from module to module , it is possible that repetition of concepts rather than any single module may have contributed to the increasing scores with increased module use . another limitation of the study is the possibility that teachers using the modules were we believe that this is unlikely because the questions for the basic test were not directed specifically to the module content but rather to the principles associated with the module content . although the advanced test questions did address specific module content , many required the students to apply what they learned from the modules to a new situation . third , the improvements in the scores associated with module usage may not be caused by the modules themselves ; other variables that we did not control could explain ( at least in part ) the higher scores . for example , suppose that a large majority of the students who used the apep modules have higher academic motivation and abilities than those who did not use the modules , and this difference , rather than beneficial effects of learning with the modules , accounts for their higher average test scores . however , it is unlikely that a systematic disparity in student abilities would be present because the disparities would also need to be present in a dose - response manner ( as were the higher scores ) , and classes of the same type taught by the same teacher were likely to be similar in terms of prematriculation abilities . nevertheless , such confounders are always possible given that we did not control for student ability . this is the fourth study in which we have demonstrated that students who use problem - based units addressing relevant topics to their lives ( e.g. , alcohol and drugs ) score better in chemistry and biology compared to students using the standard curriculum . in total , we have now tested 27 841 students using both randomized and nonrandomized controlled designs that generate the same results . the implementation of controlled research designs can provide teachers with meaningful information about curricular materials and teaching strategies most likely to help their students learn chemistry and biology better . additionally , a major goal in science education is to help students become critical thinkers and make good decisions about their daily lives . it remains to be determined whether learning the chemical and biological principles underlying alcohol s actions will impact students decisions about alcohol use . the apep modules can be accessed online free of charge for interactive use or downloaded as pdfs directly from our web site . a fifth module addressing fetal alcohol spectrum disorders has been added since the apep study was completed ; however , it was not used for any data collection .

we developed the alcohol pharmacology education partnership ( apep ) , a set of modules designed to integrate a topic of interest ( alcohol ) with concepts in chemistry and biology for high school students . chemistry and biology teachers ( n = 156 ) were recruited nationally to field - test apep in a controlled study . teachers obtained professional development either at a conference - based workshop ( nsta or ncsta ) or via distance learning to learn how to incorporate the apep modules into their teaching . they field - tested the modules in their classes during the following year . teacher knowledge of chemistry and biology concepts increased significantly following professional development , and was maintained for at least a year . their students ( n = 14 014 ) demonstrated significantly higher scores when assessed for knowledge of both basic and advanced chemistry and biology concepts compared to students not using apep modules in their classes the previous year . higher scores were achieved as the number of modules used increased . these findings are consistent with our previous studies , demonstrating higher scores in chemistry and biology after students use modules that integrate topics interesting to them , such as drugs ( the pharmacology education partnership ) .

Introduction Intervention Results Discussion Limitations Conclusions

it is no surprise that alcohol , as a topic , is inherently interesting to high school students ; it is one of the most commonly used drugs by this population . according to the center for disease control s youth risk behavior surveillance system , 71% of high school students have consumed alcohol on at least one day in their life . yet , basic principles of chemistry and biology are crucial in understanding our bodies , health , and disease . educators can take advantage of the fact that the topic of alcohol interests high school students . in fact , high school students indicate that the topics of most interest to them in their science classes include drugs , disease , and the environment . there have been relatively few studies that assess how specific topics affect student achievement in chemistry and biology . one study that used the chemistry in the community curriculum ( chemcomm ) , which focuses on areas such as the environment , industry , food , and health , demonstrated increased achievement in a small group of high school chemistry students . in several larger studies , we have shown that curricula focused on pharmacology topics such as drugs of abuse can markedly improve high school student achievement in chemistry and biology . the very nature of pharmacology , which integrates basic principles of chemistry and biology to uncover the mechanisms by which drugs and chemicals affect organisms , lends itself to a useful approach for providing relevance and context especially for teenagers . in our previous studies , teachers used a series of modules in their classrooms as part of our program called the pharmacology education partnership ( pep ) . the pep studies provided the basis for the current study , which brings the subject of alcohol pharmacology to high school teaching . the teachers who participated in pep suggested that the subject of alcohol was of great interest to their students and thought that a similar program devoted to alcohol as a context for learning chemistry and biology would be well received . moreover , teachers mentioned that there is considerable misconception among their students about the chemistry and biology of alcohol . for example , many adolescents believe that drinking alcohol once an hour can avoid legal intoxication , yet a basic understanding of chemistry and biology can help to correct this misconception . in addition , teachers reported that students have misconceptions of basic chemical and biological principles as well , such as the meaning of equilibrium and the relationship between genes and proteins . thus , a program that can challenge prior knowledge and correct misconceptions about chemistry and biology that relate to a topic such as alcohol may help students construct meaning from their everyday life and better their learning in these sciences . effective professional development aims to improve teacher knowledge , teaching practices , and ultimately student achievement . as compiled by supovitz and turner , high - quality professional development should include at least the following:inquiry - based approaches and active learning.an intensive and sustained program.a mechanism to engage teachers in concrete teaching tasks that they can use in their own classrooms.a focus on subject - matter knowledge and content skills . we addressed each of these features in our three previous pep studies , which delivered professional development at ( i ) a five - day residential workshop , ( ii ) a one - day workshop at a national meeting or ( iii ) a six - hour workshop via distance learning ( two hours per week ) . regardless of the workshop format , teacher content knowledge in chemistry and biology increased and was maintained for at least a year . additionally , in each case , there was greater student achievement compared to students who did not have the pep modules in their classes . thus , our findings support research indicating that rich content , rather than the duration of professional development , is associated with student learning . for this study , we recruited a new set of teachers from the united states to participate in the alcohol pharmacology education partnership ( apep ) . we used the more cost - effective forms of professional development , a full - day workshop onsite at an annual conference and an interactive three - session workshop delivered by distance learning technology . each of the apep modules that we developed addresses several basic principles of chemistry and biology . as we demonstrated previously using pep , students scored higher when tested for knowledge of chemistry and biology concepts after using the apep modules than students who did not use the modules . high school chemistry and biology teachers were recruited from across the united states by placing announcements in the national science teachers association ( nsta ) newspaper , nsta reports . in brief , 50% of teachers were of biology or chemistry , 88% were from public schools , 30% were teaching in schools with at least a 40% population of minorities , and 67% were from urban or suburban schools . teachers selected to attend six hours of apep professional development at either a full - day conference - based workshop conducted concurrently with the nsta or north carolina science teachers association ( ncsta ) annual meetings or a series of distance learning ( dl ) workshops equal in length to the residential sessions . of the 156 participating teachers , 100 attended the conference - based workshops and 56 attended the dl workshops . teachers students ( n = 14 014 ) who participated in the study were from chemistry and biology classes in grades 912 representing schools from urban , suburban , and rural districts . during the first year of the study , all students served as the control group ( before teachers attended any apep professional development ) ; the following year , a second set of students served as the experimental group . at the start of the second year , teachers attended the professional development workshop and then field - tested the apep curriculum in their classrooms over the ensuing school year . the formats for both the conference - based and dl workshops were based on our previous pep studies . , cell structure and function , transport of molecules across membranes , protein synthesis ) were presented within the context of four apep modules developed by the authors ( see below ) to be used in field - testing in the teachers classes the following year . thus , during the workshops , teachers not only were able to review each of these concepts and learn how to incorporate them within the apep modules but also tackled one of the apep modules themselves , working in groups , just as their students would . teachers in the dl course also participated in the laboratory exercise during the live broadcast with materials provided to them ahead of time . the team of authors created four apep modules to be field - tested in classrooms during the study . the four modules integrate chemistry and biology concepts within the context of alcohol - related pharmacology topics that have relevance to high school students . each module contains the following elements:an overview containing a description of the module with the associated education standards.a set of learning objectives.a student handout that contains the story followed by a set of questions that students must answer ( similar to a problem in problem - based learning).the content the bulk of the module that contains all the answers to the student questions , including animations.a student quiz with 46 questions about the chemistry and biology concepts relevant to the module ( and answers provided).activities ( integrating math calculations or lab activities with the module content).resources ( a list of references and url links ) . a student handout that contains the story followed by a set of questions that students must answer ( similar to a problem in problem - based learning ) . a student quiz with 46 questions about the chemistry and biology concepts relevant to the module ( and answers provided ) . these include:biology and chemistry connections a table showing the specific chemistry and biology concepts that are covered in each module and links to a review of specific chemistry and biology concepts normally covered in high school courses.explore more , a set of pages ( also linked within the modules ) that cover topics related to the modules such as alcoholism and neuroscience , and technologies such as magnetic resonance imaging .. a glossary.list of the next generation science standards and former national science education standards . a table showing the specific chemistry and biology concepts that are covered in each module and links to a review of specific chemistry and biology concepts normally covered in high school courses . explore more , a set of pages ( also linked within the modules ) that cover topics related to the modules such as alcoholism and neuroscience , and technologies such as magnetic resonance imaging .. list of the next generation science standards and former national science education standards . these include misconceptions such as ( i ) drinking one drink an hour will not get you drunk , ( ii ) you can get rid of alcohol by urinating it out , and ( iii ) beer and wine are less intoxicating than liquor . it is up to the teacher as to whether the students can use web - based literature to find the answers , or use the apep module content pages to find the answers . the general structure of the apep modules is shown in figure 1 and the story for module 4 is provided in the supporting information . the apep modules may be accessed online ( both a teacher and student version ) . teachers were instructed to field test the four apep modules in their classrooms over the year . we did not prescribe a specific approach for teachers to use the apep modules ; instead , we asked teachers to report how they incorporated the content into their courses . previously , we showed that the more pep modules used , the greater the beneficial effect on student scores in both basic and advanced science knowledge . to determine the effectiveness of the workshop on teachers knowledge gain and retention after one year , we administered a short test consisting of 20 multiple - choice questions that addressed the chemistry and biology content listed in table 2 . the test was administered to the teachers at the beginning of the workshop ( pretest ) , at the end of the workshop ( post - test ) , and again at the end of the year ( one - year follow - up ) without prior notification . at the end of the school year after field - testing the apep modules , we sent the teachers a multiple - choice content test similar to that used in our previous pep studies to give to their students ; the tests were unannounced . the tests were constructed by the authors , with input from high school chemistry and biology teachers at the north carolina school of science and math , where one of us ( m.j.h . ) the multiple choice questions assessed student knowledge of concepts in chemistry and biology according to the framework provided by the 1996 national assessment of educational progress science test . following the 20 basic knowledge questions , there were 10 questions that were specific for the new knowledge about alcohol in the context of chemistry and biology ( advanced knowledge ) . to establish reliability of the assessment instrument , we conducted a test retest with 58 high school students in chemistry and biology classes ( unrelated to the study ) . to establish validity of the assessment instrument , we asked a team of nine high school chemistry and biology teachers ( not related to any of the authors ) to rate each of the questions as to whether it was relevant and appropriate to their courses . , chemistry or biology ) , and course level ( i.e. we compiled the percent correct scores of the 14 014 students on both the basic and advanced tests . to estimate the effects of the modules , we used logistic regression models with random effects for the teachers ( which is a type of multilevel model ) , adjusting for demographic characteristics ( see table 1 ) , and number of modules as a series of indicator variables . the outcome variables are the number of correct answers out of 20 questions on the basic test and the number of correct answers out of 10 questions on the advanced test . to assess the effect of providing the pharmacology - based professional development workshop on teacher knowledge of basic chemistry and biology concepts , teachers were assessed for content knowledge at the beginning of the workshop ( pretest ) , at the end of the workshop ( post - test ) , and one year following the workshop ( one - year follow - up ) . comparisons between the pre- and post - tests revealed that average scores on both the post - test and the one - year follow - up test were significantly higher than the pretest averages ( tukey s multiple comparison test ) . there was no difference between the scores in the post - test and the one - year follow - up test for both workshops , indicating persistence of the knowledge gain . their average gain in scores on the biology questions over the year was considerably larger than their gain in scores on the chemistry questions ( mean gain in scores sd was 31 32 versus 11 26 percentage points , respectively ( n = 31 ) ; p < 0.05 , paired student s t test ) . although there was no set prescription for implementing the modules , we did collect information on how the teachers used the modules in their classes ( table 4 ) . the results revealed that the use of the apep modules was a significant predictor of greater student achievement on both the basic and advanced knowledge tests , as shown in figure 2 and in the supporting information . for example , a typical student in a class that used all four modules had 1.21 times the odds of answering basic knowledge questions correctly compared to a typical student in a class that used no modules . student basic and advanced knowledge scores were higher as more modules were used in their classroom . use of modules was a significant predictor of higher scores ( logistic regression ; n = 14 014 students ; see the supporting information , tables s4 and s5 ) . however , the higher chemistry student scores on biology and chemistry questions remained relatively flat when one , two , and three modules were used compared to no modules . thus , the steady increase in scores from biology students when using one , two , and three modules may have accounted for most of the higher scores as reported in the overall analysis ( figure 2 ) . interestingly , students in biology class whose teachers used three or four apep modules achieved chemistry scores similar to the baseline scores of students in chemistry ( see table 5 ) . use of modules was a significant predictor of higher scores ( logistic regression ; n = 14 014 students ) . students in biology class whose teachers used three or four apep modules achieved chemistry scores similar to the baseline scores of students in chemistry . in this study , we demonstrate that implementing pharmacology - based science education ( using alcohol as the context ) in high school classrooms increases student and teacher achievement in chemistry and biology . in the apep study , all of the modules focused on one drug ( alcohol ) , so it is possible that limiting the subject matter may have contributed somewhat to a smaller effect compared to our previous studies that incorporated a variety of drug topics . the format of the apep curriculum is similar to the pep curriculum , so it would be easy to combine the two , thereby increasing the number of modules ( to 10 ) that could be used to teach chemistry and biology concepts . the integration of chemistry , biology , and math concepts in apep addresses several components of the new ngss , including scientific practices and the integration of disciplinary core ideas . we surmise that integration of chemistry and biology may have been successful in our study because students in biology , who typically take chemistry a year after biology , were able to increase their chemistry scores to the same level as that achieved by control chemistry students just by using at least three apep modules . the apep curriculum has several features that may have contributed to the positive outcome , including the following:the relevance of the various alcohol - focused stories ( and the content ) to the high school population.an inquiry - based approach to learn the content.content that addresses myths and misconceptions about alcohol.repetition of core concepts among the modules . as mentioned above , we have shown in a previous pep study that use of six modules predicts even higher scores in chemistry and biology than reported here , supporting the importance of dose . in terms of professional development , both on - site conference - based workshops and the distance learning workshops helped teachers improve their knowledge in chemistry and biology and maintain that improvement for at least a year . although the results presented here replicate what we ve found in our previous studies , there are several limitations . first , although teachers used the modules in a variety of ways , we do not know which of the implementation methods was a better predictor for the stepwise increase in scores . because several teachers used multiple implementation methods in their classes at different times , it was not realistic to determine whether any method was more effective than another . however , presenting the module material over several classes was the most prevalent implementation format ( 50% of classes used this format ) , so it is reasonable to conclude that at least this particular method may have contributed significantly to the increasing scores . in contrast , only 6% of classes worked on the apep modules online , so it is unlikely that this method alone contributed significantly to the positive findings . third , the improvements in the scores associated with module usage may not be caused by the modules themselves ; other variables that we did not control could explain ( at least in part ) the higher scores . for example , suppose that a large majority of the students who used the apep modules have higher academic motivation and abilities than those who did not use the modules , and this difference , rather than beneficial effects of learning with the modules , accounts for their higher average test scores . however , it is unlikely that a systematic disparity in student abilities would be present because the disparities would also need to be present in a dose - response manner ( as were the higher scores ) , and classes of the same type taught by the same teacher were likely to be similar in terms of prematriculation abilities . , alcohol and drugs ) score better in chemistry and biology compared to students using the standard curriculum . the implementation of controlled research designs can provide teachers with meaningful information about curricular materials and teaching strategies most likely to help their students learn chemistry and biology better . the apep modules can be accessed online free of charge for interactive use or downloaded as pdfs directly from our web site .

dipeptidylpeptidase 4 ( dpp4 ) is a ubiquitously expressed cell surface protease that cleaves dipeptides from the amino terminus of peptides containing a penultimate alanine or proline ; soluble dpp4 resulting from proteolytic cleavage of the membrane form is also present in the circulation . the first selective dpp4 inhibitor , sitagliptin , was approved by the us food and drug administration in 2006 for the management of hyperglycemia in patients with type 2 diabetes mellitus . dpp4 inhibition decreases the degradation of endogenous incretin hormones , including glucagonlike peptide1 ( glp1 ) and thereby augments nutrientstimulated insulin release , suppresses glucagon secretion , and slows gastric emptying . the widespread expression of dpp4 within the vasculature and its numerous vasoactive hormone substrates also raise the possibility that dpp4 could affect vascular function . glp1 and brain natriuretic peptide ( bnp ) represent 2 vasoactive peptide hormone substrates of dpp4 . in rodent models , glp1 activates the glp1 receptor to produce mild vasodilation , inotropic action , and ischemic preconditioning . at the same time , central and peripheral administration of glp1 receptor agonists in a rat model increases blood pressure and heart rate by activating autonomic regulatory neurons . dpp4 cleaves glp1 to its metabolite glp1(936 ) , which promotes endotheliumdependent vasodilation independent of the glp1 receptor . consequently , inhibition of dpp4 may potentiate the effects of glp1 but also could also decrease the favorable effects of its metabolite glp1(936 ) . bnp(132 ) is produced by the ventricular cardiomyocytes in response to increased filling pressures and promotes vasodilation and natriuresis . bnp(132 ) is rapidly cleaved to bnp(332 ) by dpp4 in human plasma , and this is prevented by addition of the dpp4 inhibitor vildagliptin . the metabolite bnp(332 ) causes less vasodilation and natriuresis than its intact precursor in a dog model . adults with diabetes are nearly two times more likely to die from heart disease than adults without diabetes , and over twothirds of adults with diabetes have hypertension or are prescribed antihypertensive therapies . two recent placebocontrolled clinical trials demonstrated no beneficial effect of the dpp4 inhibitors alogliptin or saxagliptin on cardiovascular events , although saxagliptin use was associated with an increased risk of hospitalization for heart failure . consequently , it is important to understand the cardiovascular effects of this class of antidiabetic medications at a mechanistic level . this study tested the hypothesis that dpp4 inhibition potentiates the vasodilator responses to glp1 and bnp in the human forearm vasculature . seventeen healthy , nonobese ( body mass index < 30 kg / m ) , nonsmoking adults participated in a doubleblind , randomized , placebocontrolled , crossover study ( figure 1 ; subject characteristics are shown in table 1 ) . the study adhered to the principles of the declaration of helsinki and title 45 , us code of federal regulations , part 46 , protection of human subjects , following approval by the vanderbilt university institutional review board , and all subjects provided written informed consent prior to initiation of study procedures . patients with a history of chronic illness , including diabetes , hypertension , cardiovascular disease , and chronic renal or hepatic insufficiency , were excluded from participation . intraarterial glp1 was first infused in 3 graded doses , lasting 5 minutes each , followed by bnp . forearm blood flow measurement , followed by arterial and venous sampling , was performed at baseline and at completion of each dose of peptide . bnp indicates brain natriuretic peptide ; glp1 , glucagonlike peptide1 . each subject was studied on 2 days at least 1 week apart . subjects were assigned to treatment order ( sitagliptin or matching placebo ) using a block randomization algorithm provided by the study biostatistician . randomization was stratified by race and sex . on each study day , subjects reported to the vanderbilt clinical research center in the morning after an overnight fast . subjects were given an oral study drug ( sitagliptin 200 mg or matching placebo ) , and an arterial line was placed in the brachial artery of the nondominant arm with an adjacent peripheral intravenous line . baseline forearm blood flow ( fbf ) measurements and blood sampling were obtained 60 minutes following ingestion of the study drug and at least 30 minutes after insertion of the arterial catheter . subjects then received sequential intraarterial infusions of glp1 followed by bnp ; a 30minute washout separated each infusion . we observed a carryover effect on fbf when bnp was infused prior to glp1 in the first subject , despite the intervening washout . the affected data were excluded from the analyses , and glp1 was infused first on all subsequent study days . fbf was assessed during the last 2 minutes of each dose , and arterial and venous blood samples were then drawn simultaneously . on the second study day , the protocol was repeated using the opposite study drug ( sitagliptin or matching placebo ) . after subdermal administration of 1% lidocaine , a size 3f catheter ( cook inc ) was inserted into the brachial artery of the nondominant arm for direct intraarterial administration of peptides and arterial blood sampling . arterial catheter patency was maintained by infusion of intravenous fluid ( 0.9% sodium chloride solution ) at a rate of 1 ml / min . glp1 ( clinalfa basic ) was infused at 0.45 , 0.90 , and 1.80 pmol / min in the first 3 randomized subjects ; however , we did not see any effect of glp1 on vasodilation at these doses . glp1 infusions were increased to 0.90 , 1.80 , and 3.60 pmol / min in 11 additional subjects . after we still saw no effect of glp1 , we discontinued glp1 in the remaining 3 subjects . bnp ( nesiritide ; scios inc ) was infused at 480 , 720 , and 1440 pmol / min . drug concentrations in the infusate were adjusted to maintain an infusion volume of 1 ml / min throughout the study . the wrist was supported in a sling to raise the level of the forearm above the level of the atrium , and a strain gauge was placed around the widest part of the forearm of the nondominant hand . for each measurement , a cuff placed around the upper arm was inflated to 45 mm hg with a rapid cuff inflator ( model e20 rapid cuff inflator and ag 101 cuff inflator air source ; d.e . the hand was excluded from the measurement of blood flow by inflation of a pediatric sphygmomanometer cuff to 200 mm hg around the wrist . flow measurements were recorded for 7 seconds , and a minimum of 6 readings were analyzed using a noninvasive vascular software program ( d.e . forearm vascular resistance was calculated as mean arterial pressure ( map ) divided by fbf . arteriovenous concentration gradients were calculated by subtracting the plasma level measured in simultaneously collected venous and arterial blood . forearm plasma flow was calculated from the fbf , and hematocrit was corrected for 1% trapped plasma . net release was calculated at each time point : simultaneous arterial and venous samples were obtained from the infused arm at baseline and at completion of each dose of infused peptide . blood samples were collected on ice and centrifuged immediately , and plasma was stored at 80c in prespecified aliquots until time of assay . venous dpp4 activity was assayed by incubating sera with a colorimetric substrate , lglycyllprolyl pnitroanilide , hydrochloride ( sigma ) , at 37c . blood for measurement of venous glp1 levels was collected in tubes containing edta and protease inhibitor ( aprotinin ; roche diagnostics ) . glp1 levels were determined using a multiplex magnetic bead assay ( milliplex map human metabolic hormone magnetic bead panel ; emd millipore ) that detects active glp1(736 ) in the range of 4 to 3033 pmol / l with no crossreactivity for glp1(936 ) . arterial and venous blood for catecholamine measurement was collected in prechilled tubes containing sodium heparin . potential carryover and period effects were tested for by comparing the measures of fbf obtained prior to each infusion . wilcoxon signed rank test was used to compare baseline variables as well as venous glp1 levels . mixedeffect models were used to analyze the data with a random subject effect and with fixed effects of treatment ( placebo or sitagliptin ) , peptide dose , and their interaction . the mixedeffect model with a random subject effect allowed the inclusion of subjects with missing data at some time points . for inferences of interest , statistical analyses were performed using ibm spss software v. 21.0 , graphpad prism 5 , and r 2.15.0 ( www.r-project.org ) . seventeen healthy , nonobese ( body mass index < 30 kg / m ) , nonsmoking adults participated in a doubleblind , randomized , placebocontrolled , crossover study ( figure 1 ; subject characteristics are shown in table 1 ) . the study adhered to the principles of the declaration of helsinki and title 45 , us code of federal regulations , part 46 , protection of human subjects , following approval by the vanderbilt university institutional review board , and all subjects provided written informed consent prior to initiation of study procedures . patients with a history of chronic illness , including diabetes , hypertension , cardiovascular disease , and chronic renal or hepatic insufficiency , were excluded from participation . intraarterial glp1 was first infused in 3 graded doses , lasting 5 minutes each , followed by bnp . forearm blood flow measurement , followed by arterial and venous sampling , was performed at baseline and at completion of each dose of peptide . bnp indicates brain natriuretic peptide ; glp1 , glucagonlike peptide1 . each subject was studied on 2 days at least 1 week apart . subjects were assigned to treatment order ( sitagliptin or matching placebo ) using a block randomization algorithm provided by the study biostatistician . randomization was stratified by race and sex . on each study day , subjects reported to the vanderbilt clinical research center in the morning after an overnight fast . subjects were given an oral study drug ( sitagliptin 200 mg or matching placebo ) , and an arterial line was placed in the brachial artery of the nondominant arm with an adjacent peripheral intravenous line . baseline forearm blood flow ( fbf ) measurements and blood sampling were obtained 60 minutes following ingestion of the study drug and at least 30 minutes after insertion of the arterial catheter . subjects then received sequential intraarterial infusions of glp1 followed by bnp ; a 30minute washout separated each infusion . we observed a carryover effect on fbf when bnp was infused prior to glp1 in the first subject , despite the intervening washout . the affected data were excluded from the analyses , and glp1 was infused first on all subsequent study days . fbf was assessed during the last 2 minutes of each dose , and arterial and venous blood samples were then drawn simultaneously . on the second study day , the protocol was repeated using the opposite study drug ( sitagliptin or matching placebo ) . after subdermal administration of 1% lidocaine , a size 3f catheter ( cook inc ) was inserted into the brachial artery of the nondominant arm for direct intraarterial administration of peptides and arterial blood sampling . arterial catheter patency was maintained by infusion of intravenous fluid ( 0.9% sodium chloride solution ) at a rate of 1 ml / min . glp1 ( clinalfa basic ) was infused at 0.45 , 0.90 , and 1.80 pmol / min in the first 3 randomized subjects ; however , we did not see any effect of glp1 on vasodilation at these doses . glp1 infusions were increased to 0.90 , 1.80 , and 3.60 pmol / min in 11 additional subjects . after we still saw no effect of glp1 , we discontinued glp1 in the remaining 3 subjects . bnp ( nesiritide ; scios inc ) was infused at 480 , 720 , and 1440 pmol / min . drug concentrations in the infusate were adjusted to maintain an infusion volume of 1 ml / min throughout the study . the wrist was supported in a sling to raise the level of the forearm above the level of the atrium , and a strain gauge was placed around the widest part of the forearm of the nondominant hand . for each measurement , a cuff placed around the upper arm was inflated to 45 mm hg with a rapid cuff inflator ( model e20 rapid cuff inflator and ag 101 cuff inflator air source ; d.e . the hand was excluded from the measurement of blood flow by inflation of a pediatric sphygmomanometer cuff to 200 mm hg around the wrist . flow measurements were recorded for 7 seconds , and a minimum of 6 readings were analyzed using a noninvasive vascular software program ( d.e . forearm vascular resistance was calculated as mean arterial pressure ( map ) divided by fbf . arteriovenous concentration gradients were calculated by subtracting the plasma level measured in simultaneously collected venous and arterial blood . forearm plasma flow was calculated from the fbf , and hematocrit was corrected for 1% trapped plasma . simultaneous arterial and venous samples were obtained from the infused arm at baseline and at completion of each dose of infused peptide . all samples blood samples were collected on ice and centrifuged immediately , and plasma was stored at 80c in prespecified aliquots until time of assay . venous dpp4 activity was assayed by incubating sera with a colorimetric substrate , lglycyllprolyl pnitroanilide , hydrochloride ( sigma ) , at 37c . blood for measurement of venous glp1 levels was collected in tubes containing edta and protease inhibitor ( aprotinin ; roche diagnostics ) . glp1 levels were determined using a multiplex magnetic bead assay ( milliplex map human metabolic hormone magnetic bead panel ; emd millipore ) that detects active glp1(736 ) in the range of 4 to 3033 pmol / l with no crossreactivity for glp1(936 ) . arterial and venous blood for catecholamine measurement was collected in prechilled tubes containing sodium heparin . potential carryover and period effects were tested for by comparing the measures of fbf obtained prior to each infusion . wilcoxon signed rank test was used to compare baseline variables as well as venous glp1 levels . mixedeffect models were used to analyze the data with a random subject effect and with fixed effects of treatment ( placebo or sitagliptin ) , peptide dose , and their interaction . the mixedeffect model with a random subject effect allowed the inclusion of subjects with missing data at some time points . for inferences of interest , statistical analyses were performed using ibm spss software v. 21.0 , graphpad prism 5 , and r 2.15.0 ( www.r-project.org ) . dpp4 inhibition with sitagliptin decreased dpp4 activity by 64% ( p=0.002 ) , whereas dpp4 antigen was unchanged ( table 2 ) . dpp4 inhibition increased systolic blood pressure by 5% ( p=0.03 ) but had no effect on diastolic blood pressure ( p=0.70 ) , map ( p=0.21 ) , or heart rate ( p=0.92 ) prior to intraarterial peptide infusions . dpp4 inhibition increased fbf by 30% ( p=0.02 ) and decreased forearm vascular resistance by 18% ( p=0.03 ) prior to intraarterial peptide infusions . bpm indicates beats per minute ; dpp4 , dipeptidyl peptidase 4 ; fbf , forearm blood flow ; fvr , forearm vascular resistance . intraarterial infusion of glp1 significantly increased venous glp1 concentrations from baseline during both placebo ( p=0.01 ) and sitagliptin ( p=0.01 ) . venous glp1 levels were significantly higher during sitagliptin ( p=0.04 versus placebo at highest dose of glp1 ) and exceeded postprandial levels normally found in healthy adults ( figure 2 ) . effect of dpp4 inhibition on venous glp1 levels at baseline and during the maximum dose of glp1 . * p<0.05 vs baseline during same treatment . p<0.05 vs placebo at same peptide dose . vasodilator response is presented as fbf , because local intraarterial infusion of peptide did not significantly affect map in either treatment group , and as a percent change , given that baseline fbf differed between treatment with sitagliptin and placebo ( figure 3 ) . intraarterial infusion of glp1 did not increase fbf , even when its concentrations were increased by sitagliptin . effect of dpp4 inhibition on forearm blood flow response to intraarterial glp1 ( 14 subjects ) and to bnp ( 17 subjects ) . as noted in the methods , the first 3 subjects received lower doses of glp1 . bnp indicates brain natriuretic peptide ; dpp4 , dipeptidylpeptidase 4 ; glp1 , glucagonlike peptide1 . intraarterial infusion of bnp increased fbf in a dosedependent manner ( p<0.001 effect of dose ) ; however , treatment with sitagliptin did not affect this vasodilator response . intraarterial infusion of glp1 did not significantly affect heart rate , map , norepinephrine levels , or net vascular norepinephrine release during placebo or sitagliptin treatment ( data not shown ) . intraarterial infusion of bnp infusion increased heart rate in a dosedependent manner ( p=0.01 effect of dose ) ; treatment with sitagliptin did not affect this response . intraarterial infusion of bnp increased arterial norepinephrine levels only during sitagliptin ( p<0.001 effect of dose ) . there was no effect of intraarterial infusion of bnp on map , venous norepinephrine levels , or net norepinephrine release . three subjects did not complete the second study day due to inability to obtain adequate arterial access . one subject experienced a syncopal episode 1 hour after completion of his first study visit . he was found to be orthostatic , was given intravenous fluids , and was withdrawn from the study . other adverse events included transient lightheadedness and nausea , which resolved with increased oral fluid intake and rest ( 3 subjects ) , and neuropraxia in the instrumented arm , which resolved over a period of 2 weeks without therapy ( 1 subject ) . dpp4 inhibition with sitagliptin decreased dpp4 activity by 64% ( p=0.002 ) , whereas dpp4 antigen was unchanged ( table 2 ) . dpp4 inhibition increased systolic blood pressure by 5% ( p=0.03 ) but had no effect on diastolic blood pressure ( p=0.70 ) , map ( p=0.21 ) , or heart rate ( p=0.92 ) prior to intraarterial peptide infusions . dpp4 inhibition increased fbf by 30% ( p=0.02 ) and decreased forearm vascular resistance by 18% ( p=0.03 ) prior to intraarterial peptide infusions . bpm indicates beats per minute ; dpp4 , dipeptidyl peptidase 4 ; fbf , forearm blood flow ; fvr , forearm vascular resistance . intraarterial infusion of glp1 significantly increased venous glp1 concentrations from baseline during both placebo ( p=0.01 ) and sitagliptin ( p=0.01 ) . venous glp1 levels were significantly higher during sitagliptin ( p=0.04 versus placebo at highest dose of glp1 ) and exceeded postprandial levels normally found in healthy adults ( figure 2 ) . effect of dpp4 inhibition on venous glp1 levels at baseline and during the maximum dose of glp1 . * p<0.05 vs baseline during same treatment . p<0.05 vs placebo at same peptide dose . vasodilator response is presented as fbf , because local intraarterial infusion of peptide did not significantly affect map in either treatment group , and as a percent change , given that baseline fbf differed between treatment with sitagliptin and placebo ( figure 3 ) . intraarterial infusion of glp1 did not increase fbf , even when its concentrations were increased by sitagliptin . effect of dpp4 inhibition on forearm blood flow response to intraarterial glp1 ( 14 subjects ) and to bnp ( 17 subjects ) . as noted in the methods , the first 3 subjects received lower doses of glp1 . bnp indicates brain natriuretic peptide ; dpp4 , dipeptidylpeptidase 4 ; glp1 , glucagonlike peptide1 . intraarterial infusion of bnp increased fbf in a dosedependent manner ( p<0.001 effect of dose ) ; however , treatment with sitagliptin did not affect this vasodilator response . intraarterial infusion of glp1 did not significantly affect heart rate , map , norepinephrine levels , or net vascular norepinephrine release during placebo or sitagliptin treatment ( data not shown ) . intraarterial infusion of bnp infusion increased heart rate in a dosedependent manner ( p=0.01 effect of dose ) ; treatment with sitagliptin did not affect this response . intraarterial infusion of bnp increased arterial norepinephrine levels only during sitagliptin ( p<0.001 effect of dose ) . there was no effect of intraarterial infusion of bnp on map , venous norepinephrine levels , or net norepinephrine release . three subjects did not complete the second study day due to inability to obtain adequate arterial access . one subject experienced a syncopal episode 1 hour after completion of his first study visit . he was found to be orthostatic , was given intravenous fluids , and was withdrawn from the study . other adverse events included transient lightheadedness and nausea , which resolved with increased oral fluid intake and rest ( 3 subjects ) , and neuropraxia in the instrumented arm , which resolved over a period of 2 weeks without therapy ( 1 subject ) . this study tested the hypothesis that dpp4 inhibition potentiates the vasodilator responses to glp1 and bnp in the human forearm . we found that glp1 does not cause vasodilation in the forearm vasculature of healthy humans , even when its degradation is inhibited by sitagliptin and high concentrations are achieved . although several prior studies have examined the effect of intravenous glp1 on endothelial function , our study is unique in examining the direct vascular effect of intraarterial glp1 while blocking its degradation by dpp4 . specifically , 2 prior studies examining the effect of intravenous glp1 on endothelial function during hyperglycemic clamp suggested that glp1 improves endothelial function , as measured by flowmediated dilation during hyperglycemia in diabetic subjects but not during normoglycemia . in contrast , basu et al reported that intravenous glp1 enhanced the forearm vasodilator response to intraarterial acetylcholine but not to nitroprusside in healthy subjects . because systemic administration of glp1 increases insulin , we infused glp1 directly in the brachial artery . tesauro et al also assessed the effect of intraarterial glp1 and reported that glp1 enhanced the fbf response to acetylcholine and nitroprusside in patients with metabolic syndrome during coinfusion of insulin but not during saline . in contrast to our study , the investigators did not inhibit the degradation of glp1 by dpp4 and did not achieve concentrations of glp1 comparable to physiological concentrations achieved after a meal . these data in humans conflict with data in rodent models , which indicate that glp1 causes direct vasodilation . the lack of effect of dpp4 inhibition by sitagliptin on the vascular response to glp1 is particularly important because ban et al reported that both glp1 and its dpp4 metabolite glp1(936 ) dilate preconstricted mesenteric arteries through a glp1 receptor independent and nitric oxide synthase in contrast , tesauro et al reported no effect of intraarterial glp1(936 ) in the human forearm . likewise , if endogenous glp1(936 ) causes vasodilation in humans , we would have expected to observe an increase in forearm vascular resistance during dpp4 inhibition , but instead we observed a decrease in baseline forearm vascular resistance . activation of the glp1 receptor in the brain has also been shown to modulate sympathetic activity in animal models and humans . yamamoto et al demonstrated that systemic administration of glp1 receptor agonist increased blood pressure and heart rate in a dosedependent fashion and activated autonomic neurons responsible for sympathetic outflow in rats . bharucha et al found that intravenous infusion of glp1 in healthy subjects increased skeletal muscle sympathetic nerve activity but did not affect cardiac sympathetic indices , as assessed by spectral analysis . vasodilation in response to glp1 may be compromised by increased sympathetic activity ; however , we administered glp1 intraarterially , thereby avoiding systemic counterregulatory responses , and did not see any effect of glp1 on norepinephrine release . elevated bnp levels are characteristic of heart failure , and recombinant bnp reduces afterload and promotes natriuresis . dpp4 activity is increased in the setting of heart failure and thus may play a role in the pathophysiology of heart failure by increasing degradation of bnp to bnp(332 ) , which is a less potent vasodilator and natriuretic agent in dogs . we did not find that sitagliptin potentiated the vasodilatory response to bnp in the human forearm . we can not exclude minimal potentiation of the effects of bnp because intraarterial infusion of bnp tended to reduce map from baseline during sitagliptin treatment only and did increase arterial norepinephrine levels . it is possible that there was mild systemic vasodilation and activation of the baroreflex following bnp infusion during dpp4 inhibition , but the lack of effect of sitagliptin on the heart rate response to bnp infusion does not support this . chan et al reported that bnp promotes norepinephrine release in an ex vivo rodent model and proposed that this may account for the inability of recombinant bnp to reduce the rate of hospitalization and death from heart failure . we did not find an effect of intraarterial bnp on venous norepinephrine levels or net norepinephrine release in the presence or absence of sitagliptin . we observed an increase in basal systolic blood pressure with an accompanying increase in fbf and decrease in forearm vascular resistance during sitagliptin , consistent with our previous observation in healthy subjects . although we did not observe a significant increase in baseline heart rate or net norepinephrine release during sitagliptin in the present study , we have previously reported that dpp4 inhibition increases sympathetic activation by substance p. as noted , an effect of dpp4 inhibition on the degradation of glp1 in the central nervous system could have contributed to this observed increase in systolic blood pressure . a few study design considerations we used an acute dose of 200 mg of sitagliptin because this dose inhibits ddp4 inhibition to the same extent as the clinically approved dose of 100 mg but within a shorter time period . we can not exclude an effect of chronic dpp4 inhibition on vascular responses to glp1 and bnp or on endothelial function . we studied a small sample size due to the rigorous nature of the protocol ; this may have limited our power to detect an effect of sitagliptin on the vasodilator responses . we also studied healthy individuals in the fasting state to control for the fluctuations in other vasoactive hormones , including glp1 and insulin , and to control for medications and diseases that may affect endothelial function . it is possible that our findings would have been different in diseased subjects , but prior studies do not support this , as discussed earlier . we focused on vasodilator responses and can not exclude an effect of sitagliptin on the natriuretic response to bnp . we used an acute dose of 200 mg of sitagliptin because this dose inhibits ddp4 inhibition to the same extent as the clinically approved dose of 100 mg but within a shorter time period . we can not exclude an effect of chronic dpp4 inhibition on vascular responses to glp1 and bnp or on endothelial function . we studied a small sample size due to the rigorous nature of the protocol ; this may have limited our power to detect an effect of sitagliptin on the vasodilator responses . we also studied healthy individuals in the fasting state to control for the fluctuations in other vasoactive hormones , including glp1 and insulin , and to control for medications and diseases that may affect endothelial function . it is possible that our findings would have been different in diseased subjects , but prior studies do not support this , as discussed earlier . we focused on vasodilator responses and can not exclude an effect of sitagliptin on the natriuretic response to bnp . the recent saxagliptin assessment of vascular outcomes recorded in patients with diabetes mellitus ( savor)thrombolysis in myocardial infarction ( timi ) 53 trial revealed an increased risk for hospitalization due to heart failure with the dpp4 inhibitor saxagliptin . dpp4 inhibitor therapies may influence cardiovascular risk by affecting the degradation of peptide substrates that influence vascular function and sympathetic activity . we previously reported that the dpp4 substrate substance p increases sympathetic activity when dpp4 inhibition is combined with angiotensinconverting enzyme inhibition . in the present study , we investigated the impact of dpp4 inhibition on the vascular effects of glp1 and bnp . our data do not support the hypothesis that decreased degradation of glp1 and bnp during acute dpp4 inhibition directly regulates vascular function or sympathetic activity in healthy humans , despite data to the contrary in animal models . other mechanisms that may account for the increased risk of hospitalization for heart failure during chronic dpp4 inhibitor therapy should be explored . we thank stacy gilbert , rn ; delia woods , rn ; and carol meisch , rn , for their recruitment of volunteers and nursing assistance . we thank anthony dematteo , zuofei wang , the vanderbilt hormone assay core , and the vanderbilt clinical research center core laboratory for their technical assistance .

backgrounddipeptidylpeptidase 4 ( dpp4 ) inhibitors improve glycemic control in patients with diabetes mellitus by preventing the degradation of glucagonlike peptide1 ( glp1 ) . glp1 causes vasodilation in animal models but also increases sympathetic activity ; the effect of glp1 in the human vasculature and how it is altered by dpp4 inhibition is not known . dpp4 also degrades the vasodilator brain natriuretic peptide ( bnp ) to a less potent metabolite . this study tested the hypothesis that dpp4 inhibition potentiates the vasodilator responses to glp1 and bnp in the human forearm.method and resultsseventeen healthy subjects participated in this randomized , doubleblinded , placebocontrolled crossover study . on each study day , subjects received dpp4 inhibitor ( sitagliptin 200 mg by mouth ) or placebo . sitagliptin increased forearm blood flow and decreased forearm vascular resistance without affecting mean arterial pressure and pulse . glp1 and bnp were infused in incremental doses via brachial artery . venous glp1 concentrations were significantly higher during sitagliptin use , yet there was no effect of glp1 on forearm blood flow in the presence or absence of sitagliptin . bnp caused dosedependent vasodilation ; however , sitagliptin did not affect this response . glp1 and bnp had no effect on net norepinephrine release.conclusionsthese data suggest that glp1 does not act as a direct vasodilator in humans and does not contribute to sympathetic activation . sitagliptin does not regulate vascular function in healthy humans by affecting the degradation of glp1 and bnp.clinical trial registrationurl : www.clinicaltrials.gov/ unique identifier : nct01413542 .

Clinical Trial Registration Introduction Methods Study Protocol Insertion of Arterial Line and Intraarterial Administration of Study Drugs Forearm Blood Flow Measurements Laboratory Analyses Statistical Analysis Results Effect of Treatment on DPP4 Activity and Initial Hemodynamic Parameters Effect of DPP4 Inhibition on GLP1 Concentrations and Forearm Blood Flow Effect of DPP4 Inhibition on Mean Arterial Pressure, Heart Rate, and Norepinephrine Levels Safety Discussion Limitations Conclusions Acknowledgments

dipeptidylpeptidase 4 ( dpp4 ) is a ubiquitously expressed cell surface protease that cleaves dipeptides from the amino terminus of peptides containing a penultimate alanine or proline ; soluble dpp4 resulting from proteolytic cleavage of the membrane form is also present in the circulation . the first selective dpp4 inhibitor , sitagliptin , was approved by the us food and drug administration in 2006 for the management of hyperglycemia in patients with type 2 diabetes mellitus . dpp4 inhibition decreases the degradation of endogenous incretin hormones , including glucagonlike peptide1 ( glp1 ) and thereby augments nutrientstimulated insulin release , suppresses glucagon secretion , and slows gastric emptying . the widespread expression of dpp4 within the vasculature and its numerous vasoactive hormone substrates also raise the possibility that dpp4 could affect vascular function . glp1 and brain natriuretic peptide ( bnp ) represent 2 vasoactive peptide hormone substrates of dpp4 . at the same time , central and peripheral administration of glp1 receptor agonists in a rat model increases blood pressure and heart rate by activating autonomic regulatory neurons . consequently , inhibition of dpp4 may potentiate the effects of glp1 but also could also decrease the favorable effects of its metabolite glp1(936 ) . bnp(132 ) is rapidly cleaved to bnp(332 ) by dpp4 in human plasma , and this is prevented by addition of the dpp4 inhibitor vildagliptin . adults with diabetes are nearly two times more likely to die from heart disease than adults without diabetes , and over twothirds of adults with diabetes have hypertension or are prescribed antihypertensive therapies . two recent placebocontrolled clinical trials demonstrated no beneficial effect of the dpp4 inhibitors alogliptin or saxagliptin on cardiovascular events , although saxagliptin use was associated with an increased risk of hospitalization for heart failure . this study tested the hypothesis that dpp4 inhibition potentiates the vasodilator responses to glp1 and bnp in the human forearm vasculature . seventeen healthy , nonobese ( body mass index < 30 kg / m ) , nonsmoking adults participated in a doubleblind , randomized , placebocontrolled , crossover study ( figure 1 ; subject characteristics are shown in table 1 ) . forearm blood flow measurement , followed by arterial and venous sampling , was performed at baseline and at completion of each dose of peptide . bnp indicates brain natriuretic peptide ; glp1 , glucagonlike peptide1 . on each study day , subjects reported to the vanderbilt clinical research center in the morning after an overnight fast . subjects were given an oral study drug ( sitagliptin 200 mg or matching placebo ) , and an arterial line was placed in the brachial artery of the nondominant arm with an adjacent peripheral intravenous line . baseline forearm blood flow ( fbf ) measurements and blood sampling were obtained 60 minutes following ingestion of the study drug and at least 30 minutes after insertion of the arterial catheter . we observed a carryover effect on fbf when bnp was infused prior to glp1 in the first subject , despite the intervening washout . on the second study day , the protocol was repeated using the opposite study drug ( sitagliptin or matching placebo ) . glp1 ( clinalfa basic ) was infused at 0.45 , 0.90 , and 1.80 pmol / min in the first 3 randomized subjects ; however , we did not see any effect of glp1 on vasodilation at these doses . after we still saw no effect of glp1 , we discontinued glp1 in the remaining 3 subjects . forearm vascular resistance was calculated as mean arterial pressure ( map ) divided by fbf . blood for measurement of venous glp1 levels was collected in tubes containing edta and protease inhibitor ( aprotinin ; roche diagnostics ) . seventeen healthy , nonobese ( body mass index < 30 kg / m ) , nonsmoking adults participated in a doubleblind , randomized , placebocontrolled , crossover study ( figure 1 ; subject characteristics are shown in table 1 ) . forearm blood flow measurement , followed by arterial and venous sampling , was performed at baseline and at completion of each dose of peptide . bnp indicates brain natriuretic peptide ; glp1 , glucagonlike peptide1 . on each study day , subjects reported to the vanderbilt clinical research center in the morning after an overnight fast . subjects were given an oral study drug ( sitagliptin 200 mg or matching placebo ) , and an arterial line was placed in the brachial artery of the nondominant arm with an adjacent peripheral intravenous line . baseline forearm blood flow ( fbf ) measurements and blood sampling were obtained 60 minutes following ingestion of the study drug and at least 30 minutes after insertion of the arterial catheter . subjects then received sequential intraarterial infusions of glp1 followed by bnp ; a 30minute washout separated each infusion . we observed a carryover effect on fbf when bnp was infused prior to glp1 in the first subject , despite the intervening washout . on the second study day , the protocol was repeated using the opposite study drug ( sitagliptin or matching placebo ) . glp1 ( clinalfa basic ) was infused at 0.45 , 0.90 , and 1.80 pmol / min in the first 3 randomized subjects ; however , we did not see any effect of glp1 on vasodilation at these doses . after we still saw no effect of glp1 , we discontinued glp1 in the remaining 3 subjects . forearm vascular resistance was calculated as mean arterial pressure ( map ) divided by fbf . blood for measurement of venous glp1 levels was collected in tubes containing edta and protease inhibitor ( aprotinin ; roche diagnostics ) . glp1 levels were determined using a multiplex magnetic bead assay ( milliplex map human metabolic hormone magnetic bead panel ; emd millipore ) that detects active glp1(736 ) in the range of 4 to 3033 pmol / l with no crossreactivity for glp1(936 ) . dpp4 inhibition increased systolic blood pressure by 5% ( p=0.03 ) but had no effect on diastolic blood pressure ( p=0.70 ) , map ( p=0.21 ) , or heart rate ( p=0.92 ) prior to intraarterial peptide infusions . dpp4 inhibition increased fbf by 30% ( p=0.02 ) and decreased forearm vascular resistance by 18% ( p=0.03 ) prior to intraarterial peptide infusions . bpm indicates beats per minute ; dpp4 , dipeptidyl peptidase 4 ; fbf , forearm blood flow ; fvr , forearm vascular resistance . intraarterial infusion of glp1 significantly increased venous glp1 concentrations from baseline during both placebo ( p=0.01 ) and sitagliptin ( p=0.01 ) . venous glp1 levels were significantly higher during sitagliptin ( p=0.04 versus placebo at highest dose of glp1 ) and exceeded postprandial levels normally found in healthy adults ( figure 2 ) . effect of dpp4 inhibition on venous glp1 levels at baseline and during the maximum dose of glp1 . vasodilator response is presented as fbf , because local intraarterial infusion of peptide did not significantly affect map in either treatment group , and as a percent change , given that baseline fbf differed between treatment with sitagliptin and placebo ( figure 3 ) . intraarterial infusion of glp1 did not increase fbf , even when its concentrations were increased by sitagliptin . effect of dpp4 inhibition on forearm blood flow response to intraarterial glp1 ( 14 subjects ) and to bnp ( 17 subjects ) . as noted in the methods , the first 3 subjects received lower doses of glp1 . bnp indicates brain natriuretic peptide ; dpp4 , dipeptidylpeptidase 4 ; glp1 , glucagonlike peptide1 . intraarterial infusion of bnp increased fbf in a dosedependent manner ( p<0.001 effect of dose ) ; however , treatment with sitagliptin did not affect this vasodilator response . intraarterial infusion of glp1 did not significantly affect heart rate , map , norepinephrine levels , or net vascular norepinephrine release during placebo or sitagliptin treatment ( data not shown ) . intraarterial infusion of bnp infusion increased heart rate in a dosedependent manner ( p=0.01 effect of dose ) ; treatment with sitagliptin did not affect this response . intraarterial infusion of bnp increased arterial norepinephrine levels only during sitagliptin ( p<0.001 effect of dose ) . there was no effect of intraarterial infusion of bnp on map , venous norepinephrine levels , or net norepinephrine release . three subjects did not complete the second study day due to inability to obtain adequate arterial access . dpp4 inhibition increased systolic blood pressure by 5% ( p=0.03 ) but had no effect on diastolic blood pressure ( p=0.70 ) , map ( p=0.21 ) , or heart rate ( p=0.92 ) prior to intraarterial peptide infusions . dpp4 inhibition increased fbf by 30% ( p=0.02 ) and decreased forearm vascular resistance by 18% ( p=0.03 ) prior to intraarterial peptide infusions . bpm indicates beats per minute ; dpp4 , dipeptidyl peptidase 4 ; fbf , forearm blood flow ; fvr , forearm vascular resistance . intraarterial infusion of glp1 significantly increased venous glp1 concentrations from baseline during both placebo ( p=0.01 ) and sitagliptin ( p=0.01 ) . venous glp1 levels were significantly higher during sitagliptin ( p=0.04 versus placebo at highest dose of glp1 ) and exceeded postprandial levels normally found in healthy adults ( figure 2 ) . effect of dpp4 inhibition on venous glp1 levels at baseline and during the maximum dose of glp1 . vasodilator response is presented as fbf , because local intraarterial infusion of peptide did not significantly affect map in either treatment group , and as a percent change , given that baseline fbf differed between treatment with sitagliptin and placebo ( figure 3 ) . intraarterial infusion of glp1 did not increase fbf , even when its concentrations were increased by sitagliptin . effect of dpp4 inhibition on forearm blood flow response to intraarterial glp1 ( 14 subjects ) and to bnp ( 17 subjects ) . as noted in the methods , the first 3 subjects received lower doses of glp1 . bnp indicates brain natriuretic peptide ; dpp4 , dipeptidylpeptidase 4 ; glp1 , glucagonlike peptide1 . intraarterial infusion of bnp increased fbf in a dosedependent manner ( p<0.001 effect of dose ) ; however , treatment with sitagliptin did not affect this vasodilator response . intraarterial infusion of glp1 did not significantly affect heart rate , map , norepinephrine levels , or net vascular norepinephrine release during placebo or sitagliptin treatment ( data not shown ) . intraarterial infusion of bnp infusion increased heart rate in a dosedependent manner ( p=0.01 effect of dose ) ; treatment with sitagliptin did not affect this response . intraarterial infusion of bnp increased arterial norepinephrine levels only during sitagliptin ( p<0.001 effect of dose ) . there was no effect of intraarterial infusion of bnp on map , venous norepinephrine levels , or net norepinephrine release . three subjects did not complete the second study day due to inability to obtain adequate arterial access . this study tested the hypothesis that dpp4 inhibition potentiates the vasodilator responses to glp1 and bnp in the human forearm . we found that glp1 does not cause vasodilation in the forearm vasculature of healthy humans , even when its degradation is inhibited by sitagliptin and high concentrations are achieved . although several prior studies have examined the effect of intravenous glp1 on endothelial function , our study is unique in examining the direct vascular effect of intraarterial glp1 while blocking its degradation by dpp4 . specifically , 2 prior studies examining the effect of intravenous glp1 on endothelial function during hyperglycemic clamp suggested that glp1 improves endothelial function , as measured by flowmediated dilation during hyperglycemia in diabetic subjects but not during normoglycemia . in contrast , basu et al reported that intravenous glp1 enhanced the forearm vasodilator response to intraarterial acetylcholine but not to nitroprusside in healthy subjects . because systemic administration of glp1 increases insulin , we infused glp1 directly in the brachial artery . tesauro et al also assessed the effect of intraarterial glp1 and reported that glp1 enhanced the fbf response to acetylcholine and nitroprusside in patients with metabolic syndrome during coinfusion of insulin but not during saline . in contrast to our study , the investigators did not inhibit the degradation of glp1 by dpp4 and did not achieve concentrations of glp1 comparable to physiological concentrations achieved after a meal . these data in humans conflict with data in rodent models , which indicate that glp1 causes direct vasodilation . the lack of effect of dpp4 inhibition by sitagliptin on the vascular response to glp1 is particularly important because ban et al reported that both glp1 and its dpp4 metabolite glp1(936 ) dilate preconstricted mesenteric arteries through a glp1 receptor independent and nitric oxide synthase in contrast , tesauro et al reported no effect of intraarterial glp1(936 ) in the human forearm . likewise , if endogenous glp1(936 ) causes vasodilation in humans , we would have expected to observe an increase in forearm vascular resistance during dpp4 inhibition , but instead we observed a decrease in baseline forearm vascular resistance . activation of the glp1 receptor in the brain has also been shown to modulate sympathetic activity in animal models and humans . yamamoto et al demonstrated that systemic administration of glp1 receptor agonist increased blood pressure and heart rate in a dosedependent fashion and activated autonomic neurons responsible for sympathetic outflow in rats . bharucha et al found that intravenous infusion of glp1 in healthy subjects increased skeletal muscle sympathetic nerve activity but did not affect cardiac sympathetic indices , as assessed by spectral analysis . vasodilation in response to glp1 may be compromised by increased sympathetic activity ; however , we administered glp1 intraarterially , thereby avoiding systemic counterregulatory responses , and did not see any effect of glp1 on norepinephrine release . dpp4 activity is increased in the setting of heart failure and thus may play a role in the pathophysiology of heart failure by increasing degradation of bnp to bnp(332 ) , which is a less potent vasodilator and natriuretic agent in dogs . we did not find that sitagliptin potentiated the vasodilatory response to bnp in the human forearm . it is possible that there was mild systemic vasodilation and activation of the baroreflex following bnp infusion during dpp4 inhibition , but the lack of effect of sitagliptin on the heart rate response to bnp infusion does not support this . we did not find an effect of intraarterial bnp on venous norepinephrine levels or net norepinephrine release in the presence or absence of sitagliptin . we observed an increase in basal systolic blood pressure with an accompanying increase in fbf and decrease in forearm vascular resistance during sitagliptin , consistent with our previous observation in healthy subjects . although we did not observe a significant increase in baseline heart rate or net norepinephrine release during sitagliptin in the present study , we have previously reported that dpp4 inhibition increases sympathetic activation by substance p. as noted , an effect of dpp4 inhibition on the degradation of glp1 in the central nervous system could have contributed to this observed increase in systolic blood pressure . a few study design considerations we used an acute dose of 200 mg of sitagliptin because this dose inhibits ddp4 inhibition to the same extent as the clinically approved dose of 100 mg but within a shorter time period . we can not exclude an effect of chronic dpp4 inhibition on vascular responses to glp1 and bnp or on endothelial function . we studied a small sample size due to the rigorous nature of the protocol ; this may have limited our power to detect an effect of sitagliptin on the vasodilator responses . we also studied healthy individuals in the fasting state to control for the fluctuations in other vasoactive hormones , including glp1 and insulin , and to control for medications and diseases that may affect endothelial function . we focused on vasodilator responses and can not exclude an effect of sitagliptin on the natriuretic response to bnp . we used an acute dose of 200 mg of sitagliptin because this dose inhibits ddp4 inhibition to the same extent as the clinically approved dose of 100 mg but within a shorter time period . we can not exclude an effect of chronic dpp4 inhibition on vascular responses to glp1 and bnp or on endothelial function . we studied a small sample size due to the rigorous nature of the protocol ; this may have limited our power to detect an effect of sitagliptin on the vasodilator responses . we also studied healthy individuals in the fasting state to control for the fluctuations in other vasoactive hormones , including glp1 and insulin , and to control for medications and diseases that may affect endothelial function . we focused on vasodilator responses and can not exclude an effect of sitagliptin on the natriuretic response to bnp . the recent saxagliptin assessment of vascular outcomes recorded in patients with diabetes mellitus ( savor)thrombolysis in myocardial infarction ( timi ) 53 trial revealed an increased risk for hospitalization due to heart failure with the dpp4 inhibitor saxagliptin . dpp4 inhibitor therapies may influence cardiovascular risk by affecting the degradation of peptide substrates that influence vascular function and sympathetic activity . we previously reported that the dpp4 substrate substance p increases sympathetic activity when dpp4 inhibition is combined with angiotensinconverting enzyme inhibition . in the present study , we investigated the impact of dpp4 inhibition on the vascular effects of glp1 and bnp . our data do not support the hypothesis that decreased degradation of glp1 and bnp during acute dpp4 inhibition directly regulates vascular function or sympathetic activity in healthy humans , despite data to the contrary in animal models .

the spondyloarthropathies ( spa ) , now better denominated as spondyloarthritides ( spas ) , are a diverse group of interrelated inflammatory arthritides that share multiple clinical features and common genetic predisposing factors . this group includes not only the prototypical disease , ankylosing spondylitis ( as ) , but also reactive arthritis ( rea ) , psoriatic arthritis ( psa ) , crohn 's disease , undifferentiated spa , and juvenile - onset spondyloarthritis . the clinical features of as include inflammatory back pain , asymmetrical peripheral oligoarthritis , enthesitis , and specific organ involvement , such as anterior uveitis , psoriasis , and chronic inflammatory bowel disease . its major clinical features include sacroilitis , loss of spinal mobility , and spinal inflammation . chronic inflammation leads to fibrosis and ossification , where bridging spurs of bone known as syndesmophytes form , especially at the edges of the inter - vertebral discs , producing the ankylosing . as affects men more often than women , at a ratio of 2 : 1 . studies conducted in different countries have shown that the incidence of as varies from 0.5 to 14 per 100,000 people per year . diagnoses of as are based more on clinical features than on laboratory tests ; currently , diagnoses are made in accordance with the modified new york criteria ( table 1 ) . as is of unknown etiology but it is known to be highly heritable , as > 90% of the risk of developing the disease has been shown to be genetically determined . as in the case of most common heritable diseases , progress in identifying candidate genes associated with the disease , and their possible role in pathogenesis , is one of the challenges that must be confronted in the near future . this review discusses recent advances in hla - b27 studies , characterization of immune responses , and the identification of some genes associated with as . human leukocyte antigen ( hla)-b27 is a major histocompatibility complex ( mhc ) class i molecule that is encoded on chromosome 6p . it is ubiquitous among cell types and is highly expressed on antigen - presenting cells . after translation and tertiary folding , hla - b27 heavy chains form heterotrimeric complexes with 2-microglobulin ( 2 m ) and intracellular peptides derived from self - proteins , viruses , and bacteria . the association of hla - b27 with as was first described for hla alleles and inflammatory diseases in 1973 , and this association remains one of the best examples of a disease association with a hereditary marker [ 7 , 8 ] ; however , it does not explain the cause of the disease . reports indicate that the risk of developing as is approximately 5% in hla - b27-positive subjects , but substantially higher for hla - b27-positive relatives . more than 90% of caucasians with as are hla - b27-positive ; however , most hla - b27-positive individuals remain healthy , suggesting that other genes , both inside and outside the mhc , are involved in disease susceptibility [ 1012 ] . thus , hla - b27 may only account for perhaps 20 to 50% of overall genetic susceptibility to as [ 13 , 14 ] . though there is no question that hla - b27 is the major susceptibility gene for as there is strong evidence that different subtypes of hla - b27 have distinct strengths of association with as in specific populations . some 100 hla - b27 subtypes have been reported to date ( http://hla.alleles.org/proteins/class1.html ) , but the number is increasing rapidly . most of them differ from each other by only a few amino acids , but these changes are sufficient to alter the molecule 's peptide - binding properties . hla - b*2705 is present in all populations and appears to be the original or most of the other subtypes appear to have evolved along three pathways , defined by the pattern of amino acid substitutions in the first ( 1 ) and second ( 2 ) domains and through geographic patterns [ 15 , 16 ] . the most common subtypes reported in association with as are hla - b*2705 , b*2702 , b*2704 , and b*2707 [ 1719 ] ; whereas hla - b*2706 and b*2709 , which are common in southeast asia and sardinia , have no association with as , possibly due to amino acid differences in the b pocket of the hla antigen - binding cleft that could modify the composition of the peptides that these hla - b27 subtypes present [ 15 , 20 ] . hla - b*2706 and b*2709 differ from the as - associated subtypes at residue 116 in the second domain . hla - b*2706 differs from hla - b*2704 , which is highly disease - associated among the chinese , in only two amino acid positions ( h114d , d116y ) , both of which reside on the floor of the f pocket of the peptide - binding groove . hla - b*2709 , meanwhile , differs from b*2705 by a single amino acid substitution at position 116 ( d116h ) [ 21 , 22 ] . this position is a relevant polymorphism that gives rise to different repertoires of bound peptides and cytotoxic cd8 t cells ( ctl ) . as an example , pvipr , a self - peptide derived from type i receptor of vasoactive intestinal peptide evokes autoreactive ctl responses in hla - b*2705 individuals , mostly patients with as , but not in hla - b*2709 healthy individuals . several theories have been proposed to explain the molecular pathogenic role of hla - b27 in as , including the presentation of arthritogenic peptides , the aberrant folding of surface heavy chains , hla - b27 misfolding , and enhanced intracellular microbial survival ( figure 1 ) . the dominant paradigm ( arthritogenic peptide hypothesis ) has been that self - peptides displayed by folded hla - b27 become the target of autoreactive cd8 t cells because they resemble microbial peptides , which does not occur with other hla molecules . this hypothesis invokes the unique peptide - binding specificity of hla - b27 as the problem . in support of this concept , hla - b27-restricted cd8 t - cell clones with specificity for bacteria or possibly self - peptides have been detected in both synovial fluid and peripheral blood of patients with rea and as . additionally , the finding of a self - peptide derived from the vasoactive intestinal peptide receptor ( vipr ) that shows high sequence homology to an epstein - barr virus derived epitope of latent membrane protein 2 ( plmp2 ) peptide were reported in patients with as . although the vipr peptide was chosen as a potential target that exhibit hla - b27 subtype - dependent molecular mimicry with the ebv epitope , there was little cross - reactivity between vipr and ebv - specific cd8 t cells , but as yet there is no proof of the involvement of these peptides in the pathogenesis of as . the cell surface hla - b27 homodimers hypothesis suggests that hla - b27 heavy chain homodimers are produced on the cell surface during endosomal recycling . the formation of disulphide bonds between the cysteine residue at position 67 ( c67 ) in the b pockets of the peptide binding groove of two separate heavy chain molecules creates homodimers with no participation by 2 m . hla - b27 homodimers bind to specific receptors expressed on nk cells , t lymphocytes , and myelomonocytic cells ; therefore , they could play a role in the pathogenesis of autoimmune disorders [ 2729 ] . in support of this theory , it was found that hla - b27 positive patients showed an increased number of nk cells and cd4 t cells expressing kir3dl2 , a killer immunoglobulin - like receptor ( kir ) that recognizes homodimers of hla - b27 but not its heterodimers . however , residue c67 , which is critical for the formation of homodimers , exists in both hla - b27 subtypes those that are related to as and those that are not . as counter arguments , no association has yet been reported between free heavy chains of hla - b27 molecules and predisposition to as , and the hla - b2706 subtype , which is not related to as , also forms homodimers . the hla - b27 misfolding hypothesis proposes that as results from an accumulation of aberrantly - folded hla - b27 in the endoplasmic reticulum ( er ) , that produces an inflammatory response . er stress resulting from the accumulation of misfolded heavy chains then activates the unfolded protein response ( upr ) , triggering a series of signaling pathways that culminate in the induction of er - resident chaperones ( bip ) , which may induce cytokine production by macrophages , thereby promoting inflammation [ 34 , 35 ] . another pathway that can activate er stress is the er - overload response ( eor ) to excessive membrane protein trafficking within the er , which involves activation of the transcription of nuclear factor kappa b ( nf-b ) that can stimulate the synthesis of proinflammatory cytokines such as tnf- , il-1 and il-6 in certain cell types . the enhanced intracellular microbial survival hypothesis may also play a role in the pathogenesis of as . this mechanism is based on the inability of hla - b27-positive individuals to eliminate certain intracellular pathogens . abnormal immune system activation or modulation can occur due to ineffective peptide loading into hla - b27 , leading to excessive viral or intracellular bacterial proliferation and delayed antigenic peptide clearance . carriers of hla - b27 are defective in the killing of intracellular bacterial species of the genera yersinia , salmonella , shigella , and chlamydia [ 37 , 38 ] , all of which as is well documented are involved in the triggering of reactive arthritis ; however , an infectious trigger for as has yet to be demonstrated . it is highly likely that all of these mechanisms play some part in predisposing an individual to as . unfortunately , the precise role of hla - b27 in pathogenesis remains unclear , but features that distinguish it from other genes and differences among its many subtypes have provided the basis for several putative explanations as to how it might predispose individuals to as and mediate the disease . interleukin 17 ( il-17 ) is a proinflammatory cytokine that contributes to the pathogenesis of several inflammatory diseases . one major source of il-17 is a lineage of t cells known as t helper 17 cells ( th17 cells ) , but t cells , natural killer ( nk ) cells , mast cells , and neutrophils may also be involved . it is well established that il-17 activity contributes to various aspects of acute inflammation , because it mediates the release of il-6 and il-8 ( figure 2 ) . the role of il-17 in rheumatic diseases has been ascertained on the basis of findings that indicate that il-17 promotes cartilage damage in a murine model . it is also well known that il-23 is able to induce il-17 production and , therefore , is a crucial factor in the th17 response . in this regard , recent studies suggest that il-23r could be one of the major genetic factors involved in susceptibility to as . moreover , th17 cells have been implicated in many experimental autoimmune diseases , and in the pathogenesis of several inflammatory diseases , including rheumatoid arthritis ( ra ) , psoriasis , and inflammatory bowel disease . they also stimulate the formation of osteoclasts and , consequently , bone resorption and the recruitment of neutrophils and monocytes . several studies have been carried out in efforts to determine the role of different immune cells in the pathogenesis of as , as well as cd4 t cells producing il-17 that have been associated with autoimmune diseases , particularly with inflammatory autoimmune diseases . mast cells infiltrated into synovial joints in spas have increased the expression of il-17 , which supports the notion that they could be a source of th17 generation . moreover , th17 cells have been shown to be involved in promoting the inflammatory process in as . significantly elevated levels of th17 cells have been reported in the peripheral blood of patients with as [ 46 , 5052 ] , suggesting that they could have a role in inflammation . moreover , il-17 and il-23 have been found to be high as well in the serum of as patients [ 42 , 53 , 54 ] . the role of th17 cells in inflammation , and in as , is supported by studies that have demonstrated that anti - tnf- therapy reduces levels of il-17 and th17 cells in patients with as . though it is assumed that inflammation stimulates new bone formation , no concrete correlation between inflammation and osteoproliferation has yet been demonstrated ; moreover , inflammation and new bone formation can occur at distinct locations and , apparently , anti - tnf- therapy does not affect new bone formation in as [ 57 , 58 ] . despite the fact that the relationship between inflammation and new bone formation remains controversial , there is a clear relationship between as , th17 cell levels , and the latter 's cytokine secretion , which suggests an important role in the inflammatory process observed in as ( figure 2 ) . regulatory t cells ( tregs ) mediate peripheral tolerance by actively suppressing effector t cells and inhibiting immune - mediated tissue damage . tregs were first identified by the expression of cd25 , but now they are characterized by the expression of the intracellular transcription factor , foxp3 . in addition , they have been implicated in the regulation of almost every adaptive immune response and , therefore , also in inflammatory responses , by using appropriated mechanisms that inhibit targeted cell populations . in the case of as , few studies have been carried out to analyze the levels of tregs in the peripheral blood of patients ; however , low percentages of treg cells have been reported in the peripheral blood [ 46 , 51 , 69 , 70 ] , and in the synovial fluid of patients with as , suggesting an imbalance between tregs and the adaptive immune response . moreover , as patients treated with anti - tnf therapy showed similar levels of treg cells to those observed in healthy subjects . these data suggest a possible role of tregs in as , and th17/tregs imbalance has been proposed as playing a novel role in as . the recognition that both the adaptive and innate immune responses play key roles in as led us to focus on nk cells as a target for improving our understanding of the pathogenesis of as . nk cells are major components of innate immunity and provide surveillance during early defense against virus , intracellular bacteria , and cancer cells , but they have also been associated with autoimmunity . nk cells can be identified by the expression of cd56 and the lack of the cd3 complex . decreased numbers and impaired function of peripheral blood nk cells in patients with autoimmune diseases such as systemic lupus erythematosus ( sle ) , multiple sclerosis , diabetes , ra , and psoriasis however , the frequencies of circulating cd3cd56 nk cells have been reported to be higher in as patients [ 73 , 79 ] , as have those of the cd56cd16 subsets , but not cd56cd16 . the role of nk cells in as has been supported by the finding that the hla - b27 protein is specifically recognized by the nk - inhibitory receptor kir3dl1 . the killing activity of nk cells is balanced by the signals transduced by both inhibitory and activating receptors . surface receptors of nk cells have been evaluated in several studies , and nkp44 and nkp46 receptors have been evaluated in patients with as , while nkp44 expression has been found to be elevated in the ileum of patients with as . also , these cells secreted increased amounts of il-22 [ 73 , 81 ] . indeed , healthy hla - b27-positive subjects have similar nk cell levels to those in as patients . increasing evidence points to a role of the kirs in the development of autoimmune diseases . in particular , a positive association of kir3ds1 ( an activating receptor ) and a negative association of kir3dl1 ( an inhibitory receptor ) with as have been reported . additionally , it is known that kir3dl2 binds to free h chain forms of hla - b27 . genetic polymorphisms of kirs genes have been studied by some groups , finding that kir2dl1 , kir2dl5 , kir2ds5 , kir3ds1 , and kir3dl1 are all associated with as , though in different populations [ 82 , 8491 ] . these data suggest that nk cells could play a relevant pathogenic role in as via the expression of kirs . the chemokine ( c - c motif ) receptor 6 ( ccr6 ) is expressed on b cells , a fraction of t cells , and immature dcs , and studies have shown that it is a specific marker for th17 cells that distinguishes them from other helper t cells . moreover , ccr6 has been shown to be important for b - lineage maturation and antigen - driven b - cell differentiation and may regulate the migration and recruitment of dendritic cells ( dcs ) and t cells during inflammatory and immunological responses . ccr6 human memory t cells have a low stimulation threshold for il-10 production and , consequently , secrete il-10 after suboptimal stimulation by autologous dcs . ccr6 is considered an important receptor that guides effector t cells into inflamed tissue , thus favoring the th17 phenotype and downregulating the tregs . thus , ccr6 t cells play a central role in balancing regulatory and inflammatory processes during homeostasis and inflammation . it has also been reported that ccr6 deficient mice have altered cd4 t - cell responses , including reduced hypersensitivity and enhanced delayed type hypersensitivity responses , all of which supports the role of ccr6 in homeostasis . ccr6 are also involved in several autoimmune diseases , including psoriasis and ra [ 9698 ] ; however , ccr6 polymorphisms have not been associated with as in the few populations that have been analyzed [ 98 , 99 ] , despite the fact that some studies have demonstrated that it is expressed on the th17 cells of as patients [ 100 , 101 ] and that these patients have a higher proportion of these cells . cytotoxic t - lymphocyte antigen 4 ( ctla-4 , cd154 ) is a costimulatory molecule that is expressed by activated t cells and interacts with the b7 molecules on the surface of antigen - presenting cells to induce downregulation of t - cell activation . ctla-4 , which is encoded by the ctla4 gene located on chromosome 2p33 , is a structural homologue of cd28 . engagement of ctla-4 appears to regulate ongoing t - cell responses and induce peripheral t - cell tolerance , while the absence of this function appears to be involved in autoimmunity . in addition , ctla-4 is highly expressed by regulatory t cells and could play an important role in their functioning . theoretically , polymorphisms of ctla4 that reduce ctla-4 expression may cause autoimmune t - cell clonal proliferation , thus contributing to the pathogenesis of autoimmune diseases . the ctla4 gene has many single nucleotide polymorphisms ( snp ) , some of which are present in regulatory positions , while others appear in 3 utr , but the most important one is the leader sequence ( + 49 a / g ; rs231775 ) [ 62 , 104 , 105 ] . the + 49-a / g is located at position + 49 of the first exon of the ctla4 gene , where it provokes a threonine - to - alanine change in amino acid 17 of the leader peptide . it has been reported that + 49 a / g polymorphism in ctla4 gene alters the intracellular distribution of ctla-4 , il-2 production , and t - cell proliferation [ 103 , 106 ] , suggesting their possible role in autoimmune diseases . this snp have been analyzed in patients with as in different populations ( table 2 ) , but so far results are negative [ 6163 ] , though higher levels of circulating ctla-4 in spas and association of the + 49-gg genotype with circulatory c - reactive protein in patients with as have been found , indicating a possible role in the pathogenesis of as . the programmed cell death-1 ( pdcd1 , also known as pd1 ) gene is one of the costimulatory genes located on chromosome 2q37.3 . it encodes the surface receptor pd-1 , an inhibitory immunoreceptor expressed on activated t cells , b cells , and myeloid cells belonging to the immunoglobulin superfamily b7-cd28 . pd-1 is expressed in a variety of hematopoietic cells on the periphery after stimulation by antigen receptor signaling and cytokine receptors . two pd-1 ligands have been described ( pd - l1 and pd - l2 ) , and their expression is regulated by the inflammatory environment , cytokines such as tnf- , type 1 and 2 interferons , il-2 , il-7 , and il-15 . the co - stimulatory pathways consisting of the pd-1 receptor and its ligands deliver inhibitory signals that regulate the balance among t - cell activation , tolerance , and immune - mediated tissue damage . various snps in the pd1 gene have been identified , such as pd1.1 ( rs36084323 ) , pd1.3 ( rs11568821 ) , pd1.5 ( rs2227981 ) , and pd1.9 ( rs2227982 ) . among these , pd1.3 , pd1.5 , and pd1.9 have been associated with autoimmune disorders in different ethnic groups . in the case of as , despite controversies among studies of the polymorphism associated with the disease , it appears that pd1.3 , pd1.5 , and pd1.9 are all candidates for association ( table 2 ) [ 6468 ] . however , it is necessary to study these polymorphisms or conduct meta - analysis in additional populations , before these associations can be confirmed . erap1 is the term currently accepted by the human genome organization ( hugo ) nomenclature committee ( hgnc ) , though in the past it was known by such names as endoplasmic reticulum aminopeptidase associated with antigen processing ( eraap ) , adipocyte - derived leucine aminopeptidase ( a - lap ) , and aminopeptidase regulating tumor necrosis factor receptor i ( tnfri ) shedding ( arts-1 ) . erap1 is a zinc aminopeptidase belonging to the m1 family of the metallopeptidases that share the consensus gamen and hexxh(x)18e zinc - binding motifs . two major erap1 protein isoforms are generated : the longer isoform , a ( erap1-a ) and the shorter isoform , b ( erap1-b ) . because erap1 is highly polymorphic multiple splice variants with potential effects on biological functions have been described , for example , rs2287987 ( m349v ) located on the active site , rs17482078 ( r725q ) , and rs27044 ( q730e ) , which are exposed on the inner surface of the c - terminal cavity and could affect the substrate sequence or length specificity . other polymorphisms , such as rs26653 ( r127p ) , rs30187 ( k528r ) , and rs10050860 ( d575n ) , localized at domain junctions , reduce either specificity or aminopeptidase activity toward a synthetic peptide substrate by altering the conformational change between open and closed conformations . the association of erap1 snps with as can be explained from a functional perspective . first , in the endoplasmic reticulum , erap1 acts as a molecular ruler , trimming peptide antigens to optimal length for binding to mhc class i molecules . complex proteins are initially degraded in the cytosol by the proteasome complex to generate peptide fragments up to 25 amino acids in length . these antigenic peptides , and their n - terminal extended precursors , are subsequently transported into the er by the transporter associated with antigen processing ( tap ) that preferentially transports peptides of 816 residues in length [ 140142 ] . nascent mhc class i molecules typically bind short peptide fragments 8 - 9 residues long and transport them to the cell surface for presentation to t cells . erap1 is expressed in the lumen of the er , where peptide loading to mhc class i molecules takes place . here , erap1 preferentially trims substrates 1016 residues in length ; whereas peptides 8 - 9 residues in length are optimal for binding mhc class i molecules [ 143 , 144 ] . second , the cleavage of cell surface receptors by proinflammatory cytokines such as tnfr1 , il-1r2 , and il-6r results in the downregulation of their intracellular signaling . for this reason , the malfunctioning of erap1 would lead to either an increase or decrease in the number of cell surface receptors available for these cytokines , thus propitiating proinflammatory effects and , finally , raising disease susceptibility to as , though some polymorphisms of erap1 associated with as do not influence the cytokine receptor levels in patients with this disease . third , erap1 is involved in the activation of macrophages induced by lipopolysaccharide ( lps ) and interferon ( ifn)- . the first confirmed association of erap1 with as was reported by the wellcome trust case - control consortium and australo - anglo - american spondyloarthritis consortium ( wtccc / tasc ) in 2007 . this was the first non - mhc gene for which a definitive as - association was observed . in that study , the minor allele frequency of the erap1 snp rs30187 and rs27044 in as patients was considerably higher than in controls , and those variants have been repeatedly confirmed by nearly all population studies as conferring strong susceptibility to this disease . at the same time , this study indicates that in the mhc locus hla - b27 confers the greatest risk of as susceptibility , with an attributable risk of 50% ; erap1 is the second strongest association , with an attributable risk of 26% . recent genome - wide association studies ( gwas ) have revealed numerous erap1 polymorphisms that are associated with as and strongly related to the hla - b27 mhc i allele [ 110 , 150 ] . one gwas study found that the polymorphisms of erap1 only affect as risk in hla - b27-positive individuals . the association of as with erap1 has been replicated in multiple cohorts and ethnicities , including a family - based association study , case - control studies [ 113123 ] , and meta - analyses [ 124 , 125 ] of canadian , british , portuguese , chinese , hungarian , korean , spanish , and iranian populations . those studies were able to replicate the association of the rs30187 and rs27044 snp in all the aforementioned populations , with the sole exception of pazar 's study , where the snp rs30187 failed to show any significant connection with as susceptibility . it is important to mention that the north american spondylitis consortium ( nasc ) studied multiplex as families , finding and reporting , for the first time , a novel haplotype erap2 associated with as . in several disease models , , aberrant trimming of peptides or the presentation of erap1 and hla - b27 are involved in the pathogenesis of hla - b27-associated as . these findings suggest that erap1 participates in as pathogenesis with associated alleles that reduce the risk of disease through a mechanism that involves altered peptide presentation by mhc class i , though a great deal of additional experimental research is necessary to validate this . in table 3 , studies of association of erap1 and as are summarized . receptors for the fc fragment of igg ( fcr ) form a group of type i transmembrane glycoproteins belonging to the ig superfamily and expressed mostly on leucocytes , providing a critical link between the humoral and cellular arms of the immune response . fcr has three major functions : ( 1 ) positive and negative regulation of cell activation ; ( 2 ) ig transport and regulation of ig homeostasis ; and ( 3 ) uptake of the immune complex ( ic ) for the degradation and promotion of antigenic peptides for antigen presentation [ 151 , 152 ] that can trigger effector mechanisms , such as antibody - dependent cellular cytotoxicity ( adcc ) , phagocytosis , degranulation , and cytokine production via immune tyrosine activating or inhibitory motifs ( itam or itim ) . three classes of leukocyte fcr are currently distinguished : fcri ( cd64 ) , fcrii ( cd32 ) , and fcriii ( cd16 ) . fcri is a high - affinity receptor that binds both monomeric igg and immune complexes ; whereas the affinity of igg is low for fcrii and intermediate for fcriii . fcria , fcriia , fcriic , and fcriiia are activating receptors characterized by the presence of an immunoreceptor tyrosine - based activation motif ( itam ) ; fcriiib is unique , as it is anchored by a glycosylphosphatidylinositol ( gpi ) anchor . in contrast , fcriib is an inhibitory receptor that contains an immunoreceptor tyrosine - based inhibitory motif ( itim ) in its cytoplasmic domain . the inhibitory receptor fcriib plays a major role in controlling the antibody and immune response and the development of autoimmune diseases [ 154156 ] . fcriib includes two isoforms , fcriib1 and fcriib2 , which transduce inhibitory signals that downregulate immune functions triggered by activating receptors . for instance , fcriib engagement triggers the blockade of bcr - induced b - cell activation , once it joins with bcr . the opposed signaling pathways of activating and inhibitory fcr act in concert to determine the magnitude of the effector cell responses in immune - complex inflammation and autoimmune disease . in noninflamed tissues , the ratio of activating to inhibitory fcr is low , but it increases markedly in an inflamed environment . therefore , activating fcr promotes disease development , while inhibitory fcr contributes to protection in two different ways : first , through the downregulation of effector cell responses and , second , by maintaining peripheral tolerance . some studies have found promoter mutations that induce lower fcriib expression levels in individuals that are susceptible to autoimmune diseases [ 159 , 160 ] . likewise , it has been reported that fcrs may play a role in the pathogenesis of ra and sle . recently , based on specific locus genetic loci studies , fcriib was reported to be associated with as development in a case - control study in han chinese . that study indicated that rs10917661 may be a novel snp involved in as genetic predisposition . in a previous study , we reported the association of rs1801274 ( h131r ) of fcriia and rs396991 ( v158f ) of fcriiia with as in a small group of patients , having found that the fcriia - hh and fcriiia - vv variants are associated with as . we therefore suggested that these polymorphisms could be related to the igg3 immune response against bacterial antigens , as previously reported , and to human and bacterial hsp60 . moreover , a relation between the fcriiia - vv genotype and the response to infliximab has also been found . these discoveries are important for our understanding of the association between fcr and the pathogenesis of as , but more research is required in this field , including replication of the association of fcriib , fcriia , and fcriiia with as in multiple cohorts and ethnicities , to ascertain whether these snps are linked with predisposition to as . tumor necrosis factor - alpha is a highly potent proinflammatory molecule and a key signaling component of the immune system that is strongly induced after infection or tissue injury . this cytokine is known to be present at higher concentrations in patients with as , ra , and psa . the important role of tnf in these diseases has been proven by their successful treatment with anti - tnf drugs [ 166168 ] , particularly as patients respond well to tnf- antagonist therapy . indeed , anti - tnf- therapy has become the standard of care for as patients over the last decade . several studies have shown that tnf- blocking agents , such as infliximab , etanercept , and adalimumab are highly efficacious in controlling inflammation and improving the clinical assessment of as patients [ 167 , 170172 ] . a prospective study showed that three years of tnf- blocking therapy result in a significant increase in the bone formation marker after three months , a result that continued at a higher level up to three years , leading to a bone turnover balance that favors bone formation , in combination with continuous improvements in bone mineral density ( bmd ) in the lumbar spine . furthermore , bisphosphonates in conjunction with anti - tnf agents have a synergistic effect that provides additional increases in the bmd of as patients . this suggests that tnf- blockades may even reinforce extensive bone formation by suppressing the inflammatory component of as . the tnfa gene contains several snps , the most widely studied of which is 308 a / g snp ( rs1800629 ) , and 238 a / g ( rs361525 ) in the promoter region . it has been found to be involved in many diseases due to its ability to modify cytokine levels and clinical outcomes [ 176 , 177 ] . the 308 a / g and 238 a / g transition seems to be associated with susceptibility to autoimmune diseases , although other snp at 1031 ( rs1799964 ) , 863 ( rs1800630 ) , 857 ( rs1799724 ) , and 238 ( rs361525 ) in the promoter region of tnfa have been evaluated in as ( table 4 ) . however , there are contradictory results , because some studies did not found association between the 238 and 308 polymorphisms of tnfa with as [ 126131 ] , although the a allele and the aa genotype of tnfa ( 308 and 238 ) have been associated with as , and with a higher production of tnf- in two populations , which could leads to a state of latent inflammation and subsequent tissue damage , confirming the participation of elevated levels of this cytokine in establishing the inflammatory process in this disease [ 132 , 133 ] . in this regard , a swedish group has reported that patients with as , ar , and psa who carry the gg genotype exclusively showed a good response to anti - tnf- therapy , whereas a moderate response was associated with the 308 a / g genotype and unresponsiveness with the aa genotype . these results emphasize the importance of tnfa polymorphism as a predictor of responses to tnf- blocking agents . in contrast , a meta - analysis conducted by lee and song ( 2009 ) failed to demonstrate any association of the tnfa 308 polymorphisms with as in europeans , leaving several open questions as to the importance of the role of tnfa polymorphisms in as . tnf- is synthesized and expressed on the cell surface as a transmembrane protein ( tmtnf- ) that can be processed by the tnf- converting enzyme to generate a soluble form ( stnf- ) . both the soluble and transmembrane forms of tnf- are biologically active in their trimeric forms [ 180 , 181 ] and act by binding two different receptors , tnfr1 , which is activated by both stnf- and tmtnf- , and tnfr2 , which is activated mainly by tmtnf- [ 167 , 182 ] . tnfr-1 is the primary signaling receptor on most cell types and accounts for the majority of the proinflammatory , cytotoxic , and apoptotic effects classically attributed to tnf- [ 183 , 184 ] . therefore , at least in the inflammatory environment , the tnf-/tnfr2 pathway is critical for stabilizing the treg - cell pool that is required to restrain the magnitude and length of an inflammatory immune response and prevent damage to self - tissues [ 185 , 186 ] . moreover , several polymorphisms of the tnfrs may contribute to the development of an abnormal immune response in as . in the mexican population , the work group of corona - sanchez et al . found a high frequency of the aa genotype of the 383 tnfr1 polymorphism in patients with as . in addition , the a allele is significantly associated with a higher risk of as . hla - b27 has been associated with as since 1973 ; however , different pathways have been described to explain this association . these receptors are expressed by nk cells , but the involvement of nk cells in as takes place not only through recognition of hla - b27 , but also through the secretion of proinflammatory cytokines and their effect on tregs . new participants in the inflammatory process are th17 cells that induce the secretion of cytokines , such as tnf- , which is clearly involved in the pathogenesis of as . the genetic factor has been analyzed in studies of several of the genes involved in the immune response , and particularly in inflammatory responses . the snps included suggest that these variations could play a role in as because of their functional effect on the expression of the genes . describing the cells and genes associated with this disease is the first step towards the eventual detection of possible therapeutic targets that could be used to improve current treatments and patients ' quality of life . however , it is necessary to analyze the functional role of these genes and cells in the pathogenesis of the disease in order to reach an understanding of the mechanisms of these cells and genes in the pathogenesis of as .

ankylosing spondylitis ( as ) is a chronic inflammatory disease of unknown etiology , though it is considered an autoimmune disease . hla - b27 is the risk factor most often associated with as , and although the mechanism of involvement is unclear , the subtypes and other features of the relationship between hla - b27 and as have been studied for years . additionally , the key role of il-17 and th17 cells in autoimmunity and inflammation suggests that the latter and the cytokines involved in their generation could play a role in the pathogenesis of this disease . recent studies have described the sources of il-17 and il-23 , as well as the characterization of th17 cells in autoimmune diseases . other cells , such as nk and regulatory t cells , have been implicated in autoimmunity and have been evaluated to ascertain their possible role in as . moreover , several polymorphisms , mutations and deletions in the regulatory proteins , protein - coding regions , and promoter regions of different genes involved in immune responses have been discovered and evaluated for possible genetic linkages to as . in this review , we analyze the features of hla - b27 and the suggested mechanisms of its involvement in as while also focusing on the characterization of the immune response and the identification of genes associated with as .

1. Introduction 2. HLA-B27 3. Cells and AS 4. Molecules, Their Genes and AS

this group includes not only the prototypical disease , ankylosing spondylitis ( as ) , but also reactive arthritis ( rea ) , psoriatic arthritis ( psa ) , crohn 's disease , undifferentiated spa , and juvenile - onset spondyloarthritis . the clinical features of as include inflammatory back pain , asymmetrical peripheral oligoarthritis , enthesitis , and specific organ involvement , such as anterior uveitis , psoriasis , and chronic inflammatory bowel disease . as is of unknown etiology but it is known to be highly heritable , as > 90% of the risk of developing the disease has been shown to be genetically determined . as in the case of most common heritable diseases , progress in identifying candidate genes associated with the disease , and their possible role in pathogenesis , is one of the challenges that must be confronted in the near future . this review discusses recent advances in hla - b27 studies , characterization of immune responses , and the identification of some genes associated with as . after translation and tertiary folding , hla - b27 heavy chains form heterotrimeric complexes with 2-microglobulin ( 2 m ) and intracellular peptides derived from self - proteins , viruses , and bacteria . the association of hla - b27 with as was first described for hla alleles and inflammatory diseases in 1973 , and this association remains one of the best examples of a disease association with a hereditary marker [ 7 , 8 ] ; however , it does not explain the cause of the disease . though there is no question that hla - b27 is the major susceptibility gene for as there is strong evidence that different subtypes of hla - b27 have distinct strengths of association with as in specific populations . the most common subtypes reported in association with as are hla - b*2705 , b*2702 , b*2704 , and b*2707 [ 1719 ] ; whereas hla - b*2706 and b*2709 , which are common in southeast asia and sardinia , have no association with as , possibly due to amino acid differences in the b pocket of the hla antigen - binding cleft that could modify the composition of the peptides that these hla - b27 subtypes present [ 15 , 20 ] . several theories have been proposed to explain the molecular pathogenic role of hla - b27 in as , including the presentation of arthritogenic peptides , the aberrant folding of surface heavy chains , hla - b27 misfolding , and enhanced intracellular microbial survival ( figure 1 ) . this hypothesis invokes the unique peptide - binding specificity of hla - b27 as the problem . in support of this concept , hla - b27-restricted cd8 t - cell clones with specificity for bacteria or possibly self - peptides have been detected in both synovial fluid and peripheral blood of patients with rea and as . although the vipr peptide was chosen as a potential target that exhibit hla - b27 subtype - dependent molecular mimicry with the ebv epitope , there was little cross - reactivity between vipr and ebv - specific cd8 t cells , but as yet there is no proof of the involvement of these peptides in the pathogenesis of as . the cell surface hla - b27 homodimers hypothesis suggests that hla - b27 heavy chain homodimers are produced on the cell surface during endosomal recycling . hla - b27 homodimers bind to specific receptors expressed on nk cells , t lymphocytes , and myelomonocytic cells ; therefore , they could play a role in the pathogenesis of autoimmune disorders [ 2729 ] . in support of this theory , it was found that hla - b27 positive patients showed an increased number of nk cells and cd4 t cells expressing kir3dl2 , a killer immunoglobulin - like receptor ( kir ) that recognizes homodimers of hla - b27 but not its heterodimers . as counter arguments , no association has yet been reported between free heavy chains of hla - b27 molecules and predisposition to as , and the hla - b2706 subtype , which is not related to as , also forms homodimers . the enhanced intracellular microbial survival hypothesis may also play a role in the pathogenesis of as . carriers of hla - b27 are defective in the killing of intracellular bacterial species of the genera yersinia , salmonella , shigella , and chlamydia [ 37 , 38 ] , all of which as is well documented are involved in the triggering of reactive arthritis ; however , an infectious trigger for as has yet to be demonstrated . unfortunately , the precise role of hla - b27 in pathogenesis remains unclear , but features that distinguish it from other genes and differences among its many subtypes have provided the basis for several putative explanations as to how it might predispose individuals to as and mediate the disease . interleukin 17 ( il-17 ) is a proinflammatory cytokine that contributes to the pathogenesis of several inflammatory diseases . one major source of il-17 is a lineage of t cells known as t helper 17 cells ( th17 cells ) , but t cells , natural killer ( nk ) cells , mast cells , and neutrophils may also be involved . in this regard , recent studies suggest that il-23r could be one of the major genetic factors involved in susceptibility to as . moreover , th17 cells have been implicated in many experimental autoimmune diseases , and in the pathogenesis of several inflammatory diseases , including rheumatoid arthritis ( ra ) , psoriasis , and inflammatory bowel disease . several studies have been carried out in efforts to determine the role of different immune cells in the pathogenesis of as , as well as cd4 t cells producing il-17 that have been associated with autoimmune diseases , particularly with inflammatory autoimmune diseases . moreover , th17 cells have been shown to be involved in promoting the inflammatory process in as . significantly elevated levels of th17 cells have been reported in the peripheral blood of patients with as [ 46 , 5052 ] , suggesting that they could have a role in inflammation . moreover , il-17 and il-23 have been found to be high as well in the serum of as patients [ 42 , 53 , 54 ] . the role of th17 cells in inflammation , and in as , is supported by studies that have demonstrated that anti - tnf- therapy reduces levels of il-17 and th17 cells in patients with as . though it is assumed that inflammation stimulates new bone formation , no concrete correlation between inflammation and osteoproliferation has yet been demonstrated ; moreover , inflammation and new bone formation can occur at distinct locations and , apparently , anti - tnf- therapy does not affect new bone formation in as [ 57 , 58 ] . despite the fact that the relationship between inflammation and new bone formation remains controversial , there is a clear relationship between as , th17 cell levels , and the latter 's cytokine secretion , which suggests an important role in the inflammatory process observed in as ( figure 2 ) . in addition , they have been implicated in the regulation of almost every adaptive immune response and , therefore , also in inflammatory responses , by using appropriated mechanisms that inhibit targeted cell populations . in the case of as , few studies have been carried out to analyze the levels of tregs in the peripheral blood of patients ; however , low percentages of treg cells have been reported in the peripheral blood [ 46 , 51 , 69 , 70 ] , and in the synovial fluid of patients with as , suggesting an imbalance between tregs and the adaptive immune response . these data suggest a possible role of tregs in as , and th17/tregs imbalance has been proposed as playing a novel role in as . the recognition that both the adaptive and innate immune responses play key roles in as led us to focus on nk cells as a target for improving our understanding of the pathogenesis of as . decreased numbers and impaired function of peripheral blood nk cells in patients with autoimmune diseases such as systemic lupus erythematosus ( sle ) , multiple sclerosis , diabetes , ra , and psoriasis however , the frequencies of circulating cd3cd56 nk cells have been reported to be higher in as patients [ 73 , 79 ] , as have those of the cd56cd16 subsets , but not cd56cd16 . the role of nk cells in as has been supported by the finding that the hla - b27 protein is specifically recognized by the nk - inhibitory receptor kir3dl1 . surface receptors of nk cells have been evaluated in several studies , and nkp44 and nkp46 receptors have been evaluated in patients with as , while nkp44 expression has been found to be elevated in the ileum of patients with as . increasing evidence points to a role of the kirs in the development of autoimmune diseases . additionally , it is known that kir3dl2 binds to free h chain forms of hla - b27 . genetic polymorphisms of kirs genes have been studied by some groups , finding that kir2dl1 , kir2dl5 , kir2ds5 , kir3ds1 , and kir3dl1 are all associated with as , though in different populations [ 82 , 8491 ] . these data suggest that nk cells could play a relevant pathogenic role in as via the expression of kirs . the chemokine ( c - c motif ) receptor 6 ( ccr6 ) is expressed on b cells , a fraction of t cells , and immature dcs , and studies have shown that it is a specific marker for th17 cells that distinguishes them from other helper t cells . thus , ccr6 t cells play a central role in balancing regulatory and inflammatory processes during homeostasis and inflammation . ccr6 are also involved in several autoimmune diseases , including psoriasis and ra [ 9698 ] ; however , ccr6 polymorphisms have not been associated with as in the few populations that have been analyzed [ 98 , 99 ] , despite the fact that some studies have demonstrated that it is expressed on the th17 cells of as patients [ 100 , 101 ] and that these patients have a higher proportion of these cells . cytotoxic t - lymphocyte antigen 4 ( ctla-4 , cd154 ) is a costimulatory molecule that is expressed by activated t cells and interacts with the b7 molecules on the surface of antigen - presenting cells to induce downregulation of t - cell activation . in addition , ctla-4 is highly expressed by regulatory t cells and could play an important role in their functioning . it has been reported that + 49 a / g polymorphism in ctla4 gene alters the intracellular distribution of ctla-4 , il-2 production , and t - cell proliferation [ 103 , 106 ] , suggesting their possible role in autoimmune diseases . this snp have been analyzed in patients with as in different populations ( table 2 ) , but so far results are negative [ 6163 ] , though higher levels of circulating ctla-4 in spas and association of the + 49-gg genotype with circulatory c - reactive protein in patients with as have been found , indicating a possible role in the pathogenesis of as . various snps in the pd1 gene have been identified , such as pd1.1 ( rs36084323 ) , pd1.3 ( rs11568821 ) , pd1.5 ( rs2227981 ) , and pd1.9 ( rs2227982 ) . in the case of as , despite controversies among studies of the polymorphism associated with the disease , it appears that pd1.3 , pd1.5 , and pd1.9 are all candidates for association ( table 2 ) [ 6468 ] . erap1 is the term currently accepted by the human genome organization ( hugo ) nomenclature committee ( hgnc ) , though in the past it was known by such names as endoplasmic reticulum aminopeptidase associated with antigen processing ( eraap ) , adipocyte - derived leucine aminopeptidase ( a - lap ) , and aminopeptidase regulating tumor necrosis factor receptor i ( tnfri ) shedding ( arts-1 ) . because erap1 is highly polymorphic multiple splice variants with potential effects on biological functions have been described , for example , rs2287987 ( m349v ) located on the active site , rs17482078 ( r725q ) , and rs27044 ( q730e ) , which are exposed on the inner surface of the c - terminal cavity and could affect the substrate sequence or length specificity . other polymorphisms , such as rs26653 ( r127p ) , rs30187 ( k528r ) , and rs10050860 ( d575n ) , localized at domain junctions , reduce either specificity or aminopeptidase activity toward a synthetic peptide substrate by altering the conformational change between open and closed conformations . second , the cleavage of cell surface receptors by proinflammatory cytokines such as tnfr1 , il-1r2 , and il-6r results in the downregulation of their intracellular signaling . for this reason , the malfunctioning of erap1 would lead to either an increase or decrease in the number of cell surface receptors available for these cytokines , thus propitiating proinflammatory effects and , finally , raising disease susceptibility to as , though some polymorphisms of erap1 associated with as do not influence the cytokine receptor levels in patients with this disease . in that study , the minor allele frequency of the erap1 snp rs30187 and rs27044 in as patients was considerably higher than in controls , and those variants have been repeatedly confirmed by nearly all population studies as conferring strong susceptibility to this disease . at the same time , this study indicates that in the mhc locus hla - b27 confers the greatest risk of as susceptibility , with an attributable risk of 50% ; erap1 is the second strongest association , with an attributable risk of 26% . recent genome - wide association studies ( gwas ) have revealed numerous erap1 polymorphisms that are associated with as and strongly related to the hla - b27 mhc i allele [ 110 , 150 ] . it is important to mention that the north american spondylitis consortium ( nasc ) studied multiplex as families , finding and reporting , for the first time , a novel haplotype erap2 associated with as . in several disease models , , aberrant trimming of peptides or the presentation of erap1 and hla - b27 are involved in the pathogenesis of hla - b27-associated as . fcr has three major functions : ( 1 ) positive and negative regulation of cell activation ; ( 2 ) ig transport and regulation of ig homeostasis ; and ( 3 ) uptake of the immune complex ( ic ) for the degradation and promotion of antigenic peptides for antigen presentation [ 151 , 152 ] that can trigger effector mechanisms , such as antibody - dependent cellular cytotoxicity ( adcc ) , phagocytosis , degranulation , and cytokine production via immune tyrosine activating or inhibitory motifs ( itam or itim ) . the inhibitory receptor fcriib plays a major role in controlling the antibody and immune response and the development of autoimmune diseases [ 154156 ] . likewise , it has been reported that fcrs may play a role in the pathogenesis of ra and sle . in a previous study , we reported the association of rs1801274 ( h131r ) of fcriia and rs396991 ( v158f ) of fcriiia with as in a small group of patients , having found that the fcriia - hh and fcriiia - vv variants are associated with as . these discoveries are important for our understanding of the association between fcr and the pathogenesis of as , but more research is required in this field , including replication of the association of fcriib , fcriia , and fcriiia with as in multiple cohorts and ethnicities , to ascertain whether these snps are linked with predisposition to as . several studies have shown that tnf- blocking agents , such as infliximab , etanercept , and adalimumab are highly efficacious in controlling inflammation and improving the clinical assessment of as patients [ 167 , 170172 ] . the 308 a / g and 238 a / g transition seems to be associated with susceptibility to autoimmune diseases , although other snp at 1031 ( rs1799964 ) , 863 ( rs1800630 ) , 857 ( rs1799724 ) , and 238 ( rs361525 ) in the promoter region of tnfa have been evaluated in as ( table 4 ) . however , there are contradictory results , because some studies did not found association between the 238 and 308 polymorphisms of tnfa with as [ 126131 ] , although the a allele and the aa genotype of tnfa ( 308 and 238 ) have been associated with as , and with a higher production of tnf- in two populations , which could leads to a state of latent inflammation and subsequent tissue damage , confirming the participation of elevated levels of this cytokine in establishing the inflammatory process in this disease [ 132 , 133 ] . in this regard , a swedish group has reported that patients with as , ar , and psa who carry the gg genotype exclusively showed a good response to anti - tnf- therapy , whereas a moderate response was associated with the 308 a / g genotype and unresponsiveness with the aa genotype . in contrast , a meta - analysis conducted by lee and song ( 2009 ) failed to demonstrate any association of the tnfa 308 polymorphisms with as in europeans , leaving several open questions as to the importance of the role of tnfa polymorphisms in as . therefore , at least in the inflammatory environment , the tnf-/tnfr2 pathway is critical for stabilizing the treg - cell pool that is required to restrain the magnitude and length of an inflammatory immune response and prevent damage to self - tissues [ 185 , 186 ] . moreover , several polymorphisms of the tnfrs may contribute to the development of an abnormal immune response in as . hla - b27 has been associated with as since 1973 ; however , different pathways have been described to explain this association . these receptors are expressed by nk cells , but the involvement of nk cells in as takes place not only through recognition of hla - b27 , but also through the secretion of proinflammatory cytokines and their effect on tregs . new participants in the inflammatory process are th17 cells that induce the secretion of cytokines , such as tnf- , which is clearly involved in the pathogenesis of as . the genetic factor has been analyzed in studies of several of the genes involved in the immune response , and particularly in inflammatory responses . the snps included suggest that these variations could play a role in as because of their functional effect on the expression of the genes . describing the cells and genes associated with this disease is the first step towards the eventual detection of possible therapeutic targets that could be used to improve current treatments and patients ' quality of life . however , it is necessary to analyze the functional role of these genes and cells in the pathogenesis of the disease in order to reach an understanding of the mechanisms of these cells and genes in the pathogenesis of as .

diabetes mellitus ( dm ) is a group of metabolic diseases of prolonged hyperglycaemia due to either the pancreas not producing enough insulin , or the cells of the body not responding properly to the insulin produced . it is a major public health problem that is approaching epidemic proportions worldwide and largely associated with lifestyle changes in emerging economies , a double edged sword . the worldwide prevalence of both types 1 and 2 dm among adults was 285 million ( 6.4% ) in 2010 and is predicted to rise to around 439 million ( 7.8% ) by 2030 . although sub - saharan africa has been reported to have an estimated dm adult prevalence of 2.4% , this is probably not just an understatement but the burden is also likely to increase in a few years ' time . the progressive nature of the disease requires regular monitoring of glycaemia and , when necessary , intensification of any existing treatment . the most common form of monitoring involves pricking the fingertip to obtain a blood sample , which is tested with a glucometer to determine the patient 's blood glucose level . the results from self - glucose monitoring aid the diabetics in decision making on the food , exercise , and use of medications including dose adjustment . this allows diabetics to manage their disease and avoid associated complications of uncontrolled abnormal plasma glucose levels . for type 2 dm , self - management behaviours are an important aspect of management and should be recommended for all diabetic patients . these self - management behaviours include , but are not limited to , adherence and self - blood glucose monitoring ( sbgm ) as well as exercise and body mass index ( bmi ) monitoring . high level of glycated hemoglobin indicates poor control of diabetes and is associated with cardiovascular disease , nephropathy , and retinopathy . measuring hba1c can be used to reflect average blood glucose levels over the previous 8 to 12 weeks prior to the measurement , thus providing a useful longer - term gauge of glycaemic control . the hba1c test should be done approximately every 3 months in uncontrolled or at least twice a year in well - controlled diabetic patients . a number of studies have been conducted among diabetic patients in different parts of the world and most of them aimed to estimate the exact burden and associated factors . in a study by mahmood and aamir in pakistan , over half of the patients had poor diabetic control . et al . in venezuela reported 87% poor glycaemic control status in type 1 and 75% in type 2 diabetic patients . a six sub - saharan african countries study revealed that only 29% of the patients had good glycaemic control . the background retinopathy ( 18% ) and cataract ( 14% ) were the most common eye complications while macrovascular disease was rare , and 48% had neuropathy . in south africa reported that 20.1% of patients had good glycaemic control which was associated with obesity . rwegerera in tanzania reported 24.2% and 32.9% good glycaemic control using hba1c and fasting / random blood glucose , respectively . satisfaction with current antidiabetic treatment was associated with improved glycaemic control among non - insulin - treated type 2 diabetic patients , but gender and participation in a diabetes education program were not [ 10 , 15 ] . in addition , adherence to antidiabetic drugs significantly increased with an increase in the number of nondiabetic medications . good antidiabetic medication adherence was associated with better glycaemic control , but the results were not statistically significant . other studies elsewhere revealed poor glycaemic control status in most patients and factors such as treatment satisfaction , gender , treatment adherence , dm knowledge , exercise , and obesity were associated with glycaemic control but multiprofessional care and participation in education programs were not [ 11 , 16 , 17 ] . most studies did not assess reasons for nonadherence , sbgm , and means as influencers of glycaemic control . in zambia , diabetes prevalence is estimated to be at < 5% but the associated morbidity and mortality at university teaching hospital ( uth ) were 7.7% and 20.3% , respectively , in 2010 [ 18 , 19 ] . the reasons for the increasing morbidity and mortality from dm are unclear although poor or sedentary personal lifestyle could be among the factors contributing to this huge burden . however , there is still scarcity of information on glycaemic control patterns and factors that may be associated with it . the poor glycaemic control among diabetic patients compounded with inadequate self - care in zambia is such an emerging public health concern needing urgent response . the health care delivery system in zambia has a pyramid area based structure , with provision of primary health care ( phc ) services in lower health facilities such as health posts ( hps ) and health centres ( hcs ) covering a limited geographical area . the phc services are supported by the first- , second- , and third - level referral hospitals , through an established referral system . this is largely due to the insufficient capacities at lower levels , including shortages of health workers , erratic supply of essential drugs and medical supplies , and inequities in the distribution of essential physical infrastructure and equipment to offer services that are appropriate to their level , and also due to the limited scope of services offered by facilities at lower levels . in view of the foregoing , level two hospitals are forced to operate more as district hospitals , as many patients bypass the hps and hcs due to the observed capacity challenges . similarly , level three ( tertiary ) hospitals are mainly providing first- and second - level hospital services and this situation amounts to inappropriate use of resources , leading to inefficiencies in service delivery . thus , rightsizing and strengthening the hospital referral systems would result in reductions in congestion at higher level referral facilities and increase in the efficiency and effectiveness of health service delivery . it is believed that prevention and control strategies need to be multithronged in approach and must encompass both individual and group factors despite the evolving theory of change suggesting that individual factors are very critical and probably more than nonindividual factors . there is evidence of efforts focusing on individual factor such as information , education , and communication on sbgm , exercise , diet , and adherence to antidiabetic treatment . although these have continued to be emphasised as part of the overall management of dm patients , achieving good glycaemic control has continued to be a challenge and has been considered unattainable among some of the diabetic patients . we aimed to assess the current glycaemic control and self - management behaviours that may affect diabetic control among dm patients attending outpatients diabetic clinic at the uth in lusaka , zambia . data stem from a cross - sectional study , carried out at the uth diabetic clinic , lusaka , zambia . the uth is the main national referral health centre that treats and reviews patients with various diseases , including dm . the patients attend the diabetic clinic at appointed times as advised by the medical officers for regular monitoring of their disease . all the confirmed diabetic patients on treatment attending outpatients care for at least two years and aged 15 years and above were eligible for the study and newly diagnosed patients that started antidiabetic treatment less than two years were excluded from the study . the patients who agreed to participate in the study provided written informed consent before participating in the study . the study proposal was reviewed and approved by an independent university of zambia biomedical ethics committee . a simple random sampling method was used to accrue eligible consenting patients between september 2013 and december 2013 . the sample size and duration of accrual were calculated based on estimated 360 diabetic patients that attend outpatients diabetic clinic and fulfil appointments as well as the proportion of newly referred patients over a four - month period . based on krejcie and morgan 's formula for calculating sample size of a finite population , this gave a calculated sample size of at least 186 participants ( see sample size calculation below ) . however , to account for possible exclusions due to refusal to give consent and the need to carry out subgroup analysis , oversampling was ensured to achieve a total of 198 patients who were finally included in the study . x is the table value of chi - square for 1 degree of freedom at the desired confidence level of 0.05 ( 1.96 = 3.84 ) ( for 95% ci ) . p is the population proportion ( assumed to be 0.50 since this would provide the maximum sample size ) . d is the degree of accuracy expressed as a proportion ( 0.05 ) ( 5% accepted error ) . general characteristics : a structured interview schedule was used to capture data on demographic characteristics , self - management behaviours , and laboratory results . the interview schedule was developed based on the world health organization ( who ) stepwise survey ( steps ) instrument , version three . the data on sociodemographic information were collected using a structured interview schedule , and additional data on clinical factors were extracted from medical records and laboratory results of these patients . thereafter this data was checked for completeness and entered into the epidata version 3.0 . the weight and height of the patients were measured using a zt-160 adult weighing mechanical scale model with a height rod ( wuxi weigher factory co. , ltd . , zhejiang , china ) whose values were used to compute the bmi based on the formula developed by lambert adolphe jacques qutelet in 1835 . tokyo , japan ) was used to obtain the actual bmi figure by dividing weight in kilograms with height squared in metres which was also verified by the who bmi chart . laboratory measurement ( hba1c ) : the quantitative determination of hba1c level in the collected blood from the patients was carried out by the immunoturbidimetry method using the abx pentra 400 automated clinical chemistry analyser ( horiba abx sas , 34184 montpellier , france ) , whose technique has been certified by the national glycohemoglobin standardisation program ( ngsp ) of australia . the fpg was measured by the enzymatic determination of glucose using the trinder method using the same analyser . the therapeutic objective of hba1c has been to obtain values 48 mmol / mol as recommended by the international diabetes federation ( idf ) and american college of endocrinology ( ace ) and the target for fpg is 6 mmol / l . statistical analyses were carried out using ibm spss statistics for windows version 20.0 ( ibm corp . , armonk , ny , usa ) . pearson 's chi - squared test , fisher 's exact test , and student 's t - test were used to select potential self - management behaviours that may be associated with glycaemic control status . the odds ratio and 95% confidence interval were calculated using multivariate binary logistic regression to identify true potential predictors of glycaemic control while adjusting for confounders . this study was approved by the university of zambia biomedical research ethics committee on reference number 005 - 07 - 13 . written informed consent was obtained from all study participants randomly selected which ensured that all eligible persons were given an equal chance to participate or decline . to ensure confidentiality , the interviews were conducted in preselected private spaces within the health facility and participants ' identifiers were kept with the hospital in charge as a standard management protocol but they were ultimately delinked from all research documents except through numerical codes . venipuncture to obtain blood for testing was considered to pose minimal risk and was acceptable to patients as it was considered a standard practice in the disease management . in similar manner , the patients were not given any direct immediate benefits as they were being interviewed within the hospital environment and at the time that they came for routine referral , consultation , monitoring , or review . of all the patients enrolled for the study ( n = 198 ) , median age was 55 years ( iqr 45 , 62 ) . most ( 92.9% ) of the patients had type 2 dm in contrast to the 7.1% that had type 1 dm . in addition , majority ( 61.3% ) of the patients had poor glycaemic control status and only 38.7% had good glycemic control among those whose data was complete . the mean ( sd ) fpg of the patients was 9.65 4.96 mmol / l . most ( 73.7% ) of the patients reported not following the treatment regimen as prescribed ( adherence ) in contrast to the 52 ( 26.3% ) participants that reported adherence to the type of antidiabetic treatment they were on . only a few ( 13.1% ) patients reported sbgm at home whereas most ( 86.1% ) of them reported none . amongst the patients who reported sbgm at home , 13 ( 6.6% ) of the patients reported monitoring glucose control at the public health facility , 2.5% own glucometer , and 4.0% reported glucose monitoring at the private health facility . the majority ( 59.6% ) of the patients the glycaemic control status of the patients according to the characteristics and self - management behaviours is shown in table 2 . in bivariate analysis , there was an association between glycaemic control status and adherence to treatment and fpg . however , there was no association between age , sex , education , bmi , sbgm , means of sbgm , exercise , and glycaemic control status . the multivariate binary logistic regression model was tested for multicollinearity , hosmer and lemeshow test of model fitness for data , and omnibus test of model coefficients and classification accuracy . the dependent variable was glycaemic control status : good ( 1 ) and poor ( 0 ) . the results of the multivariate binary logistic regression analysis to predict whether the 9 variable factors , age , sex , education level , bmi , adherence to treatment , sbgm , sbgm means , and exercise , predicted glycaemic control status showed that only adherence to treatment and fpg predicted glycaemic control status of the patients ( table 3 ) . thus , the patients who do not adhere to antidiabetic treatment and those with mean ( sd ) fpg , 10.26 5.17 mmol / l , are 68% and 7% less likely to achieve good glycaemic control status ( table 3 ) . it is well established that nonadherence rates for chronic disease regimens and for lifestyle changes are generally approximately 50% , and patients with diabetes are particularly prone to regimen adherence problems especially when on multiple treatment regimens including medications , lifestyle , diet , and exercise [ 3033 ] . consequently , successful management of dm is challenging , yet patients with good self - care behaviours can achieve excellent glycaemic control and avoid frequent diabetic complications . however , many patients are devoid of optimal self - care behaviours and continue to suffer from complications of the disease . regular sbgm empowers patients to play a role in the management of diabetes , simultaneously improving their metabolic parameters . glucometers are frequently used to assess the fpg and the results can prompt and help the patient to adjust the diet ( especially carbohydrate intake ) , exercise , and improve adherence to or modify medication dosage . the consistent use and correct response to the glycaemic results have been shown to improve glycaemic control in type 2 diabetes , consequently preventing or delaying further complications of diabetes . in this study we found evidence of poor glycaemic control which was significantly associated with poor adherence to medication use among diabetic patients that regularly attend medical review at the diabetic clinic . however , poor glycaemic control was not strongly associated with age , sex , education level , sbgm and means of sbgm , exercise , and bmi , associations that have been observed elsewhere [ 3537 ] . while the present study showed higher hba1c values in patients aged over 54 years as well as those that attained at least secondary education , the results were not statistically significant . however , adherence to antidiabetic treatment and fpg were statistically significantly associated with higher hba1c values . these findings are not surprising given that as a developing nation zambia lacks the resources and capacity to manage this disease which could be associated with adherence . while developed nations have processes , strategies , and infrastructures imbedded in their health care programs , including electronic continuous monitoring technology , that allow medical management of both early and late stage disease , developing nations have inadequate capability to manage and reverse the increasing morbidity and mortality . this association with adherence to antidiabetic treatment and fpg observed in this study is a common problem among individuals with diabetes [ 4043 ] . it is therefore necessary to determine the effective behavioural interventions that can improve adherence in the zambian diabetic patients . consequently , this study has persuaded us to consider a focus for a changed interventional approach targeting selected factors including patient centered approaches such as understanding patient insight of the disease , and collaborative and clear communication between health care professionals and patients could impact positively on glycaemic control in these patients . firstly , we are aware that this was a cross - sectional study and therefore it is difficult to establish a causal relation between hba1c and the self - management behaviours . secondly , we also acknowledge that this was a small and highly selected sample from a frame of hospital attendees at a referral tertiary hospital . this limitation was augmented by the fact that the cost of purchasing laboratory materials and supplies for the study provided additional sample size limitations . thirdly and in addition , we also observe that there could be additional biological individual variations which could potentially bias our measurements . fourthly and lastly though not the least , we also report that there was incomplete data on medical records of the diabetic outpatients at the clinic , making it difficult to follow the morbidity and mortality patterns and thus reducing further the possible sample which would have improved the study power and understanding of possible determinants . notwithstanding the possible presence of such selection and measurement biases , we do not think these could have been important in explaining the findings as their effects are assumed to have been non - differentially distributed given the random selection of subjects that was used . in zambia the mortality of patients with type 2 dm is likely to continue to increase as the zambian economy improves a factor associated with increase in western life style including diet and sedentary way of life . a needs ' assessment for the noncommunicable disease program carried out by the moh identified deficiencies in diabetic control as having inadequacies in terms of drugs and laboratory reagents , diagnostic facilities , expertise , and community awareness for dm . it may thus be not surprising to find presence of individual factors associated with poor glycaemic control status where we further argue that these are also associated with not only system supply factors but also social factors , predominantly operating at an individual level . consequently , there is need to define dm disease burden and epidemiology with focus on determinants and thus determine potential strategies that could address these inadequacies and improve the disease outcome . the majority of diabetic outpatients in this study had poor glycaemic control status and this could be due to a variety of factors including lack of resources and ability to buy diabetic chips and strips that allow for more frequent checks of fpg levels and/or ability to have adequate resources to store , for example , the insulin in a refrigerator which is frequently not available to many zambians . the reasons for this were not the focus of this paper and so we may only speculate regarding possible reasons for these differential glycaemic control status associations . we , however , disagree with what other evidence suggest that this can also probably be because of poor diet and exercise habits and other multiple barriers . however , good glycaemic control was reported in japan and germany also and it has been argued that perhaps this is because of higher literacy levels in these countries resulting in improved knowledge translation about dm [ 47 , 48 ] . it was interesting to note that there was a statistically significant association between adherence to antidiabetic treatment and glycaemic control status of the diabetic outpatients in this study . this is important given that the effectiveness of drug treatment depends primarily on the efficacy of the prescribed treatment and adherence of the patient to the treatment . these findings have been supported by studies elsewhere which have shown that adherence to antidiabetic treatment among diabetic patients is poor and the possible reasons have been outlined . one of the reasons is simply failure to understand and consequently failure to comply with the prescribed clinical regimen , thereby resulting in very poor outcomes . in the present study , this was illustrated clearly where we observed that the diabetic outpatients who did not adhere to dm treatment had 68% decrease in the likelihood of achieving good glycaemic control status compared to those who adhered . in similar manner , in the study by ahmad et al . in malaysia , 53.0% of the patients were nonadherent to dm treatment and the main factor which was associated with that nonadherence was age . in another study by curkendall et al . in the us the adherence was high in the males , older patients , or patients residing in specific geographical area . the factors related to poor adherence were comorbidity , overall health level , number of drugs , and complexity of the drug regimen . there is thus a critical need to understand adherence related factors so as to design prevention and control strategies that will be operable but accounting for contextual differentials even across low income countries in general . in fact , some studies have shown that an increase in adherence by as little as 10% can decrease the levels of hba1c significantly [ 35 , 51 ] . such increase is possible if behavioural linked factors such as education attainment , which has already been shown to improve glycaemic control status , are targeted [ 51 , 52 ] , however , tackling nonadherence is not a simple matter , as it is multifactorial and might include cost adjustments , health belief transformations , dosing frequency repackaging , and assessment of the presence of potential confounders associated with personality disorders and patient - provider relationship . there is need to explore the effective behavioural interventions that can improve adherence to antidiabetic treatment among the diabetic patients in zambia . changing interventional approaches targeting selected factors including patient centered approaches such as understanding patient insight of the disease and collaborative and clear communication between health care professionals and patients could impact positively on glycaemic control in diabetic patients . thus , the role of diabetic patients in the management of their diabetes remains paramount . if this matter is critically managed , it is possible that the outcome of treatment would be much more satisfactory among diabetic outpatients and this could possibly delay the development of the complications of dm and improve the quality and length of lives for the affected individuals . there is thus need to institute prevention and control mechanism that are cost - effective , acceptable , and appropriate . thus , combined screening with fpg and hba1c used in this study may identify patients at very high risk for diabetes when fpg and hba1c are considered together . we conclude that , among diabetic patients , poor glycaemic control remains a challenge and this may to a greater extent be associated with this may suggest limitations in past prevention and control efforts for dm at individual level as well as at care level where monitoring dynamics are limited as these patients are largely outpatients except when they are admitted to hospital for one reason or another . however , finding that fpg predicted the glycaemic control opens potential opportunities to routinely examine and identify most at risk groups , which in turn and further opens possibilities to study the associated dynamics linked to care and support . this could in turn therefore inform interventional policies and control strategies and thus improve the overall care and support of such patients . furthermore , and given that the self - management behaviours of diabetic patients play an important role in the management of dm , there is thus need to target improvement of the efficacy of individual strategies in all prevention and control strategies for these patients . we further argue that if this is done properly , it may consequently reduce diabetic complications and thus improve the lives of these people . the health care providers also are critical stakeholders and need to foster and place greater emphasis on counselling and improving adherence , notwithstanding the context specific differences .

background . the control of diabetes mellitus depends on several factors that also include individual lifestyles . we assessed glycaemic control status and self - management behaviours that may influence glycaemic control among diabetic outpatients . methods . this cross - sectional study among 198 consenting randomly selected patients was conducted at the university teaching hospital diabetic clinic between september and december 2013 in lusaka , zambia . a structured interview schedule was used to collect data on demographic characteristics , self - management behaviours , and laboratory measurements . binary logistic regression analysis using ibm spss for windows version 20.0 was carried out to predict behaviours that were associated with glycaemic control status . results . the proportion of patients that had good glycaemic control status ( hba1c 48 mmol / mol ) was 38.7% compared to 61.3% that had poor glycaemic control status ( hba1c 49 mmol / mol ) . adherence to antidiabetic treatment and fasting plasma glucose predicted glycaemic control status of the patients . however , self - blood glucose monitoring , self - blood glucose monitoring means and exercise did not predict glycaemic control status of the patients . conclusion . we find evidence of poor glycaemic control status among most diabetic patients suggesting that health promotion messages need to take into account both individual and community factors to promote behaviours likely to reduce nonadherence .

1. Introduction 2. Methods 3. Results 4. Discussion 5. Conclusion

diabetes mellitus ( dm ) is a group of metabolic diseases of prolonged hyperglycaemia due to either the pancreas not producing enough insulin , or the cells of the body not responding properly to the insulin produced . it is a major public health problem that is approaching epidemic proportions worldwide and largely associated with lifestyle changes in emerging economies , a double edged sword . although sub - saharan africa has been reported to have an estimated dm adult prevalence of 2.4% , this is probably not just an understatement but the burden is also likely to increase in a few years ' time . the progressive nature of the disease requires regular monitoring of glycaemia and , when necessary , intensification of any existing treatment . the results from self - glucose monitoring aid the diabetics in decision making on the food , exercise , and use of medications including dose adjustment . this allows diabetics to manage their disease and avoid associated complications of uncontrolled abnormal plasma glucose levels . for type 2 dm , self - management behaviours are an important aspect of management and should be recommended for all diabetic patients . these self - management behaviours include , but are not limited to , adherence and self - blood glucose monitoring ( sbgm ) as well as exercise and body mass index ( bmi ) monitoring . high level of glycated hemoglobin indicates poor control of diabetes and is associated with cardiovascular disease , nephropathy , and retinopathy . measuring hba1c can be used to reflect average blood glucose levels over the previous 8 to 12 weeks prior to the measurement , thus providing a useful longer - term gauge of glycaemic control . the hba1c test should be done approximately every 3 months in uncontrolled or at least twice a year in well - controlled diabetic patients . a number of studies have been conducted among diabetic patients in different parts of the world and most of them aimed to estimate the exact burden and associated factors . in a study by mahmood and aamir in pakistan , over half of the patients had poor diabetic control . in venezuela reported 87% poor glycaemic control status in type 1 and 75% in type 2 diabetic patients . a six sub - saharan african countries study revealed that only 29% of the patients had good glycaemic control . the background retinopathy ( 18% ) and cataract ( 14% ) were the most common eye complications while macrovascular disease was rare , and 48% had neuropathy . in south africa reported that 20.1% of patients had good glycaemic control which was associated with obesity . rwegerera in tanzania reported 24.2% and 32.9% good glycaemic control using hba1c and fasting / random blood glucose , respectively . satisfaction with current antidiabetic treatment was associated with improved glycaemic control among non - insulin - treated type 2 diabetic patients , but gender and participation in a diabetes education program were not [ 10 , 15 ] . in addition , adherence to antidiabetic drugs significantly increased with an increase in the number of nondiabetic medications . good antidiabetic medication adherence was associated with better glycaemic control , but the results were not statistically significant . other studies elsewhere revealed poor glycaemic control status in most patients and factors such as treatment satisfaction , gender , treatment adherence , dm knowledge , exercise , and obesity were associated with glycaemic control but multiprofessional care and participation in education programs were not [ 11 , 16 , 17 ] . most studies did not assess reasons for nonadherence , sbgm , and means as influencers of glycaemic control . in zambia , diabetes prevalence is estimated to be at < 5% but the associated morbidity and mortality at university teaching hospital ( uth ) were 7.7% and 20.3% , respectively , in 2010 [ 18 , 19 ] . however , there is still scarcity of information on glycaemic control patterns and factors that may be associated with it . the poor glycaemic control among diabetic patients compounded with inadequate self - care in zambia is such an emerging public health concern needing urgent response . the phc services are supported by the first- , second- , and third - level referral hospitals , through an established referral system . this is largely due to the insufficient capacities at lower levels , including shortages of health workers , erratic supply of essential drugs and medical supplies , and inequities in the distribution of essential physical infrastructure and equipment to offer services that are appropriate to their level , and also due to the limited scope of services offered by facilities at lower levels . in view of the foregoing , level two hospitals are forced to operate more as district hospitals , as many patients bypass the hps and hcs due to the observed capacity challenges . it is believed that prevention and control strategies need to be multithronged in approach and must encompass both individual and group factors despite the evolving theory of change suggesting that individual factors are very critical and probably more than nonindividual factors . there is evidence of efforts focusing on individual factor such as information , education , and communication on sbgm , exercise , diet , and adherence to antidiabetic treatment . although these have continued to be emphasised as part of the overall management of dm patients , achieving good glycaemic control has continued to be a challenge and has been considered unattainable among some of the diabetic patients . we aimed to assess the current glycaemic control and self - management behaviours that may affect diabetic control among dm patients attending outpatients diabetic clinic at the uth in lusaka , zambia . data stem from a cross - sectional study , carried out at the uth diabetic clinic , lusaka , zambia . the patients attend the diabetic clinic at appointed times as advised by the medical officers for regular monitoring of their disease . all the confirmed diabetic patients on treatment attending outpatients care for at least two years and aged 15 years and above were eligible for the study and newly diagnosed patients that started antidiabetic treatment less than two years were excluded from the study . the patients who agreed to participate in the study provided written informed consent before participating in the study . a simple random sampling method was used to accrue eligible consenting patients between september 2013 and december 2013 . the sample size and duration of accrual were calculated based on estimated 360 diabetic patients that attend outpatients diabetic clinic and fulfil appointments as well as the proportion of newly referred patients over a four - month period . however , to account for possible exclusions due to refusal to give consent and the need to carry out subgroup analysis , oversampling was ensured to achieve a total of 198 patients who were finally included in the study . x is the table value of chi - square for 1 degree of freedom at the desired confidence level of 0.05 ( 1.96 = 3.84 ) ( for 95% ci ) . general characteristics : a structured interview schedule was used to capture data on demographic characteristics , self - management behaviours , and laboratory results . the interview schedule was developed based on the world health organization ( who ) stepwise survey ( steps ) instrument , version three . the data on sociodemographic information were collected using a structured interview schedule , and additional data on clinical factors were extracted from medical records and laboratory results of these patients . the weight and height of the patients were measured using a zt-160 adult weighing mechanical scale model with a height rod ( wuxi weigher factory co. , ltd . tokyo , japan ) was used to obtain the actual bmi figure by dividing weight in kilograms with height squared in metres which was also verified by the who bmi chart . laboratory measurement ( hba1c ) : the quantitative determination of hba1c level in the collected blood from the patients was carried out by the immunoturbidimetry method using the abx pentra 400 automated clinical chemistry analyser ( horiba abx sas , 34184 montpellier , france ) , whose technique has been certified by the national glycohemoglobin standardisation program ( ngsp ) of australia . the therapeutic objective of hba1c has been to obtain values 48 mmol / mol as recommended by the international diabetes federation ( idf ) and american college of endocrinology ( ace ) and the target for fpg is 6 mmol / l . statistical analyses were carried out using ibm spss statistics for windows version 20.0 ( ibm corp . pearson 's chi - squared test , fisher 's exact test , and student 's t - test were used to select potential self - management behaviours that may be associated with glycaemic control status . the odds ratio and 95% confidence interval were calculated using multivariate binary logistic regression to identify true potential predictors of glycaemic control while adjusting for confounders . this study was approved by the university of zambia biomedical research ethics committee on reference number 005 - 07 - 13 . written informed consent was obtained from all study participants randomly selected which ensured that all eligible persons were given an equal chance to participate or decline . in similar manner , the patients were not given any direct immediate benefits as they were being interviewed within the hospital environment and at the time that they came for routine referral , consultation , monitoring , or review . of all the patients enrolled for the study ( n = 198 ) , median age was 55 years ( iqr 45 , 62 ) . most ( 92.9% ) of the patients had type 2 dm in contrast to the 7.1% that had type 1 dm . in addition , majority ( 61.3% ) of the patients had poor glycaemic control status and only 38.7% had good glycemic control among those whose data was complete . the mean ( sd ) fpg of the patients was 9.65 4.96 mmol / l . most ( 73.7% ) of the patients reported not following the treatment regimen as prescribed ( adherence ) in contrast to the 52 ( 26.3% ) participants that reported adherence to the type of antidiabetic treatment they were on . amongst the patients who reported sbgm at home , 13 ( 6.6% ) of the patients reported monitoring glucose control at the public health facility , 2.5% own glucometer , and 4.0% reported glucose monitoring at the private health facility . the majority ( 59.6% ) of the patients the glycaemic control status of the patients according to the characteristics and self - management behaviours is shown in table 2 . in bivariate analysis , there was an association between glycaemic control status and adherence to treatment and fpg . however , there was no association between age , sex , education , bmi , sbgm , means of sbgm , exercise , and glycaemic control status . the multivariate binary logistic regression model was tested for multicollinearity , hosmer and lemeshow test of model fitness for data , and omnibus test of model coefficients and classification accuracy . the dependent variable was glycaemic control status : good ( 1 ) and poor ( 0 ) . the results of the multivariate binary logistic regression analysis to predict whether the 9 variable factors , age , sex , education level , bmi , adherence to treatment , sbgm , sbgm means , and exercise , predicted glycaemic control status showed that only adherence to treatment and fpg predicted glycaemic control status of the patients ( table 3 ) . thus , the patients who do not adhere to antidiabetic treatment and those with mean ( sd ) fpg , 10.26 5.17 mmol / l , are 68% and 7% less likely to achieve good glycaemic control status ( table 3 ) . it is well established that nonadherence rates for chronic disease regimens and for lifestyle changes are generally approximately 50% , and patients with diabetes are particularly prone to regimen adherence problems especially when on multiple treatment regimens including medications , lifestyle , diet , and exercise [ 3033 ] . consequently , successful management of dm is challenging , yet patients with good self - care behaviours can achieve excellent glycaemic control and avoid frequent diabetic complications . however , many patients are devoid of optimal self - care behaviours and continue to suffer from complications of the disease . regular sbgm empowers patients to play a role in the management of diabetes , simultaneously improving their metabolic parameters . glucometers are frequently used to assess the fpg and the results can prompt and help the patient to adjust the diet ( especially carbohydrate intake ) , exercise , and improve adherence to or modify medication dosage . the consistent use and correct response to the glycaemic results have been shown to improve glycaemic control in type 2 diabetes , consequently preventing or delaying further complications of diabetes . in this study we found evidence of poor glycaemic control which was significantly associated with poor adherence to medication use among diabetic patients that regularly attend medical review at the diabetic clinic . however , poor glycaemic control was not strongly associated with age , sex , education level , sbgm and means of sbgm , exercise , and bmi , associations that have been observed elsewhere [ 3537 ] . however , adherence to antidiabetic treatment and fpg were statistically significantly associated with higher hba1c values . these findings are not surprising given that as a developing nation zambia lacks the resources and capacity to manage this disease which could be associated with adherence . while developed nations have processes , strategies , and infrastructures imbedded in their health care programs , including electronic continuous monitoring technology , that allow medical management of both early and late stage disease , developing nations have inadequate capability to manage and reverse the increasing morbidity and mortality . this association with adherence to antidiabetic treatment and fpg observed in this study is a common problem among individuals with diabetes [ 4043 ] . consequently , this study has persuaded us to consider a focus for a changed interventional approach targeting selected factors including patient centered approaches such as understanding patient insight of the disease , and collaborative and clear communication between health care professionals and patients could impact positively on glycaemic control in these patients . firstly , we are aware that this was a cross - sectional study and therefore it is difficult to establish a causal relation between hba1c and the self - management behaviours . fourthly and lastly though not the least , we also report that there was incomplete data on medical records of the diabetic outpatients at the clinic , making it difficult to follow the morbidity and mortality patterns and thus reducing further the possible sample which would have improved the study power and understanding of possible determinants . in zambia the mortality of patients with type 2 dm is likely to continue to increase as the zambian economy improves a factor associated with increase in western life style including diet and sedentary way of life . a needs ' assessment for the noncommunicable disease program carried out by the moh identified deficiencies in diabetic control as having inadequacies in terms of drugs and laboratory reagents , diagnostic facilities , expertise , and community awareness for dm . it may thus be not surprising to find presence of individual factors associated with poor glycaemic control status where we further argue that these are also associated with not only system supply factors but also social factors , predominantly operating at an individual level . consequently , there is need to define dm disease burden and epidemiology with focus on determinants and thus determine potential strategies that could address these inadequacies and improve the disease outcome . the majority of diabetic outpatients in this study had poor glycaemic control status and this could be due to a variety of factors including lack of resources and ability to buy diabetic chips and strips that allow for more frequent checks of fpg levels and/or ability to have adequate resources to store , for example , the insulin in a refrigerator which is frequently not available to many zambians . the reasons for this were not the focus of this paper and so we may only speculate regarding possible reasons for these differential glycaemic control status associations . we , however , disagree with what other evidence suggest that this can also probably be because of poor diet and exercise habits and other multiple barriers . however , good glycaemic control was reported in japan and germany also and it has been argued that perhaps this is because of higher literacy levels in these countries resulting in improved knowledge translation about dm [ 47 , 48 ] . it was interesting to note that there was a statistically significant association between adherence to antidiabetic treatment and glycaemic control status of the diabetic outpatients in this study . this is important given that the effectiveness of drug treatment depends primarily on the efficacy of the prescribed treatment and adherence of the patient to the treatment . these findings have been supported by studies elsewhere which have shown that adherence to antidiabetic treatment among diabetic patients is poor and the possible reasons have been outlined . one of the reasons is simply failure to understand and consequently failure to comply with the prescribed clinical regimen , thereby resulting in very poor outcomes . in the present study , this was illustrated clearly where we observed that the diabetic outpatients who did not adhere to dm treatment had 68% decrease in the likelihood of achieving good glycaemic control status compared to those who adhered . in malaysia , 53.0% of the patients were nonadherent to dm treatment and the main factor which was associated with that nonadherence was age . the factors related to poor adherence were comorbidity , overall health level , number of drugs , and complexity of the drug regimen . there is thus a critical need to understand adherence related factors so as to design prevention and control strategies that will be operable but accounting for contextual differentials even across low income countries in general . such increase is possible if behavioural linked factors such as education attainment , which has already been shown to improve glycaemic control status , are targeted [ 51 , 52 ] , however , tackling nonadherence is not a simple matter , as it is multifactorial and might include cost adjustments , health belief transformations , dosing frequency repackaging , and assessment of the presence of potential confounders associated with personality disorders and patient - provider relationship . there is need to explore the effective behavioural interventions that can improve adherence to antidiabetic treatment among the diabetic patients in zambia . changing interventional approaches targeting selected factors including patient centered approaches such as understanding patient insight of the disease and collaborative and clear communication between health care professionals and patients could impact positively on glycaemic control in diabetic patients . thus , the role of diabetic patients in the management of their diabetes remains paramount . if this matter is critically managed , it is possible that the outcome of treatment would be much more satisfactory among diabetic outpatients and this could possibly delay the development of the complications of dm and improve the quality and length of lives for the affected individuals . there is thus need to institute prevention and control mechanism that are cost - effective , acceptable , and appropriate . we conclude that , among diabetic patients , poor glycaemic control remains a challenge and this may to a greater extent be associated with this may suggest limitations in past prevention and control efforts for dm at individual level as well as at care level where monitoring dynamics are limited as these patients are largely outpatients except when they are admitted to hospital for one reason or another . however , finding that fpg predicted the glycaemic control opens potential opportunities to routinely examine and identify most at risk groups , which in turn and further opens possibilities to study the associated dynamics linked to care and support . furthermore , and given that the self - management behaviours of diabetic patients play an important role in the management of dm , there is thus need to target improvement of the efficacy of individual strategies in all prevention and control strategies for these patients . the health care providers also are critical stakeholders and need to foster and place greater emphasis on counselling and improving adherence , notwithstanding the context specific differences .

crc cases were selected from patients who underwent surgical treatment at eulji university hospital from january 2000 to june 2005 . all cases were histologically confirmed to be primary colorectal adenocarcinoma and hematoxylin and eosin slides were re - evaluated by two independent pathologists . the tumor grade of the adenocarcinoma was classified into low grade ( 50% of tumor glands ) and high grade ( < 50% of tumor glands ) . for signet ring cell carcinoma and mucinous adenocarcinoma , tumor budding was defined as a single or group of less than five detached tumor cells and classified into two grades . tumor recurrence was designated as tumor occurring at the anastomosing site , in the regional lymph nodes , and the pelvic cavity diagnosed by radiology , colonoscopy , exploratory surgical , and/or histological examination . in addition , metastasis was defined as the presence of tumor cells outside the area of resection , including the liver , pancreas , lung , and other organs . all cases of crc tissue with accompanying normal mucosal tissue were fixed in 10% buffered formalin for 24 hours and embedded in paraffin . tissue sections of 34 m thickness were cut and mounted on probeon slides ( fisher scientific , pittsburgh , pa , usa ) . sections that contained both tumor and adjacent uninvolved colonic mucosa were selected for immunohistochemistry ( ihc ) in most cases . in a few cases , sections were trimmed in order to decrease the surface area for an even distribution of antibodies , so that only the tumor portion was included in the ihc evaluation . paraffin embedded tissue sections were deparaffinized and rehydrated through a series of xylene and alcohol . slides were then treated with 10 mm / l sodium citrate buffer ( ph 6.1 ) for 15 minutes and autoclaved at 120c for antigen retrieval . all slide sections for ihc were incubated in 3% h2o2 for 10 minutes to inactivate endogenous peroxidase , washed with 10 mm / l phosphate buffered saline buffer ( ph 7.4 ) , and then incubated with normal bovine serum to reduce false - positive staining . saco , me , usa ) and hif-1 ( 1:50 , novus biologicals , littleton , co , usa ) were used as primary antibodies . slide sections were incubated with primary antibodies overnight at 4c in a wet chamber and stained with diaminobenzidine as the substrate using an envision - hrp kit ( dako , glostrup , denmark ) . sections were counterstained with mayer s hematoxylin solution and then mounted . to evaluate the expression of thymosin 4 and hif-1 in association with various clinico - pathological factors , the immunoreactivity of both thymosin 4 and hif-1 were analyzed in a semi - quantitative manner by two independent pathologists who were blinded to outcomes . immunoreactivity for thymosin 4 and hif-1 was observed primarily in the cytoplasm and nuclei of normal mucosal epithelium and tumor cells , respectively . the intensity of immunohistochemical staining was scored as 0 to 2 ( 0 , weaker staining than the normal mucosal epithelium ; 1 , staining similar to the normal mucosal epithelium ; and 2 , stronger staining than the normal mucosal epithelium ) . the percentage of positive cells was scored as 1 ( < 25% of tumor cells ) , 2 ( 25%49% of tumor cells ) , 3 ( 50%74% of tumor cells ) , and 4 ( 75% of tumor cells ) . to evaluate the statistical significance between thymosin 4 and hif-1 expression and clinico - pathological factors , the median value ( 25% of tumor cells showing a strong positive reaction than normal epithelium ) of the series was used as the cutoff value to distinguish between tumor cells with low expression ( < 25% tumor cells ) and high expression ( 25% of tumor cells ) . the correlation between thymosin 4 and the various clinico - pathological parameters were analyzed with the pearson s chi - square test or fisher exact test . to evaluate statistical analysis , recurrence - free survival was defined as the duration from the date of surgery to the first date of recurrence or the date of last follow - up . similarly , overall survival was defined as the duration from the date of surgical therapy to the date of death or date of last follow - up . the mean follow - up duration for all patients was 53.3 months , ranging from 0.6 to 121.9 months . using the kaplan - meier method , the recurrence - free survival curve and the overall survival curve were formulated . to examine the statistical significance of the differences in survival distribution , log - rank test was utilized . multivariate analysis for overall survival and recurrence - free survival was performed using cox proportional hazard regression analysis . in all statistical analyses , p - values less than .05 were considered statistically significant . the institutional review board of eulji university hospital approved the study protocol and provided all necessary ethical permission . crc cases were selected from patients who underwent surgical treatment at eulji university hospital from january 2000 to june 2005 . all cases were histologically confirmed to be primary colorectal adenocarcinoma and hematoxylin and eosin slides were re - evaluated by two independent pathologists . the tumor grade of the adenocarcinoma was classified into low grade ( 50% of tumor glands ) and high grade ( < 50% of tumor glands ) . for signet ring cell carcinoma and mucinous adenocarcinoma , tumor budding was defined as a single or group of less than five detached tumor cells and classified into two grades . tumor recurrence was designated as tumor occurring at the anastomosing site , in the regional lymph nodes , and the pelvic cavity diagnosed by radiology , colonoscopy , exploratory surgical , and/or histological examination . in addition , metastasis was defined as the presence of tumor cells outside the area of resection , including the liver , pancreas , lung , and other organs . all cases of crc tissue with accompanying normal mucosal tissue were fixed in 10% buffered formalin for 24 hours and embedded in paraffin . tissue sections of 34 m thickness were cut and mounted on probeon slides ( fisher scientific , pittsburgh , pa , usa ) . sections that contained both tumor and adjacent uninvolved colonic mucosa were selected for immunohistochemistry ( ihc ) in most cases . in a few cases , sections were trimmed in order to decrease the surface area for an even distribution of antibodies , so that only the tumor portion was included in the ihc evaluation . paraffin embedded tissue sections were deparaffinized and rehydrated through a series of xylene and alcohol . slides were then treated with 10 mm / l sodium citrate buffer ( ph 6.1 ) for 15 minutes and autoclaved at 120c for antigen retrieval . all slide sections for ihc were incubated in 3% h2o2 for 10 minutes to inactivate endogenous peroxidase , washed with 10 mm / l phosphate buffered saline buffer ( ph 7.4 ) , and then incubated with normal bovine serum to reduce false - positive staining . saco , me , usa ) and hif-1 ( 1:50 , novus biologicals , littleton , co , usa ) were used as primary antibodies . slide sections were incubated with primary antibodies overnight at 4c in a wet chamber and stained with diaminobenzidine as the substrate using an envision - hrp kit ( dako , glostrup , denmark ) . to evaluate the expression of thymosin 4 and hif-1 in association with various clinico - pathological factors , the immunoreactivity of both thymosin 4 and hif-1 were analyzed in a semi - quantitative manner by two independent pathologists who were blinded to outcomes . immunoreactivity for thymosin 4 and hif-1 was observed primarily in the cytoplasm and nuclei of normal mucosal epithelium and tumor cells , respectively . the intensity of immunohistochemical staining was scored as 0 to 2 ( 0 , weaker staining than the normal mucosal epithelium ; 1 , staining similar to the normal mucosal epithelium ; and 2 , stronger staining than the normal mucosal epithelium ) . the percentage of positive cells was scored as 1 ( < 25% of tumor cells ) , 2 ( 25%49% of tumor cells ) , 3 ( 50%74% of tumor cells ) , and 4 ( 75% of tumor cells ) . to evaluate the statistical significance between thymosin 4 and hif-1 expression and clinico - pathological factors , the median value ( 25% of tumor cells showing a strong positive reaction than normal epithelium ) of the series was used as the cutoff value to distinguish between tumor cells with low expression ( < 25% tumor cells ) and high expression ( 25% of tumor cells ) . the correlation between thymosin 4 and the various clinico - pathological parameters were analyzed with the pearson s chi - square test or fisher exact test . to evaluate statistical analysis , recurrence - free survival was defined as the duration from the date of surgery to the first date of recurrence or the date of last follow - up . similarly , overall survival was defined as the duration from the date of surgical therapy to the date of death or date of last follow - up . the mean follow - up duration for all patients was 53.3 months , ranging from 0.6 to 121.9 months . using the kaplan - meier method , the recurrence - free survival curve and the overall survival curve were formulated . to examine the statistical significance of the differences in survival distribution , log - rank test was utilized . multivariate analysis for overall survival and recurrence - free survival was performed using cox proportional hazard regression analysis . in all statistical analyses , p - values less than .05 were considered statistically significant . the institutional review board of eulji university hospital approved the study protocol and provided all necessary ethical permission . the median age of the 143 crc patients ( 75 men and 68 women ) at surgery was 62.2 years ( range , 28 to 86 years ) and the median tumor size was 5.2 cm ( range , 0.8 to 12.0 cm ) in maximum diameter . the majority of crcs were moderately differentiated adenocarcinoma and 111 cases ( 77.6% ) were classified as low grade and 32 cases ( 22.4% ) as high grade ( poorly differentiated 19 , signet ring cell carcinoma 3 , and mucinous carcinoma 10 ) . one hundred and four cases ( 72.7% ) showed lymphovascular tumor invasion and 106 cases ( 74.1% ) exhibited high - grade tumor budding . according to the seventh edition of the ajcc tnm system , 26 patients ( 18.2% ) were diagnosed with early - stage tumor invasion ( five cases of pt1 and 21 cases of pt2 ) and 117 patients ( 81.8% ) were diagnosed with advanced - stage tumor invasion ( 105 cases of pt3 and 12 cases of pt4 ) . seventy - six patients ( 53.1% ) presented with regional lymph node metastasis and 22 patients ( 15.4% ) with distant metastasis . twenty - one patients ( 14.7% ) were at ptnm stage i , 45 patients ( 31.5% ) were at stage ii , 55 patients ( 38.5% ) were at stage iii , and 22 patients ( 15.4% ) were at stage iv . twenty - seven patients ( 18.9% ) were treated with chemoradiation after the first surgery ( data not shown ) . clinico - pathological characteristics of the 143 crc patients are summarized in table 1 . in the normal colonic epithelium , immunoreactivity for thymosin 4 and hif-1 while thymosin 4 expression was found primarily in the cytoplasm of cancer cells , hif-1 was stained predominantly in the nuclei of tumor cells ( fig . a high level of thymosin 4 and hif-1 expression was observed in 66 of the 143 patients ( 46.2% ) and in 67 of the 143 patients ( 46.9% ) , respectively . we found that predictive factors for prognosis , such as lymphovascular invasion , invasion depth ( pt ) , regional lymph node metastasis ( pn ) , distant metastasis , and tnm stage showed statistically significant correlations with thymosin 4 immunoreactivity ( table 1 ) . patients with high thymosin 4 expression levels showed a significantly greater presence of lymphovascular invasion , more frequent regional lymph node metastasis , deeper invasion depth , and more advanced tumor stage than in those with low thymosin 4 expression levels ( p<.001 ) . we found a statistically significant correlation between high hif-1 immunohistochemical expression and clinico - pathological factors such as lymphovascular invasion ( p=.006 ) , invasion depth ( p=.002 ) , regional lymph node metastasis ( p=.007 ) , distant metastasis ( p=.029 ) , and tnm stage ( p=.002 ) ( data not shown ) . we performed multivariate analysis to examine the correlation between thymosin 4 expression levels with recurrence - free survival and overall survival . forty patients ( 28.0% ) presented with cancer recurrence during follow - up and 55 patients ( 38.5% ) died of crc with or without metastasis . seven patients ( 4.9% ) died of unknown causes , 17 patients ( 11.9% ) were alive with local recurrence and/or distant metastasis , and 64 patients ( 44.8% ) remained alive and recurrence - free . kaplan - meier analysis showed that high thymosin 4 expression was significantly correlated with decreased recurrence - free survival ( p<.001 ) ( fig . recurrence - free survival was shorter in patients with high expression levels of thymosin 4 , with a mean duration of 37.3 months ( 95% confidence interval [ ci ] , 27.833 to 46.684 ) , and was longer in patients with low levels of thymosin 4 expression , with a mean duration of 84.6 months ( 95% ci , 73.538 to 95.571 ) . we also found that high thymosin 4 expression significantly correlated with worse overall survival ( p=.001 ) . thymosin 4 expression status also significantly split the cumulative overall survival curves ( fig . 2b ) . while the overall survival of crc patients with high thymosin 4 expression was mean duration of 51.7 months ( 95% ci , 41.381 to 61.939 ) , the overall survival of crc patients with low thymosin 4 expression was longer with mean duration of 96.8 months ( 95% ci , 86.952 to 106.732 ) . multivariate analysis was also performed to assess the prognostic value of thymosin 4 expression for recurrence - free survival and overall survival using various clinico - pathological parameters . statistically significant clinico - pathological factors that were correlated with overall survival were high thymosin 4 expression ( p=.005 ) , high tumor grade ( p=.013 ) , high tumor budding ( p=.047 ) , and presence of distant metastasis ( p=.001 ) . the relative risk ( rr ) of death in patients with a high expression level of thymosin 4 was more than two times greater ( rr , 2.457 ; 95% ci , 1.315 to 4.592 ) than those with low thymosin 4 expression levels . high thymosin 4 expression was also an independent and relevant factor of decreased recurrence - free survival ( p=.001 ) . the rr of recurrence for patients with high thymosin 4 expression level was 2.540 ( 95% ci , 1.479 to 4.362 ) . a statistically significant correlation between high hif-1 expression and high thymosin 4 expression was also found ( p<.001 ) . specifically , of the 66 cases exhibiting high thymosin 4 expression , 49 cases ( 74.2% ) also showed high nuclear immunoreactivity of hif-1. of the 77 cases expressing low thymosin 4 expression , 59 cases ( 76.6% ) revealed corresponding low hif-1 expression ( table 3 ) . the median age of the 143 crc patients ( 75 men and 68 women ) at surgery was 62.2 years ( range , 28 to 86 years ) and the median tumor size was 5.2 cm ( range , 0.8 to 12.0 cm ) in maximum diameter . the majority of crcs were moderately differentiated adenocarcinoma and 111 cases ( 77.6% ) were classified as low grade and 32 cases ( 22.4% ) as high grade ( poorly differentiated 19 , signet ring cell carcinoma 3 , and mucinous carcinoma 10 ) . one hundred and four cases ( 72.7% ) showed lymphovascular tumor invasion and 106 cases ( 74.1% ) exhibited high - grade tumor budding . according to the seventh edition of the ajcc tnm system , 26 patients ( 18.2% ) were diagnosed with early - stage tumor invasion ( five cases of pt1 and 21 cases of pt2 ) and 117 patients ( 81.8% ) were diagnosed with advanced - stage tumor invasion ( 105 cases of pt3 and 12 cases of pt4 ) . seventy - six patients ( 53.1% ) presented with regional lymph node metastasis and 22 patients ( 15.4% ) with distant metastasis . twenty - one patients ( 14.7% ) were at ptnm stage i , 45 patients ( 31.5% ) were at stage ii , 55 patients ( 38.5% ) were at stage iii , and 22 patients ( 15.4% ) were at stage iv . twenty - seven patients ( 18.9% ) were treated with chemoradiation after the first surgery ( data not shown ) . clinico - pathological characteristics of the 143 crc patients are summarized in table 1 . in the normal colonic epithelium , immunoreactivity for thymosin 4 and hif-1 while thymosin 4 expression was found primarily in the cytoplasm of cancer cells , hif-1 was stained predominantly in the nuclei of tumor cells ( fig . a high level of thymosin 4 and hif-1 expression was observed in 66 of the 143 patients ( 46.2% ) and in 67 of the 143 patients ( 46.9% ) , respectively . we found that predictive factors for prognosis , such as lymphovascular invasion , invasion depth ( pt ) , regional lymph node metastasis ( pn ) , distant metastasis , and tnm stage showed statistically significant correlations with thymosin 4 immunoreactivity ( table 1 ) . patients with high thymosin 4 expression levels showed a significantly greater presence of lymphovascular invasion , more frequent regional lymph node metastasis , deeper invasion depth , and more advanced tumor stage than in those with low thymosin 4 expression levels ( p<.001 ) . we found a statistically significant correlation between high hif-1 immunohistochemical expression and clinico - pathological factors such as lymphovascular invasion ( p=.006 ) , invasion depth ( p=.002 ) , regional lymph node metastasis ( p=.007 ) , distant metastasis ( p=.029 ) , and tnm stage ( p=.002 ) ( data not shown ) . we performed multivariate analysis to examine the correlation between thymosin 4 expression levels with recurrence - free survival and overall survival . forty patients ( 28.0% ) presented with cancer recurrence during follow - up and 55 patients ( 38.5% ) died of crc with or without metastasis . seven patients ( 4.9% ) died of unknown causes , 17 patients ( 11.9% ) were alive with local recurrence and/or distant metastasis , and 64 patients ( 44.8% ) remained alive and recurrence - free . kaplan - meier analysis showed that high thymosin 4 expression was significantly correlated with decreased recurrence - free survival ( p<.001 ) ( fig . recurrence - free survival was shorter in patients with high expression levels of thymosin 4 , with a mean duration of 37.3 months ( 95% confidence interval [ ci ] , 27.833 to 46.684 ) , and was longer in patients with low levels of thymosin 4 expression , with a mean duration of 84.6 months ( 95% ci , 73.538 to 95.571 ) . we also found that high thymosin 4 expression significantly correlated with worse overall survival ( p=.001 ) . while the overall survival of crc patients with high thymosin 4 expression was mean duration of 51.7 months ( 95% ci , 41.381 to 61.939 ) , the overall survival of crc patients with low thymosin 4 expression was longer with mean duration of 96.8 months ( 95% ci , 86.952 to 106.732 ) . multivariate analysis was also performed to assess the prognostic value of thymosin 4 expression for recurrence - free survival and overall survival using various clinico - pathological parameters . statistically significant clinico - pathological factors that were correlated with overall survival were high thymosin 4 expression ( p=.005 ) , high tumor grade ( p=.013 ) , high tumor budding ( p=.047 ) , and presence of distant metastasis ( p=.001 ) . the relative risk ( rr ) of death in patients with a high expression level of thymosin 4 was more than two times greater ( rr , 2.457 ; 95% ci , 1.315 to 4.592 ) than those with low thymosin 4 expression levels . high thymosin 4 expression was also an independent and relevant factor of decreased recurrence - free survival ( p=.001 ) . the rr of recurrence for patients with high thymosin 4 expression level was 2.540 ( 95% ci , 1.479 to 4.362 ) . a statistically significant correlation between high hif-1 expression and high thymosin 4 expression was also found ( p<.001 ) . specifically , of the 66 cases exhibiting high thymosin 4 expression , 49 cases ( 74.2% ) also showed high nuclear immunoreactivity of hif-1. of the 77 cases expressing low thymosin 4 expression , 59 cases ( 76.6% ) revealed corresponding low hif-1 expression ( table 3 ) . growing tumors require oxygen and nutrient delivery through neovasculature . however , intratumoral hypoxia induced by imbalance between tumor growth and insufficient angiogenesis can lead to expression of hif-1 , which is a transcriptional factor that activates tumor survival in an unstable hypoxic tumor microenvironment [ 29 - 32 ] . recent reports have shown that thymosin 4 stabilizes hif-1 in human cancer cells and that thymosin 4 also induces migration and metastasis of colon cancer cells via the ilk / iqgap1/rac1 signal transduction pathway . few studies have evaluated whether overexpression of thymosin 4 influences clinical prognosis and whether thymosin 4 is related to hif-1 in crcs . to better understand the relationship , we analyzed the clinical significance and expression status of thymosin 4 and hif-1 in crc patients . our study demonstrates that high thymosin 4 expression has a significant association with lymphovascular invasion , nodal status , distant metastasis , and tumor progression in crc patients ( p<.001 ) . this finding is consistent with our previous study showing hypoxia - induced high expression of thymosin 4 , which also significantly correlated with regional lymph node metastasis in breast cancer . in a previous study , we found thymosin 4 to be up - regulated under hypoxic conditions ( 5% o2 ) using an in vitro hypoxia - induced model to generate transcription profiles in human crc . based on these findings , for this study we examined the association between thymosin 4 expression and hif-1 expression in crc specimens . we then discovered that the overexpression of thymosin 4 in crc is closely related to the restricted overexpression of hif-1 in the crc cells ( p<.001 ) . recently , there have been reports regarding the association between thymosin 4 expression with tumor development and epithelial mesenchymal transition ( emt ) . in particular , nemolato et al . reported high expression of thymosin 4 at the invasive front in colon cancer and discussed its associated with emt as well as invasion and metastasis of tumor cells . however , from our study , since we were unable to find an association between thymosin 4 expression and tumor budding ( p=.118 ) and tumor border ( p=.560 ) , we found the direct association of thymosin 4 expression with emt to be weak . the hif complex , which involves various hypoxia - regulated genes , is a group of critical gene products in the tumor microenvironment of hypoxic adaptation and in angiogenesis . the hif complex is also an essential mediator in coordinating transcription of various factors in the tumor cells to survive in the hypoxic environment and its overexpression has been associated with increased mortality in various cancer types [ 31,35 - 37 ] . among hif whether hif-2 , hif-3 , and hif-1 also play critical roles in the hif pathway and regulate hif target genes is not yet clearly known [ 38 - 41 ] . hif-1 is frequently upregulated in various cancer cells and the overexpression of hif-1 correlates with advanced cancer progression or aggressiveness . however , the clinical significance of hif-1 in crc has not been extensively studied . in this study , we observed a significant association between thymosin 4 expression and hif-1 expression ( p<.001 ) . this result coincides with previous studies that found overexpression of hif-1 to be associated with poor prognosis . thymosin 4 has various functional roles in normal cell biology and its mechanism of action has recently been studied in various tumors . in this study , we found that high cytoplasmic expression of thymosin 4 is clinically important and an independent prognostic factor for crc patients . as our results demonstrate that high thymosin 4 expression significantly correlates with tumor recurrence and worse overall survival , we suggest that high thymosin 4 expression may be a useful prognostic factor in crc . our results demonstrate that hif-1 is correlated with overexpression of thymosin 4 in human crc . although further studies are necessary to further validate our findings , we suggest that thymosin 4 , has potential as a prognostic biomarker and has potential as a hif pathway target in human crc .

backgroundthymosin 4 is a multi - functional hormone - like polypeptide , being involved in cell migration , angiogenesis , and tumor metastasis . this study was undertaken to clarify the clinicopathologic implications of thymosin 4 expression in human colorectal cancers ( crcs).methodswe investigated tissue sections from 143 patients with crc by immunohistochemistry . in addition , we evaluated the expression patterns and the clinico - pathological significance of thymosin 4 expression in association with hypoxia inducible factor-1 ( hif-1 ) expression in the crc series.resultshigh expression of thymosin 4 was significantly correlated with lymphovascular invasion , invasion depth , regional lymph node metastasis , distant metastasis , and tnm stage . patients with high expression of thymosin 4 showed poor recurrence - free survival ( p = .001 ) and poor overall survival ( p = .005 ) on multivariate analysis . we also found that thymosin 4 and hif-1 were overexpressed and that thymosin 4 expression increased in parallel with hif-1 expression in crc.conclusionsa high expression level of thymosin 4 indicates poor clinical outcomes and may be a useful prognostic factor in crc . thymosin 4 is functionally related with hif-1 and may be a potentially valuable biomarker and possible therapeutic target for crc .

MATERIALS AND METHODS Case selection and immunohistochemistry Assessment of IHC staining Statistical analysis of prognostic parameters Ethical permission RESULTS Association of clinico-pathological characteristics with thymosin High thymosin DISCUSSION

tumor recurrence was designated as tumor occurring at the anastomosing site , in the regional lymph nodes , and the pelvic cavity diagnosed by radiology , colonoscopy , exploratory surgical , and/or histological examination . in addition , metastasis was defined as the presence of tumor cells outside the area of resection , including the liver , pancreas , lung , and other organs . tissue sections of 34 m thickness were cut and mounted on probeon slides ( fisher scientific , pittsburgh , pa , usa ) . in a few cases , sections were trimmed in order to decrease the surface area for an even distribution of antibodies , so that only the tumor portion was included in the ihc evaluation . saco , me , usa ) and hif-1 ( 1:50 , novus biologicals , littleton , co , usa ) were used as primary antibodies . to evaluate the expression of thymosin 4 and hif-1 in association with various clinico - pathological factors , the immunoreactivity of both thymosin 4 and hif-1 were analyzed in a semi - quantitative manner by two independent pathologists who were blinded to outcomes . immunoreactivity for thymosin 4 and hif-1 was observed primarily in the cytoplasm and nuclei of normal mucosal epithelium and tumor cells , respectively . to evaluate the statistical significance between thymosin 4 and hif-1 expression and clinico - pathological factors , the median value ( 25% of tumor cells showing a strong positive reaction than normal epithelium ) of the series was used as the cutoff value to distinguish between tumor cells with low expression ( < 25% tumor cells ) and high expression ( 25% of tumor cells ) . the correlation between thymosin 4 and the various clinico - pathological parameters were analyzed with the pearson s chi - square test or fisher exact test . to evaluate statistical analysis , recurrence - free survival was defined as the duration from the date of surgery to the first date of recurrence or the date of last follow - up . using the kaplan - meier method , the recurrence - free survival curve and the overall survival curve were formulated . multivariate analysis for overall survival and recurrence - free survival was performed using cox proportional hazard regression analysis . the tumor grade of the adenocarcinoma was classified into low grade ( 50% of tumor glands ) and high grade ( < 50% of tumor glands ) . tumor recurrence was designated as tumor occurring at the anastomosing site , in the regional lymph nodes , and the pelvic cavity diagnosed by radiology , colonoscopy , exploratory surgical , and/or histological examination . in addition , metastasis was defined as the presence of tumor cells outside the area of resection , including the liver , pancreas , lung , and other organs . saco , me , usa ) and hif-1 ( 1:50 , novus biologicals , littleton , co , usa ) were used as primary antibodies . to evaluate the expression of thymosin 4 and hif-1 in association with various clinico - pathological factors , the immunoreactivity of both thymosin 4 and hif-1 were analyzed in a semi - quantitative manner by two independent pathologists who were blinded to outcomes . immunoreactivity for thymosin 4 and hif-1 was observed primarily in the cytoplasm and nuclei of normal mucosal epithelium and tumor cells , respectively . to evaluate the statistical significance between thymosin 4 and hif-1 expression and clinico - pathological factors , the median value ( 25% of tumor cells showing a strong positive reaction than normal epithelium ) of the series was used as the cutoff value to distinguish between tumor cells with low expression ( < 25% tumor cells ) and high expression ( 25% of tumor cells ) . the correlation between thymosin 4 and the various clinico - pathological parameters were analyzed with the pearson s chi - square test or fisher exact test . to evaluate statistical analysis , recurrence - free survival was defined as the duration from the date of surgery to the first date of recurrence or the date of last follow - up . similarly , overall survival was defined as the duration from the date of surgical therapy to the date of death or date of last follow - up . using the kaplan - meier method , the recurrence - free survival curve and the overall survival curve were formulated . multivariate analysis for overall survival and recurrence - free survival was performed using cox proportional hazard regression analysis . the median age of the 143 crc patients ( 75 men and 68 women ) at surgery was 62.2 years ( range , 28 to 86 years ) and the median tumor size was 5.2 cm ( range , 0.8 to 12.0 cm ) in maximum diameter . according to the seventh edition of the ajcc tnm system , 26 patients ( 18.2% ) were diagnosed with early - stage tumor invasion ( five cases of pt1 and 21 cases of pt2 ) and 117 patients ( 81.8% ) were diagnosed with advanced - stage tumor invasion ( 105 cases of pt3 and 12 cases of pt4 ) . seventy - six patients ( 53.1% ) presented with regional lymph node metastasis and 22 patients ( 15.4% ) with distant metastasis . clinico - pathological characteristics of the 143 crc patients are summarized in table 1 . in the normal colonic epithelium , immunoreactivity for thymosin 4 and hif-1 while thymosin 4 expression was found primarily in the cytoplasm of cancer cells , hif-1 was stained predominantly in the nuclei of tumor cells ( fig . a high level of thymosin 4 and hif-1 expression was observed in 66 of the 143 patients ( 46.2% ) and in 67 of the 143 patients ( 46.9% ) , respectively . we found that predictive factors for prognosis , such as lymphovascular invasion , invasion depth ( pt ) , regional lymph node metastasis ( pn ) , distant metastasis , and tnm stage showed statistically significant correlations with thymosin 4 immunoreactivity ( table 1 ) . patients with high thymosin 4 expression levels showed a significantly greater presence of lymphovascular invasion , more frequent regional lymph node metastasis , deeper invasion depth , and more advanced tumor stage than in those with low thymosin 4 expression levels ( p<.001 ) . we found a statistically significant correlation between high hif-1 immunohistochemical expression and clinico - pathological factors such as lymphovascular invasion ( p=.006 ) , invasion depth ( p=.002 ) , regional lymph node metastasis ( p=.007 ) , distant metastasis ( p=.029 ) , and tnm stage ( p=.002 ) ( data not shown ) . we performed multivariate analysis to examine the correlation between thymosin 4 expression levels with recurrence - free survival and overall survival . seven patients ( 4.9% ) died of unknown causes , 17 patients ( 11.9% ) were alive with local recurrence and/or distant metastasis , and 64 patients ( 44.8% ) remained alive and recurrence - free . kaplan - meier analysis showed that high thymosin 4 expression was significantly correlated with decreased recurrence - free survival ( p<.001 ) ( fig . recurrence - free survival was shorter in patients with high expression levels of thymosin 4 , with a mean duration of 37.3 months ( 95% confidence interval [ ci ] , 27.833 to 46.684 ) , and was longer in patients with low levels of thymosin 4 expression , with a mean duration of 84.6 months ( 95% ci , 73.538 to 95.571 ) . we also found that high thymosin 4 expression significantly correlated with worse overall survival ( p=.001 ) . thymosin 4 expression status also significantly split the cumulative overall survival curves ( fig . while the overall survival of crc patients with high thymosin 4 expression was mean duration of 51.7 months ( 95% ci , 41.381 to 61.939 ) , the overall survival of crc patients with low thymosin 4 expression was longer with mean duration of 96.8 months ( 95% ci , 86.952 to 106.732 ) . multivariate analysis was also performed to assess the prognostic value of thymosin 4 expression for recurrence - free survival and overall survival using various clinico - pathological parameters . statistically significant clinico - pathological factors that were correlated with overall survival were high thymosin 4 expression ( p=.005 ) , high tumor grade ( p=.013 ) , high tumor budding ( p=.047 ) , and presence of distant metastasis ( p=.001 ) . the relative risk ( rr ) of death in patients with a high expression level of thymosin 4 was more than two times greater ( rr , 2.457 ; 95% ci , 1.315 to 4.592 ) than those with low thymosin 4 expression levels . high thymosin 4 expression was also an independent and relevant factor of decreased recurrence - free survival ( p=.001 ) . the rr of recurrence for patients with high thymosin 4 expression level was 2.540 ( 95% ci , 1.479 to 4.362 ) . a statistically significant correlation between high hif-1 expression and high thymosin 4 expression was also found ( p<.001 ) . specifically , of the 66 cases exhibiting high thymosin 4 expression , 49 cases ( 74.2% ) also showed high nuclear immunoreactivity of hif-1. of the 77 cases expressing low thymosin 4 expression , 59 cases ( 76.6% ) revealed corresponding low hif-1 expression ( table 3 ) . the median age of the 143 crc patients ( 75 men and 68 women ) at surgery was 62.2 years ( range , 28 to 86 years ) and the median tumor size was 5.2 cm ( range , 0.8 to 12.0 cm ) in maximum diameter . the majority of crcs were moderately differentiated adenocarcinoma and 111 cases ( 77.6% ) were classified as low grade and 32 cases ( 22.4% ) as high grade ( poorly differentiated 19 , signet ring cell carcinoma 3 , and mucinous carcinoma 10 ) . according to the seventh edition of the ajcc tnm system , 26 patients ( 18.2% ) were diagnosed with early - stage tumor invasion ( five cases of pt1 and 21 cases of pt2 ) and 117 patients ( 81.8% ) were diagnosed with advanced - stage tumor invasion ( 105 cases of pt3 and 12 cases of pt4 ) . seventy - six patients ( 53.1% ) presented with regional lymph node metastasis and 22 patients ( 15.4% ) with distant metastasis . clinico - pathological characteristics of the 143 crc patients are summarized in table 1 . in the normal colonic epithelium , immunoreactivity for thymosin 4 and hif-1 while thymosin 4 expression was found primarily in the cytoplasm of cancer cells , hif-1 was stained predominantly in the nuclei of tumor cells ( fig . a high level of thymosin 4 and hif-1 expression was observed in 66 of the 143 patients ( 46.2% ) and in 67 of the 143 patients ( 46.9% ) , respectively . we found that predictive factors for prognosis , such as lymphovascular invasion , invasion depth ( pt ) , regional lymph node metastasis ( pn ) , distant metastasis , and tnm stage showed statistically significant correlations with thymosin 4 immunoreactivity ( table 1 ) . patients with high thymosin 4 expression levels showed a significantly greater presence of lymphovascular invasion , more frequent regional lymph node metastasis , deeper invasion depth , and more advanced tumor stage than in those with low thymosin 4 expression levels ( p<.001 ) . we found a statistically significant correlation between high hif-1 immunohistochemical expression and clinico - pathological factors such as lymphovascular invasion ( p=.006 ) , invasion depth ( p=.002 ) , regional lymph node metastasis ( p=.007 ) , distant metastasis ( p=.029 ) , and tnm stage ( p=.002 ) ( data not shown ) . we performed multivariate analysis to examine the correlation between thymosin 4 expression levels with recurrence - free survival and overall survival . seven patients ( 4.9% ) died of unknown causes , 17 patients ( 11.9% ) were alive with local recurrence and/or distant metastasis , and 64 patients ( 44.8% ) remained alive and recurrence - free . kaplan - meier analysis showed that high thymosin 4 expression was significantly correlated with decreased recurrence - free survival ( p<.001 ) ( fig . recurrence - free survival was shorter in patients with high expression levels of thymosin 4 , with a mean duration of 37.3 months ( 95% confidence interval [ ci ] , 27.833 to 46.684 ) , and was longer in patients with low levels of thymosin 4 expression , with a mean duration of 84.6 months ( 95% ci , 73.538 to 95.571 ) . we also found that high thymosin 4 expression significantly correlated with worse overall survival ( p=.001 ) . while the overall survival of crc patients with high thymosin 4 expression was mean duration of 51.7 months ( 95% ci , 41.381 to 61.939 ) , the overall survival of crc patients with low thymosin 4 expression was longer with mean duration of 96.8 months ( 95% ci , 86.952 to 106.732 ) . multivariate analysis was also performed to assess the prognostic value of thymosin 4 expression for recurrence - free survival and overall survival using various clinico - pathological parameters . statistically significant clinico - pathological factors that were correlated with overall survival were high thymosin 4 expression ( p=.005 ) , high tumor grade ( p=.013 ) , high tumor budding ( p=.047 ) , and presence of distant metastasis ( p=.001 ) . the relative risk ( rr ) of death in patients with a high expression level of thymosin 4 was more than two times greater ( rr , 2.457 ; 95% ci , 1.315 to 4.592 ) than those with low thymosin 4 expression levels . high thymosin 4 expression was also an independent and relevant factor of decreased recurrence - free survival ( p=.001 ) . the rr of recurrence for patients with high thymosin 4 expression level was 2.540 ( 95% ci , 1.479 to 4.362 ) . a statistically significant correlation between high hif-1 expression and high thymosin 4 expression was also found ( p<.001 ) . specifically , of the 66 cases exhibiting high thymosin 4 expression , 49 cases ( 74.2% ) also showed high nuclear immunoreactivity of hif-1. of the 77 cases expressing low thymosin 4 expression , 59 cases ( 76.6% ) revealed corresponding low hif-1 expression ( table 3 ) . however , intratumoral hypoxia induced by imbalance between tumor growth and insufficient angiogenesis can lead to expression of hif-1 , which is a transcriptional factor that activates tumor survival in an unstable hypoxic tumor microenvironment [ 29 - 32 ] . recent reports have shown that thymosin 4 stabilizes hif-1 in human cancer cells and that thymosin 4 also induces migration and metastasis of colon cancer cells via the ilk / iqgap1/rac1 signal transduction pathway . few studies have evaluated whether overexpression of thymosin 4 influences clinical prognosis and whether thymosin 4 is related to hif-1 in crcs . to better understand the relationship , we analyzed the clinical significance and expression status of thymosin 4 and hif-1 in crc patients . our study demonstrates that high thymosin 4 expression has a significant association with lymphovascular invasion , nodal status , distant metastasis , and tumor progression in crc patients ( p<.001 ) . this finding is consistent with our previous study showing hypoxia - induced high expression of thymosin 4 , which also significantly correlated with regional lymph node metastasis in breast cancer . in a previous study , we found thymosin 4 to be up - regulated under hypoxic conditions ( 5% o2 ) using an in vitro hypoxia - induced model to generate transcription profiles in human crc . based on these findings , for this study we examined the association between thymosin 4 expression and hif-1 expression in crc specimens . we then discovered that the overexpression of thymosin 4 in crc is closely related to the restricted overexpression of hif-1 in the crc cells ( p<.001 ) . recently , there have been reports regarding the association between thymosin 4 expression with tumor development and epithelial mesenchymal transition ( emt ) . reported high expression of thymosin 4 at the invasive front in colon cancer and discussed its associated with emt as well as invasion and metastasis of tumor cells . however , from our study , since we were unable to find an association between thymosin 4 expression and tumor budding ( p=.118 ) and tumor border ( p=.560 ) , we found the direct association of thymosin 4 expression with emt to be weak . the hif complex , which involves various hypoxia - regulated genes , is a group of critical gene products in the tumor microenvironment of hypoxic adaptation and in angiogenesis . among hif whether hif-2 , hif-3 , and hif-1 also play critical roles in the hif pathway and regulate hif target genes is not yet clearly known [ 38 - 41 ] . hif-1 is frequently upregulated in various cancer cells and the overexpression of hif-1 correlates with advanced cancer progression or aggressiveness . however , the clinical significance of hif-1 in crc has not been extensively studied . in this study , we observed a significant association between thymosin 4 expression and hif-1 expression ( p<.001 ) . thymosin 4 has various functional roles in normal cell biology and its mechanism of action has recently been studied in various tumors . in this study , we found that high cytoplasmic expression of thymosin 4 is clinically important and an independent prognostic factor for crc patients . as our results demonstrate that high thymosin 4 expression significantly correlates with tumor recurrence and worse overall survival , we suggest that high thymosin 4 expression may be a useful prognostic factor in crc . our results demonstrate that hif-1 is correlated with overexpression of thymosin 4 in human crc . although further studies are necessary to further validate our findings , we suggest that thymosin 4 , has potential as a prognostic biomarker and has potential as a hif pathway target in human crc .

in the past two decades , the function of matrix metalloproteinases ( mmps ) in the central nervous system ( cns ) has gained much attention . mmps are calcium ( ca ) dependent zinc ( zn ) containing endopeptidases produced in latent forms . once activated , they participate in the regulation of diverse physiological and pathological processes . mmps are involved in the degradation of extracellular matrix ( ecm ) components , remodeling of tissues , shedding of cell surface receptors , and processing of various signaling molecules . mmps are essential for brain development due to their association with important neurophysiological functions , such as synaptic plasticity [ 2 , 3 ] and long - term potentiation [ 4 , 5 ] . in the adult brain under normal conditions , increased expression of mmps is observed in a variety of pathological conditions , including neurodegenerative diseases such as alzheimer 's disease ( ad ) , parkinson 's disease ( pd ) , amyotrophic lateral sclerosis ( als ) , huntington 's disease ( hd ) , and multiple sclerosis ( ms ) and in neuroinflammatory conditions such as traumatic brain injury , stroke , and meningitis . as part of the neuroinflammatory response , mmp activity at cns barriers contributes to the increase in permeability by altering the properties of ecm and tight junctions . this results in aggravation of neuroinflammation - induced brain damage . on the other hand , activation of mmps is known to help in tissue repair , angiogenesis , and neurogenesis . in this review , mmps , together with adamlysins and astacins , belong to metzincins , a family of zn - dependent , ca - containing endopeptidases ( 24 members in mammals ) . mmps are multidomain proteins consisting mostly of the following domains : n - terminal signal peptide ( which is cleaved in the secretory pathway ) , propeptide ( which maintains latency of mmps ) , catalytic domain ( holds the zn ion ) , hinge region ( connecting sequences ) , and c - terminal hemopexin - like domain ( required for substrate and timp recognition ) ( figure 1 ) . besides these common domains , some mmps have alternative peptide structures and different additional domains . mmps are produced as zymogens ( pro - mmp ) that are activated by other enzymes or free radicals through the cysteine switch mechanism . the thiol group of a cysteine residue in the n - terminal prodomain binds to and blocks the active - site zn atom ; activation occurs when the thiol group is blocked or removed . it has been shown that mmps play an important role in various physiological and pathological processes in the body . it acts as an adhesion site for various cells and serves as a storage site for different signaling molecules , growth factors , and proteins in general , thus influencing development and migration of the cells . ecm consists mainly of glycosaminoglycans , proteoglycans , and fibrous proteins ( collagen , laminin , and fibronectin ) . mmp cleavage of ecm influences cell migration , embryogenesis , and other processes during development as well as in the adult organism . in the brain , mmps are involved in tissue remodeling after injury , neurogenesis , axonal growth , angiogenesis , cns barrier disruption , myelinogenesis , and demyelination . additionally , mmps play an active role in immune processes by cleaving various molecules , including growth factors , death receptors , chemokines , and cytokines [ 9 , 10 ] . for example , several mmps can activate tumor necrosis factor ( tnf ) [ 1115 ] and transforming growth factor - beta ( tgf- ) , while other mmps degrade interleukin-1 ( il-1 ) . ultimately , mmp cleavage of chemokines and cytokines can lead to either pro- or anti - inflammatory processes . based on their domain organization , mmps are classified into four major subgroups : ( 1 ) gelatinases ( mmp-2 , mmp-9 ) , ( 2 ) matrilysins ( mmp-7 , mmp-26 ) , ( 3 ) archetypal mmps , and ( 4 ) furin - activated mmps ( figure 1 and table 1 ) . the archetypal mmps contain different types : stromelysins ( mmp-3 , mmp-10 ) , collagenases ( mmp-1 , mmp-8 , mmp-13 ) , and other mmps ( mmp-12 , mmp-18 , mmp-20 , mmp-27 ) . similarly , the furin - activated mmps are divided in secreted mmps ( mmp-11 , mmp-21 , mmp-28 ) ; type - i transmembrane mmps ( mmp-14 , mt1-mmp , mmp-15 , mt2-mmp , mmp-16 ( mt3-mmp ) , and mmp-24 ( mt5-mmp ) ) , type - ii transmembrane mmps ( mmp-23 ) , and gpi - anchored mmps ( mmp-17 ( mt4-mmp ) , mmp-25 ( mt6-mmp ) ) . mmps are modulated on several levels : transcriptional activation , removal of the prodomain , interaction with ecm components , and inhibition by endogenous inhibitors such as tissue inhibitors of metalloproteinases ( timps ) . timps are proteins of 2128 kda which bind the active site of mmps in a one - to - one ratio [ 7 , 20 ] . besides timps , also -2 macroglobulin and receptor mediated endocytosis can prevent activated mmps from exerting their effects . proinflammatory cytokines ( e.g. , tnf and il-1 ) and several growth factors can initiate an intracellular signaling cascade leading to the activation of ap-1 , nf - kb , or ets transcription factors , with consequent mmp transcription . on the level of mmp - zymogen activation , mostly serine proteases and other mmps are involved in initiating proteolytic cleavage of the prodomain ; for example , mmp-3 can cleave pro - mmp-9 . additionally , high levels of reactive oxygen species ( ros ) and reactive nitrogen species can induce activation of mmps . it has been observed that mmps in the cns are secreted by microglia , astrocytes , and neurons . in physiological conditions , mmps are either absent or present at undetectable levels in the mature brain , and deregulation of their activity could shift the balance and induce perpetuation of chronic inflammation . this has been shown in different peripheral chronic diseases , such as atherosclerosis and rheumatoid arthritis [ 25 , 26 ] , and neuroinflammatory diseases , such as cerebral ischemia , stroke , and bacterial meningitis . mmps are known to be involved in cns barrier maintenance and to increase the permeability of the barriers in inflammation . the proposed mechanism involves degradation of ecm components ( e.g. , laminin and collagen ) , which normally support cellular structures and limit the passage of different molecules and cells into the blood through the barriers [ 2830 ] . additionally , increased mmp activity is known to negatively affect tight junction functionality at the cns barriers [ 3033 ] . alternatively , several mmps have been shown to activate proinflammatory cytokines and free radicals , which enhances inflammation and subsequently induces disruption of the cns barriers [ 27 , 34 , 35 ] . mmps are secreted upon inflammation , for example , by peripheral neutrophils , which thereby contribute to aggravation of the inflammation and disruption of the barriers . compromise of cns barrier integrity due to activation of mmps has been observed in cerebral ischemia , traumatic brain injury ( tbi ) , and other diseases associated with neuroinflammation . in this review , we focus on the role of different mmps in the major neurodegenerative diseases , ad , pd , als , hd , and ms ( figure 2 ; tables 2 and 3 ) . during the previous century , prolongation of the human lifespan led to an increase in the proportion of elderly people in the population , which has given rise to an increased incidence of age - related diseases such as neurodegenerative disorders . neurodegenerative diseases share common features , such as progressive loss of neurons and deterioration of the structure and function of the central and/or peripheral nervous system . these chronic illnesses impose a heavy economic and social burden and affect both patients and caregivers . since all neurodegenerative diseases are incurable , the outcome in most cases is death . worldwide , 47 million people are living with ad and other dementias , including more than 5 million in the united states alone , where the number is expected to rise to 7.1 million in 2025 . furthermore , there are large numbers of patients with other neurodegenerative diseases , including ad , pd , als , hd , ms , and frontotemporal dementia . the major problem in the management of neurodegenerative diseases is the lack of adequate information on their pathogenesis and absence of mechanism - based treatments . however , interaction of genetic and environmental factors , as well as advanced age , is known to contribute to disease onset . generally , all neurodegenerative diseases share certain molecular and cellular mechanisms , including protein aggregation and formation of inclusion bodies . the traditional classification of neurodegenerative disorders is based on clinicopathological features and has been formulated through consensus criteria , that is , a set of diagnostic criteria on which experts in the field agree , along with specific molecular characteristics . this traditional classification was established during the early stages of the research on neurodegenerative diseases and was based on a rather small number of cases . one of the categories used for classification is the presence of certain molecules in brain lesions associated with the disorder , for example , -amyloid ( a ) for ad and -synuclein for pd . for several neurodegenerative diseases , mutations in specific genes have been found , for example , presenilin genes ( psen 1 and 2 ) for ad . however , in certain cases diagnosis is difficult due to the coincidence of clinicopathological features of several neurodegenerative diseases [ 9799 ] . nowadays , with the increasing amount of molecular and genetic data and the continuing efforts to find other common characteristics of neurodegenerative diseases , questions have arisen about the distinction between these diseases , making their classification even more problematic [ 99101 ] . the early classifications divided neurodegenerative diseases into either taupathies ( including ad , pick 's disease ( pid ) , argyrophilic grain disease ( agd ) , progressive supranuclear palsy ( psp ) , corticobasal degeneration ( cbd ) , and ftdp-17 ) and synucleinopathies ( including pd , dementia with lewy bodies ( dlb ) , and multiple system atrophy ( msa ) ) . today , different , nosological classifications of neurodegenerative diseases exist , taking into account clinical presentation , affected brain regions and cell types , altered proteins involved in the pathogenesis of the disease , genetics , and possible overlaps between diseases [ 96 , 103 ] . neuroinflammation is defined as inflammation of nervous tissue , which occurs as a biological response to different signals , such as infection , cns injury , autoimmunity , and toxic compounds . although it is initiated primarily as a beneficial reaction of the cns to the harmful stimulus , it could eventually aggravate the disease . neuroinflammation is characterized by the initiation of a cascade of events , including production of cytokines and chemokines accompanied by the release of free radicals and proteases , resulting in a chronic inflammatory state in the organism . four cns barriers separate blood from cns parenchyma to block entrance of immune cells and various molecules into the brain from the periphery : blood - brain barrier ( bbb ) , blood - cerebrospinal fluid barrier ( bcsfb ) , arachnoid barrier , and blood - spinal cord barrier ( bscb ) [ 104 , 105 ] . all cns barriers are dynamic structures that allow passage of immune - related molecules and cells upon specific stimuli ( e.g. , proinflammatory cytokines ) [ 106 , 107 ] . furthermore , immune functions in the brain are conveyed by microglia , innate immune cells that are constitutively present in the brain , but astrocytes can secrete many molecular mediators of the immune response . microglia are of myeloid origin and normally reside in a ramified , resting state in the cns , where they monitor the surrounding environment in the brain and spinal cord . opposite to immune cells in the periphery , microglia are predominantly involved in limiting inflammation . when triggered by various immunological stimuli , microglia morphologically transform into ameboid cells and start to proliferate and secrete inflammatory mediators [ 109 , 110 ] . besides , they start to mimic antigen presenting cells ( apc ) , which are immune cells in the periphery , by upregulating major histocompatibility complex ( mhc ) class ii and becoming phagocytic . the presence of the mhc is important for prolongation of the immune response in the cns . it has been shown that microglia are involved in neuron survival , neurogenesis , facilitation of brain repair via guidance of stem - cell migration to the site of inflammation or injury , and clearance of cell debris . nevertheless , when overactive , microglia can inflict severe damage to the brain by excessive production of molecules such as mmps , proinflammatory cytokines and chemokines , and cytotoxic molecules such as ros and nitric oxide ( no ) [ 116118 ] . they also produce different inflammatory mediators , including cytokines , chemokines , and complement components . for example , both microglia and astrocytes secrete il-2 and il-1 , thereby stimulating cd4 + t helper cells to produce gm - csf , which contributes to perpetuation of the inflammation processes by recruitment of cd11b - positive myeloid cells . also t cells can secrete cytokines and mmps that are known to disrupt the bbb and permit the entry of immune cells from periphery , converting acute inflammation into a chronic inflammatory state . although microglia have been implicated in neurodegenerative diseases , the mechanisms responsible for activating microglia are unknown . all neurodegenerative diseases are multifactorial and caused by complex interactions of genetic and environmental factors . by far the most prominent risk factor for most neurodegenerative diseases is aging , so the prevalence of these disorders is high above the age of 65 years . aging is influenced by interaction and balance between various protective and harmful factors over the lifespan of an individual . these factors include genetics , nutrition , psychosocial influences , and exposure to toxic compounds . however , it is not yet clear how aging acts on the development of neurodegenerative diseases and other age - related diseases . two of the most prominent characteristics of aging are immunosenescence ( deterioration of immune responses ) and inflammaging ( presence of chronic low grade inflammation ) [ 124 , 125 ] . physiological aging is associated with a progressive increase in the number of activated microglial cells in the brain and spinal cord [ 126128 ] accompanied by transition from normal microglial morphology to microglial dystrophy . it has been shown that aged microglia secrete il-1 and increase mhc class ii expression . low - level systemic inflammation during aging has also been linked to the presence of active microglia and increased proinflammatory cytokines levels in the brain [ 132134 ] . one of the characteristics of aging is accumulation of advanced glycation end products ( age ) , which are also elevated in ad . activation of the receptor for age ( rage ) leads to release of proinflammatory cytokines and free radicals , further contributing to inflammatory processes . nevertheless , changes in the physiological balance between mmps and timps have been related to age - related vascular diseases . one theory of aging suggests that vascular - derived insults initiate and/or contribute to aging and to some of the age - related diseases , such as ad [ 137 , 138 ] . in a comparative study , rna expression of 22,626 genes was monitored in the heart and cerebellum of young and aged mice of several strains . two potential biomarkers of aging present in both structures were identified : complement component c4 and timp-2 . using magnetic resonance imaging , romero et al . found that upregulation of mmp-9 was associated with aging and circulating levels of mmp-9 and timp-1 in patients with brain ischemia and aging . liu et al . observed that mmp-12 increased in the aging brain and that mmp-12 deficiency led to a reduction of neuroinflammaging . this finding is linked to aggravation of neuroinflammation associated with aging via the induction of the migration of bone marrow derived microglia to the brain . furthermore , safciuc et al . showed that microvessels in the brain of aged rats exhibit decreased mmp-2 activity and appearance of mmp-9 . its prominent characteristics include brain atrophy , caused by neuronal cell death , and decreased dendritic arborization in the cerebral cortex and other subcortical areas . the hallmarks of ad include presence of amyloid plaques and neurofibrillary tangles , which are linked to cerebral atrophy . amyloid plaques , also called senile plaques , appear in brain parenchyma as extracellular deposits consisting of a fibrils of 3743 amino acids , originating from alternative processing of app protein . although a deposition is considered a signature lesion for ad , it also occurs in down 's syndrome , possibly due to triple multiplication of amyloid precursor protein ( app ) , as well as in certain cases of dementia with lewy bodies [ 145 , 146 ] . additionally , occurrence of a pathology has been observed in several other neurodegenerative diseases , such as pd , pick 's disease ( pid ) , progressive supranuclear palsy ( psp ) , and corticobasal degeneration , as well as in als . besides , deposition of a occurs in the cerebral vasculature in both cerebral amyloid angiopathy ( caa ) and in 90% of patients with ad . a originates from the transmembrane protein app , which undergoes amyloidogenic processing by -secretase to produce -c terminal fragments ( ctfs ) . these fragments are cleaved by -secretase to release a in the extracellular space and app intracellular domain ( aicd ) into the cytoplasm . in the physiological nonamyloidogenic pathway , these fragments are cleaved by -secretase , resulting in the cytoplasmic peptide fragment aicd and the extracellular p3 peptide . in contrast to a peptides , p3 peptides have a low propensity to assemble into stable oligomers and they have no known harmful effects on brain cells . it has been shown that some of the members of the metalloproteinase family , including the adam ( a disintegrin and metalloproteinase ) proteins adam-17 ( also called tumor necrosis factor--converting enzyme or tace ) , adam-9 and adam-10 , can cleave app at the -secretase cleaving site . a oligomers are nonfibrillar structures , and further aggregation of a oligomers results in the formation of protofibrils and eventually fibrils . a deposits are typically surrounded by dystrophic neurites , reactive astrocytes , and activated microglia , forming dense - core plaques in the brain parenchyma . also considered hallmarks of ad are neurofibrillary tangles ( nft ) , intercellular deposits of a hyperphosphorylated form of tau protein . physiologically , tau protein plays a role in the assembly and stabilization of microtubules in neurons . cases of dementia with abundance of nfts and a few amyloid plaques have been classified as a nonspecific type of neurodegenerative disorders , called nft dementia . however , the etiology of ad is complex , and neither a nor nft alone should be considered responsible for the disease manifestations . therefore , other proposed mechanisms and manifestations of the disease should also be taken into account . some authors point to the role of an imbalance in ros formation and cellular antioxidant activity in ad . the idea is that overproduction of free radicals could be a driving force behind neurodegeneration , and given the large number of possible stressors , such as aging , inflammation , hypoxia , and cerebral hypoperfusion , there is ample opportunity for overproduction of free radicals . on the other hand , advocates of the inflammatory hypothesis propose a central role for activated microglia nearby amyloid plaques in the brain . their notion is that activated microglia produce large amounts of inflammatory cytokines and chemokines , which sustain a chronic inflammation in the brain that ultimately leads to neuronal cell death . furthermore , it has been shown that besides the established direct neurotoxic effect , a can exert indirect proinflammatory effects via microglial activation , which results in secretion of no , tnf , and superoxides [ 167 , 168 ] . interestingly , clustering of activated microglia around a aggregates has been observed even before the development of ad symptoms [ 169171 ] . in view of the relationship between mmps and ad ( figure 3 ) and in order to distinguish ad from vascular dementia , bjerke et al . proposed mmp-9 and timp-1 as biomarkers of ad , next to t - tau , p - tau , a142 , and white matter lesions . strikingly , a correlation between cognitive impairment and mmp-9 activity was observed in patients with mild cognitive impairment . in agreement with that correlation , mmp-9 expression was shown to be induced in ad patients in neuronal cytoplasm , neurofibrillary tangles , amyloid plaques , and vascular tissue , as well as in astrocytes upon a stimulation . additionally , mmp-9 was found in pyramidal neurons of the brains of ad patients , and near amyloid plaques and it was shown that mmp-9 is able to cleave a140 . moreover , yan et al . showed that mmp-9 can degrade a fibrils in vitro , as well as amyloid plaques in brain slices from app / ps1 mice . using intracerebroventricular ( icv ) injections of different a peptides in animal models , mizoguchi et al . showed an increase in mmp-9 activity in hippocampus , related this increase to a-induced cognitive impairment , and confirmed the results using mmp inhibitors and mmp-9 knockout mice . notably , another study showed that mmp-3 , by remodeling the ecm , is crucial for synaptic plasticity and learning . it has been shown that mmp-9 can act through nmda receptor signaling via an integrin 1 dependent pathway . li et al . showed in primary astrocyte cultures insignificant levels of mmp-9 in medium after treatment with a oligomers , accompanied with a decrease in mmp-2 activity . on the contrary , in the brain of app / ps1 ad mice , they observed increased mmp-2 and proinflammatory cytokine levels . they proposed that a can decrease the expression and activation of mmp-2 in astrocytes directly , while stimulating microglia to produce proinflammatory cytokines , which in turn again induce mmp-2 expression and aggravate the disease . , no elevation of mmp-2 activity was observed in ad patients . in a transgenic mouse model of ad , expression of mmp-2 and mt1-mmp , a potent mmp-2 activator , was found in reactive astrocytes around amyloid plaques , and higher levels of a142 increased the production of mmp-3 , mmp-12 , and mmp-13 in microglia . additionally , mmp-12 exacerbates the cascade of proteolytic processes by subsequent activation of other mmps such as mmp-2 and mmp-3 . looked into the effects of a on endothelial cells and bbb integrity and linked this to mmp activity . they observed increased bbb permeability in cultured endothelial cells linked to decreased zonula occludens-1 ( zo1 ) levels , one of the major components of tjs . moreover , there was an increase in mmp-9 and mmp-2 activity , and broad - spectrum mmp inhibition reversed the a-induced bbb disruption . additionally , they confirmed these results in a transgenic mouse model of ad by showing enhanced immunoreactivity of mmp-9 near cerebral capillaries and alterations in tight junction components . the proposed mechanism of a activity is through activation of rage , which is physiologically expressed on endothelial cells and activates the intracellular calcineurin ( can ) signaling pathway , which ultimately results in activation of mmps and tj cleavage . in the same year , another group showed that interaction of a with rage induces mmp-2 via the erk and jnk pathways in brain endothelial cells . besides aging , the most prominent genetic risk factor for developing late onset ad is the presence of apolipoprotein e 4 allele ( apoe 4 ) in the genome . the group of zlokovic reported that , both in transgenic mice and in humans , apoe 4 leads to bbb breakdown by activating the proinflammatory cyclophilin a ( cypa)/mmp-9 pathway in brain pericytes , which are important components of the neurovascular unit and guardians of bbb integrity [ 61 , 174 ] . this eventually results in degradation of the bbb tight junctions and basement membrane proteins [ 61 , 174 ] . as far as the role of stromelysins in ad is concerned , mmp-3 levels were significantly upregulated in plasma , similar to what was observed in csf of ad patients . in contrast , mlekusch and humpel observed downregulation of mmp-3 and mmp-2 in csf of ad patients , but it should be noted that these patients had lower a levels . in other studies , it has been shown that mmp-3 is expressed in microglia , astrocytes , and endothelial cells in the brain , as well as near senile plaques in ad . deb and gottschall showed that mmp-3 was induced and its activity increased in astrocyte and neuronal cell cultures upon a140 stimulation . as reported for mmp-9 moreover , a correlation was found between mmp35a and apoe 4 alleles , and the presence of both is a risk factor for developing ad . interestingly , we recently reported that icv injection of a142 oligomers induces loss of barrier integrity at the blood - csf barrier , and this was linked to increased mmp-3 expression and mmp activity . moreover , the a142 oligomer - induced leakage of the bcsfb could be prevented by a broad - spectrum mmp inhibitor and did not occur in mmp-3 deficient mice . leake et al . reported a notable increase in mmp-1 in brain of ad patients . found upregulated microglial / macrophage expression in tissues from ad patients , as well as in a mouse model of ad . finally , levels of timp-1 and c - reactive protein ( crp ) were found to be increased in ad patients , and they decreased remarkably after treatment with acetylcholinesterase inhibitors ( acheis ) , one of the few available therapies of ad . pd is the second most prevalent neurodegenerative disease and the most common neurodegenerative movement disorder , with an estimated 710 million people worldwide suffering from it . like ad , its prevalence increases with age , and due to the severity and long duration of the disease , its costs in the us alone are estimated at 25 billion dollars per year . a prominent characteristic of pd is the presence of intracellular protein inclusions called lewy bodies in affected brain areas . these inclusions are formed of fibrillar , misfolded proteins composed of -synuclein , parkin , synphilin , synaptic vesicle proteins , and neurofilaments . interestingly , inclusions similar to lewy bodies were found in 22% of cases of familial ad , and they occur also in dementia with lewy bodies ( dlb ) and in multiple system atrophy ( msa ) . another hallmark of pd is selective and progressive loss of dopaminergic neurons in substantia nigra pars compacta . the substantia nigra , being part of basal ganglia together with the striatum , globus pallidus , and subthalamic nucleus , modulates motor activity in the brain . thus , due to dopaminergic neuron cell death , one of the clear manifestations of the disease is loss of control over movements , resting tremor , bradykinesia , and rigidity . additionally , pd is accompanied by sensory dysfunction , mood and sleep disorders , dementia , and partial autonomic nervous system impairment . this multifactorial disorder is known to be influenced by genetic factors , such as multiplication or missense mutations in the -synuclein gene , which is a major risk factor for familial pd . additionally , mutations in parkin , pink1 , and lrrk2 have been identified as possible causes of other cases of familial pd [ 186 , 187 ] . nevertheless , most cases of pd are sporadic , and the onset of the disease has been shown to be caused by complex interaction of environmental and genetic factors . thus , some hypotheses propose that neuroinflammation could play a pivotal role in promotion and aggravation of the disease [ 188190 ] . studies on postmortem tissue of pd patients , as well as in vivo imaging , revealed astrogliosis , overactivation of microglia , and infiltration of peripheral immune cells into brain regions affected by pd [ 191195 ] . in pd patients , active microglia have been observed in most of the regions with lewy bodies , and in vivo imaging revealed microglial activation throughout the different stages of the disease , indicating chronic microglial activation . the substantia nigra seems to be particularly susceptible to inflammation due to the presence of the largest number of microglia in this brain area , so relevant stimuli lead to the activation of large numbers of microglia . in agreement with these findings , activated microglia have been found in substantia nigra in patients with sporadic or familial pd [ 196 , 198 ] as well as after exposure of humans to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine ( mptp ) . the same effect was seen in the substantia nigra and striatum in animal models of pd based on mptp injection [ 199202 ] . microglial activation was also found in other pd models and in other brain regions in pd patients , including putamen , hippocampus , transentorhinal cortex , cingulate cortex , and temporal cortex . hirsch and hunot described early microglial activation after mptp injection , followed by later neuronal cell death and infiltration of t cells and astrogliosis . other evidence for the role of microglia in pd comes from studies using anti - inflammatory drugs to inhibit microglial activation , which was protective against neurodegeneration induced by mptp or 6-ohda . active microglia are known to secrete inflammatory mediators , and accordingly , increased proinflammatory cytokine levels were increased in the substantia nigra [ 206 , 207 ] and csf in pd . furthermore , il-1 and il-6 were found to be elevated in csf of parkinson 's disease patients [ 209 , 210 ] . it has been speculated that prolonged overactivation of microglia and production of proinflammatory cytokines could lead to neuronal degeneration in pd [ 207 , 211 ] . it is also speculated that oxidative stress could be generated from dopaminergic metabolism , mitochondrial dysfunction , and microglial activation , which could be influenced beforehand by various toxins and mutations in parkin , pink1 , dj-1 , or htra1 , which are important for physiological mitochondrial functioning . other evidence for the involvement of active microglia in triggering neurodegeneration of dopaminergic neurons in the substantia nigra comes from studies on injection of lipopolysaccharide ( lps ) systemically or directly in the substantia nigra [ 213215 ] . this could be explained by specific susceptibility of dopaminergic neurons to oxidative stress due to the presence of tyrosine hydroxylase and monoamine oxidase , which are ros - generating enzymes , as well as excessive production of easily oxidized cytosolic dopamine [ 217 , 218 ] . however , it is speculated that initial neurotoxic insult to dopaminergic neurons results in the release of certain factors that activate microglia and convert them from beneficial into harmful . it has been shown that damaged dopaminergic neurons can activate microglia by releasing -synuclein and neuromelanin , causing them to produce ros . mmp-3 , produced by neurotoxin - stressed dopaminergic neurons , seems to be a self - sufficient player in microglial activation in the absence of any other inflammatory molecule . it has been suggested that this mechanism plays an important role in apoptosis . on the one hand , mmp-3 could cleave the connections between apoptotic cells and ecm , thereby facilitating subsequent phagocytosis . on the other hand , it could activate microglia , leading to the release of cytokines and receptors for phagocytosis of apoptotic cells . in an in vitro study , kim et al . additionally , they hypothesized that both active mmp-3 and catalytically active recombinant mmp-3 could activate microglia to produce proinflammatory cytokines , which in turn aggravate neuronal apoptosis of damaged cells , leading to further induction of apoptosis in neighboring dopaminergic neurons . this hypothesis is supported by postmortem studies describing progressive dopaminergic neuronal degeneration in humans and monkeys treated with mptp for 10 years [ 65 , 221 ] . using mmp-3 deficient mice and a broad spectrum mmp inhibitor , it has been shown that depletion of mmp-3 can significantly reduce mptp - induced degeneration of nigrostriatal dopaminergic neurons in the brain . furthermore , mmp-3 activated microglia produce superoxide , known to be involved in facilitation of dopaminergic neuronal cell death in vitro [ 222 , 223 ] and in vivo . using sirna , also upregulated mmp-9 activity , produced by neurons and microglia , was found in both striatum and substantia nigra after mptp treatment , and pharmacologic inhibition of mmps protected against mptp neurotoxicity . earlier , the same group analyzed postmortem brain tissue from pd patients and found no change in the activities of mmp-9 and mmp-1 in substantia nigra , cortex , or hippocampus , whereas mmp-2 was significantly reduced in the substantia nigra . additionally , they showed that mmp-9 was localized primarily in neurons and mmp-2 in astrocytes and microglia . in the same study , timp-2 levels did not change , whereas timp-1 was upregulated in substantia nigra but not in the cortex and hippocampus . the increase in mmp-9 expression in substantia nigra was later confirmed by annese et al . the data showed that mmp-9 is expressed in reactive microglia and astrocytes , pinpointing mmp-9 as a key molecule for the onset of neuroinflammation in pd . experiments done in mmp-9 deficient mice confirm that active glia diminish neuronal survival since decreased numbers of active microglia correlated with increased numbers of functional dopaminergic neurons . in a primate model of pd ( mptp - injected macaques ) , an increase in mmp-9 labeled striatal neurons and astrocytes was also found . bbb leakage was found in animal models of pd solely in brain regions and was associated with microglial activation and dopaminergic neurodegeneration [ 225 , 226 ] . recent evidence suggests that the proteolytic activity of mmps might be involved in alteration of -synuclein protein conformation , thus contributing to aggregation , lewy body formation , and microglial activation . in an in vitro study on a dopaminergic neuronal cell line , sung et al . observed mmp - dependent proteolysis of -synuclein , followed by increased aggegate formation . in this process , mmp-3 was particularly efficient , but mmp-1 , mmp-2 , and mmp-14 showed similar properties . levin et al . further studied the mmp - specific -synuclein cleavage and showed that both mmp-1 and mmp-3 mediate increased -synuclein aggregation in comparison to trypsin and proteinase k . als , also known as lou gehrig 's disease , is characterized by degeneration of motor neurons in the brain , brainstem , and spinal cord . all voluntary muscles are affected , and the muscle weakness and atrophy are followed by paralysis , and finally respiratory failure and death . some other neurodegenerative diseases share similar etiology , such as progressive lateral sclerosis ( pls ) , progressive muscular atrophy ( pma ) , als dementia , and als frontal lobe dementia . interestingly , one - third of all als patients exhibit symptoms or pathology resembling those of ad . ( 2.08 people per 100,000 in europe ) , and the prevalence is mostly in people between the ages of 45 and 65 years . familial occurrence of the disease is only 510% of all als cases , and the cause of the sporadic form of als is still unknown . some familial and sporadic cases are caused by mutation in the gene for copper - zinc superoxide dismutase 1 ( sod1 ) . additionally , als is associated with protein inclusions composed mostly of transactive response dna - binding protein 43 ( tdp-43 ) in the cytoplasm in the affected areas of the brain and spinal cord . however , occurrence of tdp-43 is not characteristic of only als but was found in several patients with frontotemporal lobar degeneration with tdp proteinopathy ( ftld - tdp ) , as well as in frontotemporal dementia , ad , and some other neurodegenerative diseases . the etiology of the disease is unknown , but various mechanisms have been proposed , including neuroinflammation , glutamate excitotoxicity , oxidative stress damage and mitochondrial dysfunction , protein misfolding and aggregation , and deficits in neurotrophic factors . besides , in als , active microglia were also observed in brain areas such as motor cortex , pons , dorsolateral prefrontal cortex , and thalamus . interestingly , activation of microglia was correlated with progression of the disease . in the in vitro work of swarup et al . , microglia overexpressing tdp-43 increased their secretion of proinflammatory cytokines upon lps treatment in comparison to wild type microglia . one of the theories suggests that bbb and bscb breakdown could contribute to the motor neuron damage , due to the importance of these barriers in maintenance of homeostasis in the cns . indications of the involvement of mmps , key players in barrier alteration , come from early studies on neocortex and spinal cord of als patients , in which mmp-2 was found in astrocytes , and mmp-9 was found in pyramidal neurons in the motor cortex and motor neurons in the spinal cord . additionally , mmp-2 activity was decreased in motor cortex whereas mmp-9 activity was increased in spinal cord . since bscb disruption in als [ 237239 ] is accompanied by downregulation of mrna for tight junction proteins , miyazaki et al . speculated that mmp-9 is involved in barrier disruption . another group showed reduced mmp-9 activity during disease progression , with the peak at the onset of als , and described a similar profile for mmp-2 . two separate groups found significant increases in both pro - mmp-9 and active mmp-9 in serum of als patients relative to healthy controls [ 242 , 243 ] . reported that in mild cases of als , expressions of mt - mmp-1 , mmp-2 , mmp-9 , and timp-1 are elevated in serum compared to csf , where mt - mmp-1 , mmp-2 , and timp-1 were upregulated or unchanged while mmp-9 levels were decreased . furthermore , fang et al . found increased levels of mmp-9 in csf from patients suffering from rapidly progressing als . they speculated that this finding is associated with progression of the disease , poor survival of the patients , and neuronal degeneration . nevertheless , mmp-2 showed a slow but progressive decrease with the development of the disease . in the study by kaplan et al . , diminishing mmp-9 function by genetic , viral , or pharmacological intervention was shown to prolong survival in a sod1 mouse model of als . moreover , mmp-9 was preexpressed only in fast motor neurons , which have been shown to be particularly susceptible to degeneration in patients suffering from als . these results show that mmp-9 is a key player in the onset of the disease and point to it as a therapeutic target . kaplan et al . focused on the early stage of the disease and expression of mmp-9 by neurons , whereas kiaei et al . studied later stages of the disease and observed expression of mmp-9 by activated microglia , giving support to the hypothesis that the pathology is mediated by cytokines secreted by microglia . since the depletion of mmp-9 gene does not rescue transgenic sod1 mice from death , als indeed has complex background . there are about 2.5 million cases worldwide , with approximately 400,000 in the us alone , and ms is twice as common in women as in men . in contrast to most neurodegenerative disorders that are prevalent in aged individuals , ms occurs in people between 20 and 45 years of age . the cause of the disease is unknown , but genetic and environmental factors contribute to its development . interestingly , epidemiological studies revealed a correlation with smoking , exposure to uvb radiation , and intake of unsaturated fatty acids . ( 1 ) relapsing - remitting ms ( rrms ) occurs in about 85% of patients suffering from ms . the disease alternates between remission ( periods of improvement ) and relapses ( periods of deterioration ) . ( 2 ) secondary progressive ms ( spms ) , which is characterized by continuous worsening of the symptoms , affects some patients suffering from rrms . ( 3 ) primary progressive ms ( ppms ) is manifested in about 10% of ms patients . ( 4 ) progressive - relapsing ms ( prms ) is the rarest type , present in less than 5% of patients . although it is progressive from the beginning , it shows relapses occasionally , but without periods of remission . at onset , rrms is characterized as a neuroinflammatory state . however , with the progression of the disease and occurrence of relapses , certain residual disability develops . over ten years , most patients enter spms , which is then observed more as neurodegeneration state resulting in permanent disability . although inflammation is considered as primary in ms , this disease is recently being acknowledged as a neurodegenerative disorder too because of recent findings . it has been observed that disability related to ms is correlated with axonal damage and neuronal cell loss more than with inflammation . the new hypothesis that emerged resembles the previously proposed mechanism for the other neurodegenerative disorders : perpetuated inflammation leads to triggering of neurodegenerative processes . interestingly , certain case reports have noted patients suffering from ms and als at the same time . in ms , the bbb is disrupted , leading to peripheral blood leukocyte infiltration , followed by focal degradation of myelin , and finally axonal disruption and neuronal cell loss . it has been shown that the bbb function in ms is lost in both relapsing - remitting and progressive phases . nevertheless , bbb dysfunction is a temporary event , although recurrence is highly possible . in fact , local bbb changes that follow the pattern of the lesions help diagnose ms by observing brains using magnetic resonance imaging ( mri ) with contrast agents that easily leak into the affected parts of the brain . some of the data suggest that bbb breakdown precedes infiltration of the immune cells , but this event is not definitively the primary cause of lesion formation . . showed in a common animal model of ms called experimental autoimmune encephalomyelitis ( eae ) that occludin dephosphorylation preceded visible signs of disease onset , which indicates that bbb breakdown is one of the first events in ms . it is known that in ms , various brain and immune cells can secrete mmps , thus contributing to the bbb breakdown [ 260 , 261 ] . cossins et al . observed expression of mmp-7 by macrophages and mmp-9 in blood vessels in active lesion sites of postmortem brain samples . another group confirmed this finding and also showed expression of mmp-3 in endothelial cells , mmp-1 , mmp-2 , mmp-3 and mmp-9 in macrophages , and to a lesser extent in astrocytes , around active and necrotic lesions . examined csf samples from patients suffering from both rrms and ppms and found an increase in mmp-9 in all the rrms cases throughout both phases of the disease . however , in ppms patients , mmp-9 was increased in only about half of the samples and in significantly smaller amounts than in the relapsing - remitting form . they argued that this points out that t - cells and macrophages are responsible for the secretion of mmp-9 in ms . additionally , they proposed that constant elevation of mmp-9 throughout the progress of the disease could contribute to the surrounding tissue damage and neuronal cell loss . in their work using the eae model , kieseier et al . showed that the increase in mmp-9 and mmp-7 expression in blood vessels and parenchyma strongly correlated with the peak of the disease . elevated levels of mmp-9 were also observed in serum of ms patients , together with an increase in timp-1 and timp-2 . in the same study , the authors pointed out an association of these increases with the number of lesions observed by mri . however , the study of waubant et al . found increased levels of mmp-9 in serum but no elevation of timp-1 levels . also , by univariant analysis they found that an increase in mmp-9 and a decrease in timp-1 levels preceded the appearance of new lesions . another group compared the mrna levels of mmp-1 , mmp-3 , mmp-7 , mmp-9 , mmp-14 , and timp-1 in blood monocytes of ms patients with those of controls . they found that all except mmp-14 were upregulated . in an interesting study by althoff et al . using the eae model , transgenic mice that constitutively express timp-1 in the cns had a normal phenotype but eae symptoms were diminished . interestingly , other studies done using the eae model showed limited bbb restoration and amelioration of the clinical picture after administration of broad - spectrum mmp inhibitors [ 90 , 265 ] . finally , mmp-9 knockout mice were shown to be less susceptible to induction of eae . besides the occurrence of the leakage , the bbb also becomes activated , meaning that cells making up the bbb , including endothelial cells , astrocytes , and potentially pericytes , start expressing and secreting various factors involved in the recruitment and functioning of leukocytes . constant leukocyte migration occurs through the bbb in active ms lesions and this migration is normally strictly regulated by a number of molecules , such as cell adhesion molecules ( cam ) , integrins , cytokines , and chemokines . the leukocyte infiltration further aggravates bbb breakdown , as shown in in vitro studies . in one in vitro study , interferon treatment downregulated mmp-9 expression and abolished mmp-2 expression , thereby diminishing subsequent migration of t - cells . newman et al . showed that microinjection of activated mmps into white matter leads to axonal injury . of the several mmps tested , the most potent was mmp-9 , followed by mmp-2 , and finally mmp-7 . the proposed mechanism of mmp action is via degradation of the ecm , since mmps have an established role in the apoptosis of different cell types by the same mechanism . additionally , mmp - activated axonal degeneration was hardly observed in peripheral nervous system , possibly due to the presence of resilient ecm and higher timps expression . it has been speculated that this could be a cause of reduced remyelination and decreased number of mature oligodendrocytes in mmp-9 and mmp-9/-12 null mice . huntington 's disease ( hd ) is an inherited neurodegenerative disorder that decreases muscle coordination and mental ability . the disease has been linked to a mutation on chromosome 4 in a gene coding for a protein called huntingtin ( htt ) . while the exact role of htt is not clear yet , it has been speculated that proteolysis of mutant htt participates in the pathology . besides caspases and calpains acting as proteases in hd miller et al . have shown that knocking down mmp-10 , mmp-14 , and mmp-23 in cultured striatal cells expressing mutant htt diminishes toxicity . additionally , mmp-10 can directly cleave htt , and the production of toxic htt fragments is reduced upon silencing of mmp-10 . analysis of deceased patients with hd revealed an increase in mmp-9 in comparison to controls , as well as upregulation of cytokine levels ( il-6 , il-8 ) in cortex and cerebellum . in striatum , the main area affected in hd , only ccl2 and il-10 were upregulated . other evidence for involvement of mmp-9 in hd comes from the 3-nitropropionic acid animal model of the disease . the authors showed that mmp-9 is accountable for the bbb disruption that occurs in the disease . moreover , significantly elevated levels of mmp-9 were found in plasma of patients suffering from hd , as well as in the r6/2 mouse hd model . the authors proposed mmp-9 ( along with il-6 , vegf , and tgf- ) as a potential biomarker of hd . as far as timps are concerned , lorenzl et al . found increased timp-1 and timp-2 levels in csf of patients suffering from hd . as far as other neurodegenerative diseases are concerned , different mmps were found to be dysregulated in people suffering from dementias . decreased levels of timp-2 were found in serum of patients with frontotemporal dementia , and downregulated timp-1 was shown in patients with vascular dementia . intriguingly , patients with vascular dementia have been noted to have higher levels of mmp-9 in csf even compared to patients suffering from ad . in csf from patients suffering from creuztfeldt - jakob disease , a rare type of dementia , there were increased levels of pro - mmp-9 and active mmp-2 , as well as timp-1 and timp-2 . for decades , conventional wisdom has taught us that mmps play a pivotal role in the dissemination of cancer : they degrade the connective tissue between the cells and allow the cancer cells to leak from the primary site of tumor formation . during the past decade , more than a dozen of mmps have been shown to be involved in progression of neurodegenerative disorders , thereby opening up the possibility of therapeutically targeting mmps . in most neurodegenerative disorders , neuroinflammation is observed either before or during the development of the pathological characteristics of the disease . during neuroinflammation , mmps often increase the permeability of the cns barriers by destroying the stability of the tight junction proteins or degrading the ecm , which in turn leads to infiltration of immune cells into the brain and cell death . on the other hand , in certain neurodegenerative diseases such as ad and mmps , and , in particular , mmp-3 and mmp-9 , were shown to degrade a plaques [ 50 , 58 ] , justifying the view of mmps as a double - edged sword . therefore , drugs that inhibit mmps could have unforeseen effects that need to be well understood and avoided before we employ them for therapy ( figure 4 ) . mmp expression is usually triggered by an inflammatory stimulus ( e.g. , infection , burns , or protein aggregates ) , which induces an inflammatory cascade . at this stage , anti - inflammatory drugs will be effective in eliminating the expression or activation of mmps . subsequently , the available synthetic broad - spectrum inhibitors might be used to inhibit mmps ( table 3 ) . however indeed , selective mmp inhibition might avoid the unwanted side effects of broad - spectrum mmp inhibition . mmp inhibitors can be broadly classified as macromolecular inhibitors ( including timps and monoclonal antibodies ) and both synthetic and natural small molecules [ 280 , 281 ] . in general , early studies conducted using broad spectrum mmp inhibitors such as batimastat on mice injected with human cancer cells gave compelling results of extending the life of the mice from six- to sevenfold , thereby paving the way for the potential use of mmp inhibitors in other diseases . mmp inhibitors yielded beneficial results in animal studies of lung inflammatory diseases , multiple sclerosis , meningitis [ 283285 ] , vascular dementia , stroke , acute cerebral ischemia [ 287 , 288 ] , and sepsis [ 1 , 15 , 289 , 290 ] . finally , interfering with the substrates downstream of mmps might also have therapeutic value . the potential use of mmp inhibitors in ad is very speculative and is based on the seemingly beneficial effect of mmp-9 , due to its role in degradation of amyloid plaques and hence its contribution to the clearance of a from the brain . furthermore , it has been reported that mmp-2 can cleave a at the -secretase site . in another similar study , it was reported that mmp-2 also can cleave full - length app , indicating that it can produce -apps at the plasma membrane or degrade a in the ecm , which leads to reduction of a burden in the brain . in contrast to those reports , other authors were unable to show a similar mmp-2 activity , but they found that mmp-2 might possess -secretase like activity , which might shift the balance towards the amyloidogenic pathway . mice deficient in mmp-2 or mmp-9 appeared to have higher levels of a than wild type animals . likewise , treatment with the broad - spectrum mmp inhibitor gm6001 resulted in an increase in a in transgenic mice overexpressing the swedish variant of app . in an in vitro study , gm6001 was shown to block the a-induced alterations in zo-1 expression and bbb permeability . similarly , gm6001 was also able to prevent the a oligomer - induced degradation of the blood - csf barrier integrity . moreover , in another study on a transgenic mouse model of ad , it was reported that inhibition of mmps with gm6001 reduced the oxidative stress associated with caa . timps , the endogenous inhibitors of mmps , were found to be localized near the a plaques and neurofibrillary tangles of ad - affected brain samples . it has been speculated that mmps and timps contribute to the evolution of these lesions . similarly , it has been shown that mmps are produced in excess at lesion sites by the immune cells surrounded by the effected regions , and that timps might be localized in these places as well to control the activity of mmps . thus , to validate mmps and timps as possible candidates for therapeutics development , it is important to investigate whether they are amyloidogenic or prevent a accumulation . this is mainly because of potential harmful side effects of broad spectrum mmp inhibitor activities , which pose a big hurdle . as far as therapeutic opportunities of mmp inhibition in pd are concerned , lorenzl et al . reported the expression of mmps such as mmp-1 , mmp-2 , and mmp-9 and also timp-1 and timp-2 in substantia nigra of postmortem pd brain samples . hence , mmp inhibitors might hold promise for management of pd because death of dopaminergic neurons seems to be linked with release of mmps . apoptotic dopaminergic neurons release mmp-3 , which in turn activates microglia in vitro , indicating that mmp-3 could serve as a signaling molecule as well . the activated microglia release proinflammatory cytokines , such as tnf , that lead to neuronal cell death . treatment of mouse mesencephalic cells with tetrahydrobiopterin ( bh4 ) , a selective dopaminergic neuronal toxin , decreased cell survival . however , when cells were exposed to a selective mmp-3 inhibitor , nngh ( n - isobutyl - n-[4-methoxyphenylsulfonyl]-glycylhydroxamic acid ) , cell survival was extended via the decrease of tnf- release from activated microglial cells . similarly , several hypotheses were proposed regarding the role of mmps in the development of als . reported that by crossing g93a sod1 mice with mmp-9 knockout mice , immunoreactivity was increased and expression of mmp-9 was elevated in spinal cord tissue of g93a sod1 mice , a model of familial als . reduced mmp-9 activity was shown to prolong survival in the als mouse model expressing mutant sod1 , pointing to mmp-9 as a potential therapeutic target . generally , mmp-9 stimulates neuronal tnf- by cleaving it from its membrane - bound form , and it also contributes to neuronal cell death by activating other proinflammatory cytokines . abnormally high levels of mmp-9 and possible degradation of the matrix components contribute to als progression . there are several reports on the use of synthetic mmp inhibitors to ameliorate the symptoms of eae , and protease inhibitors were used to treat eae as early as 1982 . mmp activity was shown to increase threefold in the csf in two acute models of eae . broad - spectrum mmp inhibitors such as gm6001 [ 87 , 89 ] , ro31 - 9790 , uk221,316 , d - penicillamine , and bb1101 were shown to be beneficial in eae . mmp-9 was shown to be elevated at the lesion sites and in the csf of ms patients . similarly , association of mmp-9 with the disruption of the bbb was also reported in mri studies . when gm6001 was administered after the clinical onset of the disease , it inhibited the development of eae , and it also reversed the clinical symptoms in sjl / j mice . it was also speculated that mmp inhibition results in restoration of damaged bbb , thereby decreasing the inflammation rather than inhibiting demyelination . in a similar study by hewson et al . , using ro31 - 9790 reduced the clinical signs in the eae model of ms when given on the day of disease induction or three days after induction . ro31 - 9790 was less effective in controlling the disease in animals with more severe clinical signs . bb1101 , another broad - spectrum inhibitor , reduced disease severity in lewis rats and reversed acute symptoms in sjl / j mice . bb1101 was also shown to be effective in chronic relapsing eae in sjl / j mice , in which bb1101 treatment reduced the glial scar and demyelination . further , b1101 treatment shifted the cytokine profile from a proinflammatory to an anti - inflammatory state . an antirheumatic drug , d - penicillamine , was also shown to partially protect against eae in sjl / j mice , but in chronic relapsing eae in biozzo mice , treatment with d - penicillamine resulted in attenuation of disease progression after disease induction . the therapeutic efficacy of minocycline , a semisynthetic derivative of tetracycline , was tested in mog35_55 peptide - induced eae in c57bl/6 mice . it reduced both the activity and expression of mmp-9 in t - cells and reduced disease severity . in the same study , interestingly , an antioxidant molecule , -lipoic acid , was found to be beneficial in suppressing eae and reducing disease severity after disease induction . this effect was linked with reduced infiltration of t cells into the cns , which led to speculation that -lipoic acid could be inhibiting mmp-9 . so far , no molecular mechanism has been identified to explain how mmp inhibition ameliorates ms disease symptoms , but it is speculated that broad - spectrum mmp inhibition inhibits transmigration of immune cells into the cns via the bbb . that would result in reduced demyelination and reduced levels of tnf through inhibition of adam17 . therefore , novel strategies are needed to harness the ability of neuroprotective mechanisms to slow down or stop the progression of the disease in order to prolong the healthy lifespan of patients . more basic research is required to fully understand the diverse roles of mmps in the pathophysiology of neurodegenerative diseases in order to design specific mmp inhibitors and therapeutic strategies for these chronic diseases of the nervous system . although the potential causes and etiology of neurodegenerative diseases remain largely elusive , mmps clearly have a pivotal role in the progression of neurodegenerative diseases such as ad , pd , als , hd , and ms , and their functions in these diseases seem to be much more complex than previously thought . targeting mmps will be of much interest for the treatment of these disorders . in most clinical cases , hence , it is necessary to explore the role of the different mmps in depth to be able to develop more therapeutic options . more than a dozen mmps were shown to be involved in neurodegenerative diseases , including mmp-2 , mmp-3 , and mmp-9 , and they seem to be important players in most of the diseases mentioned in this review . the mechanisms of action by which they contribute to the aggravation of neurodegenerative diseases are slowly starting to unfold . mmps participate in a common pathway of pathological changes in the cns homeostasis , that is , accumulation of proinflammatory molecules or aggregated proteins and peptides , leading to increased permeability of cns barriers and consequently to cell death . on the other hand , the dual roles of mmps hinder efforts to use broad - spectrum mmp inhibitors as therapeutics . however , a strong indication that selective mmp inhibitors could have therapeutic opportunities already exists . consequently , investigation of mmps and timps as potential biomarkers and therapeutics in neurodegenerative diseases needs to be continued .

neurodegeneration is a chronic progressive loss of neuronal cells leading to deterioration of central nervous system ( cns ) functionality . it has been shown that neuroinflammation precedes neurodegeneration in various neurodegenerative diseases . matrix metalloproteinases ( mmps ) , a protein family of zinc - containing endopeptidases , are essential in ( neuro)inflammation and might be involved in neurodegeneration . although mmps are indispensable for physiological development and functioning of the organism , they are often referred to as double - edged swords due to their ability to also inflict substantial damage in various pathological conditions . mmp activity is strictly controlled , and its dysregulation leads to a variety of pathologies . investigation of their potential use as therapeutic targets requires a better understanding of their contributions to the development of neurodegenerative diseases . here , we review mmps and their roles in neurodegenerative diseases : alzheimer 's disease ( ad ) , parkinson 's disease ( pd ) , amyotrophic lateral sclerosis ( als ) , huntington 's disease ( hd ) , and multiple sclerosis ( ms ) . we also discuss mmp inhibition as a possible therapeutic strategy to treat neurodegenerative diseases .

1. Introduction 2. Matrix Metalloproteinases (MMPs) 3. Neurodegenerative Diseases and Neuroinflammation 4. Role of MMPs in Neurodegenerative Diseases 5. Therapeutic Opportunities 6. Conclusion

in the past two decades , the function of matrix metalloproteinases ( mmps ) in the central nervous system ( cns ) has gained much attention . mmps are calcium ( ca ) dependent zinc ( zn ) containing endopeptidases produced in latent forms . mmps are involved in the degradation of extracellular matrix ( ecm ) components , remodeling of tissues , shedding of cell surface receptors , and processing of various signaling molecules . mmps are essential for brain development due to their association with important neurophysiological functions , such as synaptic plasticity [ 2 , 3 ] and long - term potentiation [ 4 , 5 ] . in the adult brain under normal conditions , increased expression of mmps is observed in a variety of pathological conditions , including neurodegenerative diseases such as alzheimer 's disease ( ad ) , parkinson 's disease ( pd ) , amyotrophic lateral sclerosis ( als ) , huntington 's disease ( hd ) , and multiple sclerosis ( ms ) and in neuroinflammatory conditions such as traumatic brain injury , stroke , and meningitis . as part of the neuroinflammatory response , mmp activity at cns barriers contributes to the increase in permeability by altering the properties of ecm and tight junctions . in this review , mmps , together with adamlysins and astacins , belong to metzincins , a family of zn - dependent , ca - containing endopeptidases ( 24 members in mammals ) . mmps are multidomain proteins consisting mostly of the following domains : n - terminal signal peptide ( which is cleaved in the secretory pathway ) , propeptide ( which maintains latency of mmps ) , catalytic domain ( holds the zn ion ) , hinge region ( connecting sequences ) , and c - terminal hemopexin - like domain ( required for substrate and timp recognition ) ( figure 1 ) . it has been shown that mmps play an important role in various physiological and pathological processes in the body . it acts as an adhesion site for various cells and serves as a storage site for different signaling molecules , growth factors , and proteins in general , thus influencing development and migration of the cells . in the brain , mmps are involved in tissue remodeling after injury , neurogenesis , axonal growth , angiogenesis , cns barrier disruption , myelinogenesis , and demyelination . based on their domain organization , mmps are classified into four major subgroups : ( 1 ) gelatinases ( mmp-2 , mmp-9 ) , ( 2 ) matrilysins ( mmp-7 , mmp-26 ) , ( 3 ) archetypal mmps , and ( 4 ) furin - activated mmps ( figure 1 and table 1 ) . the archetypal mmps contain different types : stromelysins ( mmp-3 , mmp-10 ) , collagenases ( mmp-1 , mmp-8 , mmp-13 ) , and other mmps ( mmp-12 , mmp-18 , mmp-20 , mmp-27 ) . similarly , the furin - activated mmps are divided in secreted mmps ( mmp-11 , mmp-21 , mmp-28 ) ; type - i transmembrane mmps ( mmp-14 , mt1-mmp , mmp-15 , mt2-mmp , mmp-16 ( mt3-mmp ) , and mmp-24 ( mt5-mmp ) ) , type - ii transmembrane mmps ( mmp-23 ) , and gpi - anchored mmps ( mmp-17 ( mt4-mmp ) , mmp-25 ( mt6-mmp ) ) . mmps are modulated on several levels : transcriptional activation , removal of the prodomain , interaction with ecm components , and inhibition by endogenous inhibitors such as tissue inhibitors of metalloproteinases ( timps ) . on the level of mmp - zymogen activation , mostly serine proteases and other mmps are involved in initiating proteolytic cleavage of the prodomain ; for example , mmp-3 can cleave pro - mmp-9 . it has been observed that mmps in the cns are secreted by microglia , astrocytes , and neurons . in physiological conditions , mmps are either absent or present at undetectable levels in the mature brain , and deregulation of their activity could shift the balance and induce perpetuation of chronic inflammation . this has been shown in different peripheral chronic diseases , such as atherosclerosis and rheumatoid arthritis [ 25 , 26 ] , and neuroinflammatory diseases , such as cerebral ischemia , stroke , and bacterial meningitis . mmps are known to be involved in cns barrier maintenance and to increase the permeability of the barriers in inflammation . mmps are secreted upon inflammation , for example , by peripheral neutrophils , which thereby contribute to aggravation of the inflammation and disruption of the barriers . compromise of cns barrier integrity due to activation of mmps has been observed in cerebral ischemia , traumatic brain injury ( tbi ) , and other diseases associated with neuroinflammation . in this review , we focus on the role of different mmps in the major neurodegenerative diseases , ad , pd , als , hd , and ms ( figure 2 ; tables 2 and 3 ) . neurodegenerative diseases share common features , such as progressive loss of neurons and deterioration of the structure and function of the central and/or peripheral nervous system . the traditional classification of neurodegenerative disorders is based on clinicopathological features and has been formulated through consensus criteria , that is , a set of diagnostic criteria on which experts in the field agree , along with specific molecular characteristics . however , in certain cases diagnosis is difficult due to the coincidence of clinicopathological features of several neurodegenerative diseases [ 9799 ] . the early classifications divided neurodegenerative diseases into either taupathies ( including ad , pick 's disease ( pid ) , argyrophilic grain disease ( agd ) , progressive supranuclear palsy ( psp ) , corticobasal degeneration ( cbd ) , and ftdp-17 ) and synucleinopathies ( including pd , dementia with lewy bodies ( dlb ) , and multiple system atrophy ( msa ) ) . today , different , nosological classifications of neurodegenerative diseases exist , taking into account clinical presentation , affected brain regions and cell types , altered proteins involved in the pathogenesis of the disease , genetics , and possible overlaps between diseases [ 96 , 103 ] . neuroinflammation is defined as inflammation of nervous tissue , which occurs as a biological response to different signals , such as infection , cns injury , autoimmunity , and toxic compounds . although it is initiated primarily as a beneficial reaction of the cns to the harmful stimulus , it could eventually aggravate the disease . it has been shown that microglia are involved in neuron survival , neurogenesis , facilitation of brain repair via guidance of stem - cell migration to the site of inflammation or injury , and clearance of cell debris . nevertheless , when overactive , microglia can inflict severe damage to the brain by excessive production of molecules such as mmps , proinflammatory cytokines and chemokines , and cytotoxic molecules such as ros and nitric oxide ( no ) [ 116118 ] . however , it is not yet clear how aging acts on the development of neurodegenerative diseases and other age - related diseases . two of the most prominent characteristics of aging are immunosenescence ( deterioration of immune responses ) and inflammaging ( presence of chronic low grade inflammation ) [ 124 , 125 ] . it has been shown that aged microglia secrete il-1 and increase mhc class ii expression . one of the characteristics of aging is accumulation of advanced glycation end products ( age ) , which are also elevated in ad . activation of the receptor for age ( rage ) leads to release of proinflammatory cytokines and free radicals , further contributing to inflammatory processes . this finding is linked to aggravation of neuroinflammation associated with aging via the induction of the migration of bone marrow derived microglia to the brain . additionally , occurrence of a pathology has been observed in several other neurodegenerative diseases , such as pd , pick 's disease ( pid ) , progressive supranuclear palsy ( psp ) , and corticobasal degeneration , as well as in als . it has been shown that some of the members of the metalloproteinase family , including the adam ( a disintegrin and metalloproteinase ) proteins adam-17 ( also called tumor necrosis factor--converting enzyme or tace ) , adam-9 and adam-10 , can cleave app at the -secretase cleaving site . furthermore , it has been shown that besides the established direct neurotoxic effect , a can exert indirect proinflammatory effects via microglial activation , which results in secretion of no , tnf , and superoxides [ 167 , 168 ] . interestingly , clustering of activated microglia around a aggregates has been observed even before the development of ad symptoms [ 169171 ] . additionally , mmp-9 was found in pyramidal neurons of the brains of ad patients , and near amyloid plaques and it was shown that mmp-9 is able to cleave a140 . it has been shown that mmp-9 can act through nmda receptor signaling via an integrin 1 dependent pathway . in a transgenic mouse model of ad , expression of mmp-2 and mt1-mmp , a potent mmp-2 activator , was found in reactive astrocytes around amyloid plaques , and higher levels of a142 increased the production of mmp-3 , mmp-12 , and mmp-13 in microglia . moreover , there was an increase in mmp-9 and mmp-2 activity , and broad - spectrum mmp inhibition reversed the a-induced bbb disruption . the proposed mechanism of a activity is through activation of rage , which is physiologically expressed on endothelial cells and activates the intracellular calcineurin ( can ) signaling pathway , which ultimately results in activation of mmps and tj cleavage . in other studies , it has been shown that mmp-3 is expressed in microglia , astrocytes , and endothelial cells in the brain , as well as near senile plaques in ad . as reported for mmp-9 moreover , a correlation was found between mmp35a and apoe 4 alleles , and the presence of both is a risk factor for developing ad . interestingly , we recently reported that icv injection of a142 oligomers induces loss of barrier integrity at the blood - csf barrier , and this was linked to increased mmp-3 expression and mmp activity . finally , levels of timp-1 and c - reactive protein ( crp ) were found to be increased in ad patients , and they decreased remarkably after treatment with acetylcholinesterase inhibitors ( acheis ) , one of the few available therapies of ad . like ad , its prevalence increases with age , and due to the severity and long duration of the disease , its costs in the us alone are estimated at 25 billion dollars per year . thus , due to dopaminergic neuron cell death , one of the clear manifestations of the disease is loss of control over movements , resting tremor , bradykinesia , and rigidity . additionally , pd is accompanied by sensory dysfunction , mood and sleep disorders , dementia , and partial autonomic nervous system impairment . nevertheless , most cases of pd are sporadic , and the onset of the disease has been shown to be caused by complex interaction of environmental and genetic factors . the substantia nigra seems to be particularly susceptible to inflammation due to the presence of the largest number of microglia in this brain area , so relevant stimuli lead to the activation of large numbers of microglia . it has been speculated that prolonged overactivation of microglia and production of proinflammatory cytokines could lead to neuronal degeneration in pd [ 207 , 211 ] . it is also speculated that oxidative stress could be generated from dopaminergic metabolism , mitochondrial dysfunction , and microglial activation , which could be influenced beforehand by various toxins and mutations in parkin , pink1 , dj-1 , or htra1 , which are important for physiological mitochondrial functioning . it has been shown that damaged dopaminergic neurons can activate microglia by releasing -synuclein and neuromelanin , causing them to produce ros . it has been suggested that this mechanism plays an important role in apoptosis . additionally , they hypothesized that both active mmp-3 and catalytically active recombinant mmp-3 could activate microglia to produce proinflammatory cytokines , which in turn aggravate neuronal apoptosis of damaged cells , leading to further induction of apoptosis in neighboring dopaminergic neurons . using mmp-3 deficient mice and a broad spectrum mmp inhibitor , it has been shown that depletion of mmp-3 can significantly reduce mptp - induced degeneration of nigrostriatal dopaminergic neurons in the brain . recent evidence suggests that the proteolytic activity of mmps might be involved in alteration of -synuclein protein conformation , thus contributing to aggregation , lewy body formation , and microglial activation . als , also known as lou gehrig 's disease , is characterized by degeneration of motor neurons in the brain , brainstem , and spinal cord . some other neurodegenerative diseases share similar etiology , such as progressive lateral sclerosis ( pls ) , progressive muscular atrophy ( pma ) , als dementia , and als frontal lobe dementia . familial occurrence of the disease is only 510% of all als cases , and the cause of the sporadic form of als is still unknown . however , occurrence of tdp-43 is not characteristic of only als but was found in several patients with frontotemporal lobar degeneration with tdp proteinopathy ( ftld - tdp ) , as well as in frontotemporal dementia , ad , and some other neurodegenerative diseases . one of the theories suggests that bbb and bscb breakdown could contribute to the motor neuron damage , due to the importance of these barriers in maintenance of homeostasis in the cns . nevertheless , mmp-2 showed a slow but progressive decrease with the development of the disease . these results show that mmp-9 is a key player in the onset of the disease and point to it as a therapeutic target . it has been shown that the bbb function in ms is lost in both relapsing - remitting and progressive phases . another group confirmed this finding and also showed expression of mmp-3 in endothelial cells , mmp-1 , mmp-2 , mmp-3 and mmp-9 in macrophages , and to a lesser extent in astrocytes , around active and necrotic lesions . additionally , they proposed that constant elevation of mmp-9 throughout the progress of the disease could contribute to the surrounding tissue damage and neuronal cell loss . besides the occurrence of the leakage , the bbb also becomes activated , meaning that cells making up the bbb , including endothelial cells , astrocytes , and potentially pericytes , start expressing and secreting various factors involved in the recruitment and functioning of leukocytes . additionally , mmp - activated axonal degeneration was hardly observed in peripheral nervous system , possibly due to the presence of resilient ecm and higher timps expression . huntington 's disease ( hd ) is an inherited neurodegenerative disorder that decreases muscle coordination and mental ability . the disease has been linked to a mutation on chromosome 4 in a gene coding for a protein called huntingtin ( htt ) . while the exact role of htt is not clear yet , it has been speculated that proteolysis of mutant htt participates in the pathology . during the past decade , more than a dozen of mmps have been shown to be involved in progression of neurodegenerative disorders , thereby opening up the possibility of therapeutically targeting mmps . in most neurodegenerative disorders , neuroinflammation is observed either before or during the development of the pathological characteristics of the disease . on the other hand , in certain neurodegenerative diseases such as ad and mmps , and , in particular , mmp-3 and mmp-9 , were shown to degrade a plaques [ 50 , 58 ] , justifying the view of mmps as a double - edged sword . the potential use of mmp inhibitors in ad is very speculative and is based on the seemingly beneficial effect of mmp-9 , due to its role in degradation of amyloid plaques and hence its contribution to the clearance of a from the brain . furthermore , it has been reported that mmp-2 can cleave a at the -secretase site . it has been speculated that mmps and timps contribute to the evolution of these lesions . similarly , it has been shown that mmps are produced in excess at lesion sites by the immune cells surrounded by the effected regions , and that timps might be localized in these places as well to control the activity of mmps . as far as therapeutic opportunities of mmp inhibition in pd are concerned , lorenzl et al . treatment of mouse mesencephalic cells with tetrahydrobiopterin ( bh4 ) , a selective dopaminergic neuronal toxin , decreased cell survival . however , when cells were exposed to a selective mmp-3 inhibitor , nngh ( n - isobutyl - n-[4-methoxyphenylsulfonyl]-glycylhydroxamic acid ) , cell survival was extended via the decrease of tnf- release from activated microglial cells . when gm6001 was administered after the clinical onset of the disease , it inhibited the development of eae , and it also reversed the clinical symptoms in sjl / j mice . so far , no molecular mechanism has been identified to explain how mmp inhibition ameliorates ms disease symptoms , but it is speculated that broad - spectrum mmp inhibition inhibits transmigration of immune cells into the cns via the bbb . more basic research is required to fully understand the diverse roles of mmps in the pathophysiology of neurodegenerative diseases in order to design specific mmp inhibitors and therapeutic strategies for these chronic diseases of the nervous system . although the potential causes and etiology of neurodegenerative diseases remain largely elusive , mmps clearly have a pivotal role in the progression of neurodegenerative diseases such as ad , pd , als , hd , and ms , and their functions in these diseases seem to be much more complex than previously thought . more than a dozen mmps were shown to be involved in neurodegenerative diseases , including mmp-2 , mmp-3 , and mmp-9 , and they seem to be important players in most of the diseases mentioned in this review . the mechanisms of action by which they contribute to the aggravation of neurodegenerative diseases are slowly starting to unfold . consequently , investigation of mmps and timps as potential biomarkers and therapeutics in neurodegenerative diseases needs to be continued .

india had started its universal immunization program ( uip ) in 1985 focusing more on infants and pregnant mothers . the success of this program depends highly on the level of cold chain maintenance of the vaccines right from the site of manufacturing up to its administration . urban health centers ( uhcs ) , set up under various municipal corporations , have been the backbone for delivering services related to immunization in urban areas in india . health workers involved in immunization are also part of the cold chain system . therefore , good knowledge of these health workers in the maintenance of the cold chain system is a pre - requisite in the correct delivery of immunization services . it is also imperative that the condition of cold chain equipment , i.e. , deep freezer and ice - lined refrigerator ( ilr ) is maintained appropriately and emergency plan in case of cold chain failure is followed adequately . this study also attempted to evaluate the loop holes in the maintenance of cold chain of vaccines and assessed the knowledge and practices adopted by the vaccinators for the same in surat city , western india . study design and setting : surat , situated 270 km north to mumbai , is the fourth fastest growing city in the world . better known as the diamond city , surat has seen a lot of urbanization in recent years . the total population ( both rural and urban ) of surat city has increased from 49.95 lakhs in 2001 to 60.79 lakhs in 2011 according to census data . surat municipal corporation has been working incessantly for improving the overall health of the citizens of surat . the city has a network of 36 uhcs under smc in a total of 7 administrative zones . the zone - wise population of surat city according to census 2001 data is given in table 1 . zone - wise distribution of urban population of surat city according to census ( 2001 ) each uhc has a designated vaccinator who looks after the vaccine cold chain system and the activities related to immunization . a cross - sectional study was conducted in 20 uhcs of municipal corporation of surat city . sample size and sampling design : as data from any prior study in surat was not available , we have included at least 50% of the uhcs of smc in our study . thus , out of 36 uhcs , we decided to include 20 uhcs for our study . at least 50% of health centers were selected from each zone so as to make suitable representation of all the health centers of the city . one maternity home from each zone was deliberately selected as it was considered as a higher center providing emergency obstetric care together with primary health care . in each zone , the remaining uhcs ( non - maternity ) were selected randomly by lottery method . selection of uhcs from 7 administrative zones data collection : permission for carrying out the study was obtained from the medical officer of health ( moh ) of smc . data collection was carried out from june 2011 to july 2011 . in this study , cold chain equipment was observed by the researchers , with regard to their condition and practices adopted by vaccinators for cold chain maintenance . the alternatives available in the uhc in case of cold chain failure were also assessed . a vaccination session was observed to assess the cold chain practices at the time of vaccination . a pre - designed and pre - tested questionnaire was used to interview the vaccinators regarding their knowledge and awareness of cold chain practices , management and handling . we assessed the knowledge of the vaccinators on a 3-pointer scale with those knowing exactly ( exactly in its stated form ) , knowing but not properly ( not exactly in its stated form , but having the concept behind it ) and those who had not heard anything about the point in question . the questionnaire was pre - tested in 2 uhcs selected purposively out of the remaining 16 uhcs not included in the study . changes were made in the questionnaire accordingly after discussing the issues that we came across in the pilot study . the observations with regard to deep freezers and ilr are tabulated in tables 3 and 4 . observations related to deep freezer and ilr ( n=20 ) observations related to ilr ( n=20 ) in this study , a separate voltage stabilizer was attached each to deep freezer and ilr in 3 ( 15% ) health centers . it was refreshing to find that in our study , temperature was being recorded twice a day for both deep freezer and ilr in all the health centers . information regarding defrosting of deep freezers and ilrs was recorded on the temperature - record register in 10 ( 50% ) health centers . thickness of ice on the side walls was > 5 mm in the deep freezers and ilrs of 10 ( 50% ) health centers . it was also found that ice packs were arranged in a criss - cross manner in only 7 ( 35% ) health centers . the present study found water bottles kept inside deep freezer in 2 ( 10% ) health centers . and , at 2 ( 10% ) health centers , things other than vaccines / diluents , like empty water bottles , were kept inside the ilr . vaccines were arranged according to temperature sensitivity in ilr in 14 ( 70% ) health centers . on observing the alternatives to cold chain failure ( n = 20 ) , we noticed that emergency contact number was written in the same room nearby in only 3 ( 15% ) health centers . we found that 3 ( 15% ) uhcs each had either a working inverter or a generator . though , an alternate working refrigerator was available in 13 ( 65% ) health centers . on observing the vaccination sessions ( n = 20 ) , we found that the vaccinators were reading vvm at the time of vaccination at 13 ( 65% ) health centers . the time of reconstitution was noted on the vaccine vial by the vaccinator at 15 ( 75% ) health centers . the reconstituted vaccine was discarded after the recommended time limits in 19 ( 95% ) health centers . it was good to see that 15 ( 75% ) vaccinators knew the definition of cold chain exactly in its stated form , whereas 5 ( 25% ) did not know it exactly . it was encouraging to see that 18 ( 90% ) vaccinators knew the full form of vvm and stages of vvm correctly . it was reassuring to see that almost all ( 95% ) vaccinators knew the correct arrangement of vaccines in the ilr according to temperature sensitivity . knowledge of the vaccinators regarding the timing of defrosting of ilr and deep freezer was poor , with only 11 ( 55% ) knowing exactly , the criteria for defrosting . it was found in the present study that 12 ( 60% ) vaccinators knew exactly about shake test . only 5 ( 25% ) vaccinators knew the exact temperature range for deep freezer , though , 16 ( 80% ) knew the exact temperature range for ilr . on being asked about the steps incumbent to refrigerator failure , 8 ( 40% ) vaccinators told to put the vaccines in cold boxes ; 5 ( 25% ) told to put it in alternate storage refrigerator ; 5 ( 25% ) told to use the generator ; 1 ( 5% ) told to call the higher authority and 1 ( 5% ) told to shift the vaccines in a nearby ice - cream vendor 's refrigerator . a separate voltage stabilizer should be attached each to deep freezer and ilr , except in emergency , when one stabilizer per two small equipment is acceptable . the function of the voltage stabilizer is to monitor the range of fluctuations in the main voltage of 90 - 280 v and maintain voltage in a required range of 220 + 10 v. stabilizer protects the cold chain equipment against voltage fluctuations and is an essential pre - requisite . the present study claimed that a separate voltage stabilizer was attached each to deep freezer and ilr in only 15% of the health centers . medical officer of the respective uhcs should ensure adequate supply and maintenance of voltage stabilizer and report to higher authority in case scarcity persists . deep freezer and ilr should be placed in such a way in the room that they are not exposed to sunlight any time during the day . exposure to sunlight will lead to increase in core temperature of the cold chain equipment , which would break the cold chain by causing an increase in the temperature of the vaccines in ilr and ice packs would not be prepared in deep freezer . the present study stated that deep freezers were exposed to sunlight in 2 ( 10% ) health centers . this finding was similar to a study by sachdeva , et al . which reported that the cold chain equipment were kept away from sunlight in all the health centers . the temperatures in the ilr and deep freezer must be monitored twice daily . a break in the cold chain is indicated if temperature rises above + 8c or falls below + 2c in the ilr ; and above 15c in the deep freezer . the ilr and deep freezers each should have a separate thermometer and temperature record book . it was refreshing to find that in our study , temperature was being recorded twice a day for both deep freezer and ilr in all the health centers in separate temperature record books . also , in the present study , working separate thermometers were found in deep freezer in 95% health centers and in ilr in all the health centers . the temperature in the ilr / freezer can rise if there is a thick layer of ice around the freezer or along the walls and bottom of ilrs . this should be done if the ice in the freezer is > 5 mm thick . the present study showed that information regarding defrosting of deep freezers and ilrs was recorded on the temperature - record register in only half of the health centers . thickness of ice on the side walls was > 5 mm in the deep freezers and ilrs of half of the health centers . ( 37.5% ) , though they kept the cut - off for thickness > 6 mm . this suggests lack of regular defrosting by the cold chain handlers , which need to be improved and monitored subsequently by the medical officer of the respective uhc . ice packs should be stacked on the floor of the deep freezer horizontally ( not flat ) on its edge by keeping 1 - 2 mm space from each other for air circulation , in a criss - cross manner . yet , in our study , we found this in only one - third ( 35% ) of the health centers . this was probably due to the increased demand of ice packs in health centers , especially during sub - national immunization day ( snid ) rounds , which are frequently conducted in urban areas of surat city . the requirement for a large number of ice packs can be easily met with proper planning like transferring around 50 ice packs into a large cold box . fifty more ice packs can be frozen in two days and continuing the procedure till the required number of ice packs are frozen . deep freezers should be used to prepare and store ice packs only ; uip vaccines should not be stored in it . in spite of these guidelines , in our study , we found water bottles kept inside deep freezer in 2 ( 10% ) health centers . it is customary , not to keep other drugs and vaccines not used in uip , in ilr . in our study , at 2 ( 10% ) health centers , things other than vaccines / diluents , like empty water bottles , were kept inside the ilr . this was much less than what sachdeva , et al . claimed in their study ( 53.12% ) . opv and measles vaccines can be kept at bottom of the basket while bcg , dpt , dt and tt vaccines should be kept in upper part of the baskets . in our study , we found that the vaccines were arranged accordingly in more than two - third of the health centers . stated in their study that heat - sensitive vaccines were stored correctly in all , while , freeze - sensitive vaccines were stored correctly in 62.5% health facilities only . vaccinators need to understand the importance of this arrangement , as impotent vaccines give a false sense of security against diseases , which a child would contract later on despite the vaccination . the present study found that emergency contact number was written nearby in the same room in only 3 ( 15% ) health centers . mallik , et al . argued in their study that no organization maintained a chart in case of cold chain failure . this is in contrast to our study , where we found 30% uhcs had either a working inverter or a generator . though , an alternate working refrigerator was available in just less than two - thirds of the health centers . the cold chain is dependent on electrically operated machines on which one can not depend completely . suitable posters should be designed and pasted on machines with clear instructions in local languages on how to handle such emergency situations , especially during electricity failure . it would be prudent to identify more than one alternative at each uhc for tackling the situation . on observing the vaccination sessions ( n = 20 ) , we found that the vaccinators were reading vvm at the time of vaccination at 13 ( 65% ) health centers . the time of reconstitution was noted on the vaccine vial by the vaccinator at 15 ( 75% ) health centers . the reconstituted vaccine was discarded after the recommended time limits in 19 ( 95% ) health centers . reconstituted bcg and measles vaccines should not be used beyond 4 hours from the time of its reconstitution . if not utilized completely , these reconstituted vaccines should be discarded within 4 hours in the case of bcg and measles . it was good to see that three - fourth of the vaccinators knew the definition of cold chain exactly in its stated form , while one - fourth of the vaccinators knew the concept behind it suggesting that they were aware of cold chain . most of the vaccinators knew the full form of vvm and stages of vvm correctly , suggesting that vvm is now a matter of common knowledge for the vaccinators . thakur , et al . also highlighted similar results with 94.4% staff members being aware that vvm is present on vaccine itself and 71.7% knowing how to read it . almost all vaccinators knew the correct arrangement of vaccines in the ilr according to temperature sensitivity . knowledge of the vaccinators regarding the timing of defrosting of ilr and deep freezer was poor , which needs improvement . shake test , for testing whether a vaccine is frozen or not , should be a matter of common knowledge for the vaccinators . but , we found in the present study that just less than two - thirds of the vaccinators knew exactly about shake test . it is imperative that vaccinators are well conversant with the temperature range to be maintained in ilr and deep freezer so that a break in cold chain can be easily detected and reported to the higher authority . in the present study , only one - fourth of the vaccinators knew the exact temperature range for deep freezer . yuan , et al . found in their study that 57.3% individuals correctly specified the optimal temperature range for vaccine storage . jeremijenko , et al . reported that educating a staff member on correct vaccine storage conditions and nominating that person to be responsible for monitoring refrigerator temperatures improves adherence to vaccine storage guidelines . on being asked about the steps incumbent to refrigerator failure , more than one - third of vaccinators told to put the vaccines in cold boxes , one - fourth told to put it in alternate storage refrigerator , one - fourth told to use the generator , 5% told to call the higher authority and 5% told to shift the vaccines in a nearby ice - cream vendor 's refrigerator . irrespective of the step that is followed in case of refrigerator failure by the vaccinators , a fixed and pre - decided plan of action should be individualized for each uhc . a separate voltage stabilizer should be attached each to deep freezer and ilr , except in emergency , when one stabilizer per two small equipment is acceptable . the function of the voltage stabilizer is to monitor the range of fluctuations in the main voltage of 90 - 280 v and maintain voltage in a required range of 220 + 10 v. stabilizer protects the cold chain equipment against voltage fluctuations and is an essential pre - requisite . the present study claimed that a separate voltage stabilizer was attached each to deep freezer and ilr in only 15% of the health centers . medical officer of the respective uhcs should ensure adequate supply and maintenance of voltage stabilizer and report to higher authority in case scarcity persists . deep freezer and ilr should be placed in such a way in the room that they are not exposed to sunlight any time during the day . exposure to sunlight will lead to increase in core temperature of the cold chain equipment , which would break the cold chain by causing an increase in the temperature of the vaccines in ilr and ice packs would not be prepared in deep freezer . the present study stated that deep freezers were exposed to sunlight in 2 ( 10% ) health centers . this finding was similar to a study by sachdeva , et al . which reported that the cold chain equipment were kept away from sunlight in all the health centers . the temperatures in the ilr and deep freezer must be monitored twice daily . a break in the cold chain is indicated if temperature rises above + 8c or falls below + 2c in the ilr ; and above 15c in the deep freezer . the ilr and deep freezers each should have a separate thermometer and temperature record book . it was refreshing to find that in our study , temperature was being recorded twice a day for both deep freezer and ilr in all the health centers in separate temperature record books . also , in the present study , working separate thermometers were found in deep freezer in 95% health centers and in ilr in all the health centers . the temperature in the ilr / freezer can rise if there is a thick layer of ice around the freezer or along the walls and bottom of ilrs . this should be done if the ice in the freezer is > 5 mm thick . the present study showed that information regarding defrosting of deep freezers and ilrs was recorded on the temperature - record register in only half of the health centers . thickness of ice on the side walls was > 5 mm in the deep freezers and ilrs of half of the health centers . ( 37.5% ) , though they kept the cut - off for thickness > 6 mm . this suggests lack of regular defrosting by the cold chain handlers , which need to be improved and monitored subsequently by the medical officer of the respective uhc . ice packs should be stacked on the floor of the deep freezer horizontally ( not flat ) on its edge by keeping 1 - 2 mm space from each other for air circulation , in a criss - cross manner . yet , in our study , we found this in only one - third ( 35% ) of the health centers . this was probably due to the increased demand of ice packs in health centers , especially during sub - national immunization day ( snid ) rounds , which are frequently conducted in urban areas of surat city . the requirement for a large number of ice packs can be easily met with proper planning like transferring around 50 ice packs into a large cold box . fifty more ice packs can be frozen in two days and continuing the procedure till the required number of ice packs are frozen . deep freezers should be used to prepare and store ice packs only ; uip vaccines should not be stored in it . in spite of these guidelines , in our study , we found water bottles kept inside deep freezer in 2 ( 10% ) health centers . it is customary , not to keep other drugs and vaccines not used in uip , in ilr . in our study , at 2 ( 10% ) health centers , things other than vaccines / diluents , like empty water bottles , were kept inside the ilr . this was much less than what sachdeva , et al . claimed in their study ( 53.12% ) . opv and measles vaccines can be kept at bottom of the basket while bcg , dpt , dt and tt vaccines should be kept in upper part of the baskets . in our study , we found that the vaccines were arranged accordingly in more than two - third of the health centers . stated in their study that heat - sensitive vaccines were stored correctly in all , while , freeze - sensitive vaccines were stored correctly in 62.5% health facilities only . vaccinators need to understand the importance of this arrangement , as impotent vaccines give a false sense of security against diseases , which a child would contract later on despite the vaccination . the present study found that emergency contact number was written nearby in the same room in only 3 ( 15% ) health centers . mallik , et al . argued in their study that no organization maintained a chart in case of cold chain failure . this is in contrast to our study , where we found 30% uhcs had either a working inverter or a generator . though , an alternate working refrigerator was available in just less than two - thirds of the health centers . the cold chain is dependent on electrically operated machines on which one can not depend completely . suitable posters should be designed and pasted on machines with clear instructions in local languages on how to handle such emergency situations , especially during electricity failure . it would be prudent to identify more than one alternative at each uhc for tackling the situation . on observing the vaccination sessions ( n = 20 ) , we found that the vaccinators were reading vvm at the time of vaccination at 13 ( 65% ) health centers . the time of reconstitution was noted on the vaccine vial by the vaccinator at 15 ( 75% ) health centers . the reconstituted vaccine was discarded after the recommended time limits in 19 ( 95% ) health centers . reconstituted bcg and measles vaccines should not be used beyond 4 hours from the time of its reconstitution . if not utilized completely , these reconstituted vaccines should be discarded within 4 hours in the case of bcg and measles . it was good to see that three - fourth of the vaccinators knew the definition of cold chain exactly in its stated form , while one - fourth of the vaccinators knew the concept behind it suggesting that they were aware of cold chain . most of the vaccinators knew the full form of vvm and stages of vvm correctly , suggesting that vvm is now a matter of common knowledge for the vaccinators . thakur , et al . also highlighted similar results with 94.4% staff members being aware that vvm is present on vaccine itself and 71.7% knowing how to read it . almost all vaccinators knew the correct arrangement of vaccines in the ilr according to temperature sensitivity . knowledge of the vaccinators regarding the timing of defrosting of ilr and deep freezer was poor , which needs improvement . shake test , for testing whether a vaccine is frozen or not , should be a matter of common knowledge for the vaccinators . but , we found in the present study that just less than two - thirds of the vaccinators knew exactly about shake test . it is imperative that vaccinators are well conversant with the temperature range to be maintained in ilr and deep freezer so that a break in cold chain can be easily detected and reported to the higher authority . in the present study , only one - fourth of the vaccinators knew the exact temperature range for deep freezer . yuan , et al . found in their study that 57.3% individuals correctly specified the optimal temperature range for vaccine storage . jeremijenko , et al . reported that educating a staff member on correct vaccine storage conditions and nominating that person to be responsible for monitoring refrigerator temperatures improves adherence to vaccine storage guidelines . on being asked about the steps incumbent to refrigerator failure , more than one - third of vaccinators told to put the vaccines in cold boxes , one - fourth told to put it in alternate storage refrigerator , one - fourth told to use the generator , 5% told to call the higher authority and 5% told to shift the vaccines in a nearby ice - cream vendor 's refrigerator . irrespective of the step that is followed in case of refrigerator failure by the vaccinators , a fixed and pre - decided plan of action should be individualized for each uhc . cold chain maintenance and practices were not up to the mark in the uhcs of smc . the present study contemplates for periodic refresher training and capacity building for cold chain maintenance of all the cold chain handlers . medical officers should be actively involved in the monitoring and supervision of the cold chain system .

background : the success of immunization depends highly on the level of cold chain maintenance . the aim of the study was to assess the condition of cold chain equipment , practices adopted for cold chain maintenance and knowledge of the vaccinators.methods:it was a cross - sectional study conducted in 20 uhcs of surat municipal corporation ( smc ) . cold chain equipment were observed with regards to their condition , along with the practices adopted by vaccinators for cold chain maintenance . a pre - designed and pre - tested questionnaire was used to interview the vaccinators regarding their knowledge and awareness regarding cold chain practices , management and handling . data were entered and analyzed using epi info v 3.5.1 . simple proportions were calculated.results:absence of separate stabilizer for deep freezers and ilrs ( 85% ) , ill - maintained temperature - record register , lack of criss - cross pattern of ice packs in deep freezer ( 65% ) , presence of things other than ice packs in deep freezer ( 10% ) and things other than vaccines in ilr ( 10% ) indicate poor cold chain maintenance . in addition to this , expired vaccines in ilr ( 5% ) , vaccines in the unusable stages of vvm ( 15% ) , lack of emergency contact number nearby in case of cold chain failure ( 85% ) , lack of inverter ( 85% ) , lack of generator ( 85% ) and failure to note time of reconstitution on the vaccine vial at the time of vaccination ( 25% ) indicate poor cold chain practices . lack of knowledge of defrosting of ilr and deep freezer ( 45% ) , lack of knowledge about shake test ( 40% ) , lack of knowledge of temperature range to be maintained in deep freezer ( 70% ) and in ilr ( 15% ) indicate poor knowledge of vaccinators.conclusion:cold chain maintenance and practices need improvement . knowledge of vaccinators was overall unsatisfactory .

INTRODUCTION METHODS Statistical analysis RESULTS DISCUSSION Observations of ILR and deep freezer Observations related to alternatives to cold chain failure Observations of vaccination sessions Knowledge of vaccinators CONCLUSIONS

the success of this program depends highly on the level of cold chain maintenance of the vaccines right from the site of manufacturing up to its administration . health workers involved in immunization are also part of the cold chain system . therefore , good knowledge of these health workers in the maintenance of the cold chain system is a pre - requisite in the correct delivery of immunization services . it is also imperative that the condition of cold chain equipment , i.e. , deep freezer and ice - lined refrigerator ( ilr ) is maintained appropriately and emergency plan in case of cold chain failure is followed adequately . this study also attempted to evaluate the loop holes in the maintenance of cold chain of vaccines and assessed the knowledge and practices adopted by the vaccinators for the same in surat city , western india . study design and setting : surat , situated 270 km north to mumbai , is the fourth fastest growing city in the world . the total population ( both rural and urban ) of surat city has increased from 49.95 lakhs in 2001 to 60.79 lakhs in 2011 according to census data . surat municipal corporation has been working incessantly for improving the overall health of the citizens of surat . the zone - wise population of surat city according to census 2001 data is given in table 1 . zone - wise distribution of urban population of surat city according to census ( 2001 ) each uhc has a designated vaccinator who looks after the vaccine cold chain system and the activities related to immunization . a cross - sectional study was conducted in 20 uhcs of municipal corporation of surat city . sample size and sampling design : as data from any prior study in surat was not available , we have included at least 50% of the uhcs of smc in our study . thus , out of 36 uhcs , we decided to include 20 uhcs for our study . at least 50% of health centers were selected from each zone so as to make suitable representation of all the health centers of the city . selection of uhcs from 7 administrative zones data collection : permission for carrying out the study was obtained from the medical officer of health ( moh ) of smc . in this study , cold chain equipment was observed by the researchers , with regard to their condition and practices adopted by vaccinators for cold chain maintenance . the alternatives available in the uhc in case of cold chain failure were also assessed . a vaccination session was observed to assess the cold chain practices at the time of vaccination . a pre - designed and pre - tested questionnaire was used to interview the vaccinators regarding their knowledge and awareness of cold chain practices , management and handling . we assessed the knowledge of the vaccinators on a 3-pointer scale with those knowing exactly ( exactly in its stated form ) , knowing but not properly ( not exactly in its stated form , but having the concept behind it ) and those who had not heard anything about the point in question . the questionnaire was pre - tested in 2 uhcs selected purposively out of the remaining 16 uhcs not included in the study . changes were made in the questionnaire accordingly after discussing the issues that we came across in the pilot study . the observations with regard to deep freezers and ilr are tabulated in tables 3 and 4 . observations related to deep freezer and ilr ( n=20 ) observations related to ilr ( n=20 ) in this study , a separate voltage stabilizer was attached each to deep freezer and ilr in 3 ( 15% ) health centers . it was refreshing to find that in our study , temperature was being recorded twice a day for both deep freezer and ilr in all the health centers . information regarding defrosting of deep freezers and ilrs was recorded on the temperature - record register in 10 ( 50% ) health centers . thickness of ice on the side walls was > 5 mm in the deep freezers and ilrs of 10 ( 50% ) health centers . it was also found that ice packs were arranged in a criss - cross manner in only 7 ( 35% ) health centers . the present study found water bottles kept inside deep freezer in 2 ( 10% ) health centers . and , at 2 ( 10% ) health centers , things other than vaccines / diluents , like empty water bottles , were kept inside the ilr . vaccines were arranged according to temperature sensitivity in ilr in 14 ( 70% ) health centers . on observing the alternatives to cold chain failure ( n = 20 ) , we noticed that emergency contact number was written in the same room nearby in only 3 ( 15% ) health centers . we found that 3 ( 15% ) uhcs each had either a working inverter or a generator . though , an alternate working refrigerator was available in 13 ( 65% ) health centers . on observing the vaccination sessions ( n = 20 ) , we found that the vaccinators were reading vvm at the time of vaccination at 13 ( 65% ) health centers . the time of reconstitution was noted on the vaccine vial by the vaccinator at 15 ( 75% ) health centers . it was good to see that 15 ( 75% ) vaccinators knew the definition of cold chain exactly in its stated form , whereas 5 ( 25% ) did not know it exactly . it was encouraging to see that 18 ( 90% ) vaccinators knew the full form of vvm and stages of vvm correctly . it was reassuring to see that almost all ( 95% ) vaccinators knew the correct arrangement of vaccines in the ilr according to temperature sensitivity . knowledge of the vaccinators regarding the timing of defrosting of ilr and deep freezer was poor , with only 11 ( 55% ) knowing exactly , the criteria for defrosting . it was found in the present study that 12 ( 60% ) vaccinators knew exactly about shake test . only 5 ( 25% ) vaccinators knew the exact temperature range for deep freezer , though , 16 ( 80% ) knew the exact temperature range for ilr . on being asked about the steps incumbent to refrigerator failure , 8 ( 40% ) vaccinators told to put the vaccines in cold boxes ; 5 ( 25% ) told to put it in alternate storage refrigerator ; 5 ( 25% ) told to use the generator ; 1 ( 5% ) told to call the higher authority and 1 ( 5% ) told to shift the vaccines in a nearby ice - cream vendor 's refrigerator . a separate voltage stabilizer should be attached each to deep freezer and ilr , except in emergency , when one stabilizer per two small equipment is acceptable . the function of the voltage stabilizer is to monitor the range of fluctuations in the main voltage of 90 - 280 v and maintain voltage in a required range of 220 + 10 v. stabilizer protects the cold chain equipment against voltage fluctuations and is an essential pre - requisite . the present study claimed that a separate voltage stabilizer was attached each to deep freezer and ilr in only 15% of the health centers . medical officer of the respective uhcs should ensure adequate supply and maintenance of voltage stabilizer and report to higher authority in case scarcity persists . deep freezer and ilr should be placed in such a way in the room that they are not exposed to sunlight any time during the day . exposure to sunlight will lead to increase in core temperature of the cold chain equipment , which would break the cold chain by causing an increase in the temperature of the vaccines in ilr and ice packs would not be prepared in deep freezer . the present study stated that deep freezers were exposed to sunlight in 2 ( 10% ) health centers . which reported that the cold chain equipment were kept away from sunlight in all the health centers . the temperatures in the ilr and deep freezer must be monitored twice daily . a break in the cold chain is indicated if temperature rises above + 8c or falls below + 2c in the ilr ; and above 15c in the deep freezer . the ilr and deep freezers each should have a separate thermometer and temperature record book . it was refreshing to find that in our study , temperature was being recorded twice a day for both deep freezer and ilr in all the health centers in separate temperature record books . also , in the present study , working separate thermometers were found in deep freezer in 95% health centers and in ilr in all the health centers . the temperature in the ilr / freezer can rise if there is a thick layer of ice around the freezer or along the walls and bottom of ilrs . the present study showed that information regarding defrosting of deep freezers and ilrs was recorded on the temperature - record register in only half of the health centers . thickness of ice on the side walls was > 5 mm in the deep freezers and ilrs of half of the health centers . ( 37.5% ) , though they kept the cut - off for thickness > 6 mm . this suggests lack of regular defrosting by the cold chain handlers , which need to be improved and monitored subsequently by the medical officer of the respective uhc . ice packs should be stacked on the floor of the deep freezer horizontally ( not flat ) on its edge by keeping 1 - 2 mm space from each other for air circulation , in a criss - cross manner . yet , in our study , we found this in only one - third ( 35% ) of the health centers . this was probably due to the increased demand of ice packs in health centers , especially during sub - national immunization day ( snid ) rounds , which are frequently conducted in urban areas of surat city . the requirement for a large number of ice packs can be easily met with proper planning like transferring around 50 ice packs into a large cold box . fifty more ice packs can be frozen in two days and continuing the procedure till the required number of ice packs are frozen . deep freezers should be used to prepare and store ice packs only ; uip vaccines should not be stored in it . in spite of these guidelines , in our study , we found water bottles kept inside deep freezer in 2 ( 10% ) health centers . in our study , at 2 ( 10% ) health centers , things other than vaccines / diluents , like empty water bottles , were kept inside the ilr . in our study , we found that the vaccines were arranged accordingly in more than two - third of the health centers . the present study found that emergency contact number was written nearby in the same room in only 3 ( 15% ) health centers . argued in their study that no organization maintained a chart in case of cold chain failure . though , an alternate working refrigerator was available in just less than two - thirds of the health centers . the cold chain is dependent on electrically operated machines on which one can not depend completely . on observing the vaccination sessions ( n = 20 ) , we found that the vaccinators were reading vvm at the time of vaccination at 13 ( 65% ) health centers . the time of reconstitution was noted on the vaccine vial by the vaccinator at 15 ( 75% ) health centers . reconstituted bcg and measles vaccines should not be used beyond 4 hours from the time of its reconstitution . if not utilized completely , these reconstituted vaccines should be discarded within 4 hours in the case of bcg and measles . it was good to see that three - fourth of the vaccinators knew the definition of cold chain exactly in its stated form , while one - fourth of the vaccinators knew the concept behind it suggesting that they were aware of cold chain . most of the vaccinators knew the full form of vvm and stages of vvm correctly , suggesting that vvm is now a matter of common knowledge for the vaccinators . almost all vaccinators knew the correct arrangement of vaccines in the ilr according to temperature sensitivity . knowledge of the vaccinators regarding the timing of defrosting of ilr and deep freezer was poor , which needs improvement . shake test , for testing whether a vaccine is frozen or not , should be a matter of common knowledge for the vaccinators . but , we found in the present study that just less than two - thirds of the vaccinators knew exactly about shake test . it is imperative that vaccinators are well conversant with the temperature range to be maintained in ilr and deep freezer so that a break in cold chain can be easily detected and reported to the higher authority . in the present study , only one - fourth of the vaccinators knew the exact temperature range for deep freezer . on being asked about the steps incumbent to refrigerator failure , more than one - third of vaccinators told to put the vaccines in cold boxes , one - fourth told to put it in alternate storage refrigerator , one - fourth told to use the generator , 5% told to call the higher authority and 5% told to shift the vaccines in a nearby ice - cream vendor 's refrigerator . irrespective of the step that is followed in case of refrigerator failure by the vaccinators , a fixed and pre - decided plan of action should be individualized for each uhc . a separate voltage stabilizer should be attached each to deep freezer and ilr , except in emergency , when one stabilizer per two small equipment is acceptable . the function of the voltage stabilizer is to monitor the range of fluctuations in the main voltage of 90 - 280 v and maintain voltage in a required range of 220 + 10 v. stabilizer protects the cold chain equipment against voltage fluctuations and is an essential pre - requisite . the present study claimed that a separate voltage stabilizer was attached each to deep freezer and ilr in only 15% of the health centers . medical officer of the respective uhcs should ensure adequate supply and maintenance of voltage stabilizer and report to higher authority in case scarcity persists . deep freezer and ilr should be placed in such a way in the room that they are not exposed to sunlight any time during the day . exposure to sunlight will lead to increase in core temperature of the cold chain equipment , which would break the cold chain by causing an increase in the temperature of the vaccines in ilr and ice packs would not be prepared in deep freezer . the present study stated that deep freezers were exposed to sunlight in 2 ( 10% ) health centers . which reported that the cold chain equipment were kept away from sunlight in all the health centers . the temperatures in the ilr and deep freezer must be monitored twice daily . a break in the cold chain is indicated if temperature rises above + 8c or falls below + 2c in the ilr ; and above 15c in the deep freezer . the ilr and deep freezers each should have a separate thermometer and temperature record book . it was refreshing to find that in our study , temperature was being recorded twice a day for both deep freezer and ilr in all the health centers in separate temperature record books . also , in the present study , working separate thermometers were found in deep freezer in 95% health centers and in ilr in all the health centers . the temperature in the ilr / freezer can rise if there is a thick layer of ice around the freezer or along the walls and bottom of ilrs . this should be done if the ice in the freezer is > 5 mm thick . the present study showed that information regarding defrosting of deep freezers and ilrs was recorded on the temperature - record register in only half of the health centers . thickness of ice on the side walls was > 5 mm in the deep freezers and ilrs of half of the health centers . ( 37.5% ) , though they kept the cut - off for thickness > 6 mm . this suggests lack of regular defrosting by the cold chain handlers , which need to be improved and monitored subsequently by the medical officer of the respective uhc . ice packs should be stacked on the floor of the deep freezer horizontally ( not flat ) on its edge by keeping 1 - 2 mm space from each other for air circulation , in a criss - cross manner . this was probably due to the increased demand of ice packs in health centers , especially during sub - national immunization day ( snid ) rounds , which are frequently conducted in urban areas of surat city . the requirement for a large number of ice packs can be easily met with proper planning like transferring around 50 ice packs into a large cold box . fifty more ice packs can be frozen in two days and continuing the procedure till the required number of ice packs are frozen . deep freezers should be used to prepare and store ice packs only ; uip vaccines should not be stored in it . in spite of these guidelines , in our study , we found water bottles kept inside deep freezer in 2 ( 10% ) health centers . in our study , at 2 ( 10% ) health centers , things other than vaccines / diluents , like empty water bottles , were kept inside the ilr . opv and measles vaccines can be kept at bottom of the basket while bcg , dpt , dt and tt vaccines should be kept in upper part of the baskets . in our study , we found that the vaccines were arranged accordingly in more than two - third of the health centers . the present study found that emergency contact number was written nearby in the same room in only 3 ( 15% ) health centers . argued in their study that no organization maintained a chart in case of cold chain failure . though , an alternate working refrigerator was available in just less than two - thirds of the health centers . the cold chain is dependent on electrically operated machines on which one can not depend completely . on observing the vaccination sessions ( n = 20 ) , we found that the vaccinators were reading vvm at the time of vaccination at 13 ( 65% ) health centers . the time of reconstitution was noted on the vaccine vial by the vaccinator at 15 ( 75% ) health centers . reconstituted bcg and measles vaccines should not be used beyond 4 hours from the time of its reconstitution . if not utilized completely , these reconstituted vaccines should be discarded within 4 hours in the case of bcg and measles . it was good to see that three - fourth of the vaccinators knew the definition of cold chain exactly in its stated form , while one - fourth of the vaccinators knew the concept behind it suggesting that they were aware of cold chain . most of the vaccinators knew the full form of vvm and stages of vvm correctly , suggesting that vvm is now a matter of common knowledge for the vaccinators . almost all vaccinators knew the correct arrangement of vaccines in the ilr according to temperature sensitivity . knowledge of the vaccinators regarding the timing of defrosting of ilr and deep freezer was poor , which needs improvement . shake test , for testing whether a vaccine is frozen or not , should be a matter of common knowledge for the vaccinators . but , we found in the present study that just less than two - thirds of the vaccinators knew exactly about shake test . it is imperative that vaccinators are well conversant with the temperature range to be maintained in ilr and deep freezer so that a break in cold chain can be easily detected and reported to the higher authority . in the present study , only one - fourth of the vaccinators knew the exact temperature range for deep freezer . found in their study that 57.3% individuals correctly specified the optimal temperature range for vaccine storage . on being asked about the steps incumbent to refrigerator failure , more than one - third of vaccinators told to put the vaccines in cold boxes , one - fourth told to put it in alternate storage refrigerator , one - fourth told to use the generator , 5% told to call the higher authority and 5% told to shift the vaccines in a nearby ice - cream vendor 's refrigerator . irrespective of the step that is followed in case of refrigerator failure by the vaccinators , a fixed and pre - decided plan of action should be individualized for each uhc . cold chain maintenance and practices were not up to the mark in the uhcs of smc . the present study contemplates for periodic refresher training and capacity building for cold chain maintenance of all the cold chain handlers . medical officers should be actively involved in the monitoring and supervision of the cold chain system .

type 2 diabetes ( t2d ) involves insulin resistance in skeletal muscle , the liver and adipose tissue . one of the most contentious issues in metabolic research is the role of mitochondrial dysfunction in the development of insulin resistance . the term mitochondrial dysfunction can describe impairments in numerous mitochondrial function indices , including respiration , atp production , membrane potential , proton leak and reactive oxygen species ( ros ) production . impaired mitochondrial function has been observed in skeletal muscle [ 36 ] , the liver [ 79 ] and adipose tissue [ 1012 ] of t2d patients and animal models of t2d . similar impairments have been observed in the skeletal muscle of insulin resistant offspring of t2d patients . this could suggest a role for impaired mitochondrial function in the development of insulin resistance . this is supported by observations of insulin resistance in humans with mitochondrial dna mutations that result in impaired mitochondrial function [ 1416 ] . mitochondrial dysfunction has been proposed to induce insulin resistance through ectopic lipid accumulation secondary to reduced -oxidation , which impairs insulin signalling . more recently , production of ros has emerged as a direct link between mitochondrial dysfunction and insulin resistance driven by numerous insults such as saturated fatty acids , inflammatory cytokines and glucocorticoids . however , numerous animal models with impaired mitochondrial function have either unchanged or increased insulin sensitivity [ 2123 ] , questioning both the causality of this relationship and whether mitochondrial dysfunction is necessary and/or sufficient for insulin resistance . further adding to controversy on this issue , anti - diabetic agents such as the biguanide and thiazolidinedione family of compounds , which enhance insulin action primarily in the liver and adipose tissue respectively , have been reported to inhibit complex i of the electron transport chain and/or the mitochondrial pyruvate carrier ( mpc ) , which impairs mitochondrial function [ 2426 ] . these counterintuitive findings have been balanced by evidence that biguanides can prevent ros production by complex i under conditions of electron backflow from complex ii , such as during high fat feeding . however , given that most of our knowledge regarding the role of mitochondria in the regulation of insulin action has been generated from studies of skeletal muscle , coupled with the fact that the primary tissues of action of these anti - diabetic drugs are not skeletal muscle , could raise the possibility that there are cell / tissue type - specific responses in this relationship that are not yet fully understood . indeed , studies of either insulin resistant humans or animal models of mitochondrial dysfunction have not been able to mechanistically dissect this relationship with any certainty . this is due to reasons such as the non - physiological nature of gene ablation , the markedly different mitochondrial respiratory rates and metabolic function of tissues involved in whole body insulin action , the complexity in controlling substrate flux to individual insulin - sensitive tissues and inter - tissue cross - talk in vivo . while these factors are important for the development of the whole body metabolic phenotype in insulin resistance , they are nonetheless confounding variables when examining the fundamental link between mitochondrial dysfunction and insulin action . the fundamental biology underpinning these paradoxical findings describing the relationship between mitochondria and insulin action is poorly understood . indeed , the potential importance of specific mitochondrial enzyme impairments and the tissue / cell type in which impairments occur are unknown . furthermore , it is unknown how alterations in many of the interdependent indices of mitochondrial function impact on cellular insulin action . as in vivo studies have been unable to dissect these mechanisms , fundamental studies in cellular systems that define the biological complexity in the relationship between mitochondrial function and insulin action in multiple cell types are required before our understanding of the physiological role of mitochondria in the development of insulin resistance can advance . determine whether mitochondrial dysfunction is necessary and/or sufficient for cellular insulin resistance ; 2 . establish whether specific mitochondrial enzyme impairment is important for this response in a cell type - dependent manner ; and 3 . assess whether ros production is a universal link between impaired mitochondrial function and insulin resistance . we used 3t3l1 adipocytes and fao hepatoma cells , as models of adipocytes and hepatocytes , respectively , to address these aims , assessing glucose uptake and suppression of glucose production as measures of insulin action . these cell lines have been used extensively to study insulin action and these cell types are characterised by impaired mitochondrial function in insulin resistant states . however , few studies have utilised these cell types to mechanistically examine the role of mitochondria in insulin action . mouse immortalised 3t3l1 fibroblasts were cultured in 10% co2 at 37 c in growth media consisting of dmem ( 4.5 g / l glucose ; invitrogen ) , 10% heat - inactivated foetal bovine serum ( hi - fbs ; thermo scientific ) and antibiotics ( 100 units / ml penicillin and 100 g / ml streptomycin ; life technologies ) . cells were induced to differentiate 2 days after reaching confluence ( day 0 ) , by supplementing growth media with 3 nm insulin ( humulin r ; eli lilly ) , 0.25 m dexamethasone ( sigma aldrich ) and 0.5 mm 1-methyl-3-isobutyl - xanthine ( sigma aldrich ) . from day 3 until day 7 , cells were maintained in growth media supplemented with 3 nm insulin after which the mature adipocytes were maintained in growth media . rat fao immortalised hepatoma hepatocytes were cultured in 5% co2 at 37 c in growth media consisting of rpmi 1640 medium ( 2 g / l glucose ; invitrogen ) and 10% foetal bovine serum ( fbs ; thermo scientific ) . all treatments of hepatocytes were for 24 h in glucose- and serum - free rpmi 1640 media supplemented with 2 mm sodium pyruvate , 20 mm sodium l - lactate and 0.1% bsa ( glucose production media ; gp media ) , except where indicated . for 3t3l1 models of insulin resistance , cells were treated with 25 mu / ml glucose oxidase ( g.o . ; sigma aldrich ) , 10 ng / ml tumour necrosis factor- ( tnf ; peprotech ) or 10 nm chronic insulin . for chronic insulin treatments , cells were returned to growth media containing no insulin 2 h before beginning glucose uptake assays or protein collection . treatment doses for oligomycin ( sigma aldrich ) and rotenone ( sigma aldrich ) models of mitochondrial dysfunction as well as antimycin a ( sigma aldrich ) and fccp ( carbonyl cyanide 4-(trifluoromethoxy ) phenylhydrazone ; sigma aldrich ) in both 3t3l1 and fao cells are as stated in figures 2 and 3 , figures s2 and s3 . the doses of rosiglitazone and phenformin are stated in figure 4 and figure s4 . mntbap ( manganese ( iii ) tetrakis ( 4-benzoic acid ) porphyrin chloride ; enzo life sciences ) co - treatments were at a dose of 300 m and wortmannin ( wort ; sigma aldrich ) co - treatments were at a dose of 100 nm . the cellular bioenergetics profile of 3t3l1 adipocytes and fao hepatocytes was assessed using the seahorse xf24 flux analyzer ( seahorse bioscience ) . 3t3l1 fibroblasts were seeded into a 24-well xf24 cell culture microplate ( seahorse bioscience ) and were differentiated to maturity , as described above , at which time the cells were treated for 24 h. fao hepatocytes were also seeded into a xf24 microplate at a density of 50,000 cells per well and 4 h later , 24 h treatments were begun in growth media . cells were washed and incubated in 600 l unbuffered dmem ( containing 25 mm glucose , 1 mm pyruvate and 1 mm glutamate ) ph 7.4 , at 37 c in a non - co2 incubator ( 1 h prior to bioenergetics assessment ) . three basal oxygen consumption rate ( ocr ) measurements were performed using the seahorse analyzer , and measurements were repeated following injection of oligomycin ( 1 m ) , fccp ( 1 m ) and antimycin a ( 1 m ) . basal extracellular acidification rate ( ecar ) was determined from data collected at basal measurement points . calculations of respiratory parameters of mitochondrial function were performed as previously described and included subtraction non - mitochondrial respiration from all mitochondrial respiration parameters . following completion of the assay cell number was determined using the cyquant cell proliferation assay kit ( molecular probes ) according to manufacturer 's instructions . mature adipocytes in 24-well plates were treated for 24 h with insults , mitochondrial inhibitors or anti - diabetic agents , as described above , in serum - starve media consisting of dmem ( 4.5 g / l glucose ) supplemented with 0.2% fatty acid - free bovine serum albumin ( bsa ; usb corporation ) . to begin the assay , cells were washed twice in 1 dulbecco 's phosphate - buffered saline , ph 7.4 ( life technologies ) , containing 0.5 mm mgcl2 , 0.5 mm cacl2 and 0.2% fatty acid - free bsa , and then incubated in the presence or absence of 10 nm insulin at 37 c for 30 min . uptake of 50 m 2-deoxyglucose and 0.2 ci 2-deoxy - d-[h]-glucose ( perkinelmer ) per well was measured over the final 10 min of this incubation . cells were washed twice in ice - cold phloretin ( 80 g / ml ) in pbs and lysed in 0.03% sds before being analysed by scintillation counting . protein content per well was measured and results expressed as pmol glucose transported per minute per g of protein . fao hepatocytes in 48-well plates were treated for 24 h with mitochondrial inhibitors or insulin sensitising agents in gp media in the presence or absence of 0.1 nm insulin . to measure the glucose produced by the cells , 40 l of media was collected from each well and combined with 250 l of assay buffer consisting of 0.12 m nah2po42h2o ph 7.0 , 1 mg / ml phenol , 0.5 mg / ml 4-aminoantipyrine , 1.6 units / ml peroxidise and 10 units / ml glucose oxidase . this was incubated for 25 min at 37 c after which absorbance was measured at 490 nm . cells were lysed in 100 l 0.03% sds and protein was quantified using a bca protein assay kit ( pierce ) . an alternative method was used to measure glucose production in hepatocytes co - treated with mntbap as the colour of mntbap interferes with any colorimetric measurement . cells were seeded and treated as in the colorimetric assay and instead glucose production was measured by a fluorometric assay kit ( cayman chemical company ) following the manufacturer 's instructions . cells were lysed in 100 l 0.03% sds and protein was quantified using a bca protein assay kit ( pierce ) . cells in 6-well plates were treated for 24 h as described above , in the absence or presence of insulin for the final 15 min ( 3t3l1 10 nm insulin in serum - starve media ; fao 0.1 nm insulin ) . proteins were extracted using protein lysis buffer ( 50 mm tris ph7.5 , 1 mm edta , 1 mm egta , 10% glycerol , 1% triton x-100 , 50 mm naf , 5 mm na4p2o7 , 1 mm na3vo4 , 1 mm dtt , protease inhibitor cocktail ) and protein concentration quantified using a bca protein assay kit ( pierce ) . 30 g of protein was separated by sds - page and transferred onto pvdf membrane using standard protocols . membranes were blocked for 1 h at room temperature ( rt ) in tbst ( tbs with 0.05% tween 20 , ph 7.4 ) containing 1% bsa , and incubated overnight at 4 c with the following primary antibodies , at the dilutions indicated , in tbst : anti - py612 irs1 ( 1:1000 , upstate ) , anti - irs1 ( 1:1000 , upstate ) , anti - ps473 akt ( 1:1000 , cell signaling ) , anti - akt ( 1:1000 , cell signaling ) , anti - ps462 as160 ( 1:1000 , cell signaling ) , anti - as160 ( 1:1000 , upstate ) , anti - pt24 foxo1 ( 1:1000 , cell signaling ) , anti - foxo1 ( 1:1000 , cell signaling ) , anti--tubulin ( 1:1,000 ; sigma membranes were washed with tbst before being exposed to the appropriate hrp conjugated secondary antibody at a dilution of 1:10,000 in tbst for 1 h at rt . the reaction was developed using chemiluminescence ( invitrogen ) and visualised using the chemidoc xrs+ with image lab software . lactate dehydrogenase ( ldh ) is released from cells upon loss of membrane integrity due to apoptosis or necrosis and can therefore be used as a measure of cell viability . cells in 48-well plates were treated as described above ( 3t3l1 in serum - starve media ) . after 24 h the cytotoxic effects of our treatments were assessed using the cytotox 96 non - radioactive cytotoxicity assay kit ( promega ) as per manufacturer 's instructions . viability was expressed as ldh in the media normalised to total ldh from the media and cell lysate . mitochondrial transmembrane potential was measured using the membrane - permeable jc-1 dye ( invitrogen ) . cells in black - walled 96-well plates were treated for 24 h as described above . in the final 10 min of treatment , cells were incubated with 10 g / ml jc-1 dye at 37 c . after three washes with pbs , both green and red fluorescence emissions were measured using an excitation wavelength of 488 nm and emission wavelengths of 522 and 605 nm , respectively . cells in black - walled 96-well plates were treated for 24 h as described above . in the final 30 min of treatment after two washes with pbs , fluorescence was measured using an excitation wavelength of 510 nm and an emission wavelength of 580 nm . one - way anova with tukey post - hoc test was used where more than two groups were compared within a single treatment , and p values less than 0.05 were considered significant . in vivo studies have not been able to determine whether mitochondrial dysfunction is necessary for insulin resistance with any certainty . to establish whether mitochondrial dysfunction is necessary for insulin resistance , we assessed mitochondrial function indices in diverse cellular models of insulin resistance in 3t3l1 adipocytes . these included glucose oxidase , tnf and chronic insulin treatment , which all impaired insulin - stimulated glucose uptake ( figure 1a ) . cellular insulin resistance in these models was not attributed to a common defect in insulin signalling at the level of irs1 , akt or as160 ( figure 1b ) . there was a small but significant increase in ldh release with glucose oxidase ( figure 1c ) , indicating compromised cell viability . as impaired mitochondrial respiration has been linked to the development of insulin resistance , we measured multiple mitochondrial respiration parameters including basal respiration , respiration due to atp turnover and respiration due to proton leak . glucose oxidase decreased respiration due to atp turnover ( figure 1d ) and increased mitochondrial ros production ( figure 1f ) . chronic insulin increased basal respiration , respiration for atp turnover ( figure 1d ) and decreased mmp ( figure 1e ) . as tnf had no effect on any of the parameters measured and there was no consistent mitochondrial perturbation across all insults , these data suggest that a common defect in mitochondrial function , and mitochondrial dysfunction more generally , is not necessary for all forms of cellular insulin resistance . we sought to clarify these findings by investigating whether physiological impairment of mitochondrial function was sufficient to induce insulin resistance . as reduced adp - stimulated respiration has been observed in insulin resistant states , we used the atp synthase inhibitor oligomycin to mimic aspects of this defect . in 3t3l1 adipocytes , 24 h treatment with 50 nm and 100 nm oligomycin reduced basal respiration by 20% and 40% , respectively ( figure 2a ) , similar to that observed in insulin resistant states . three - variable ( respiration , membrane potential and ros production ) surface plots were generated to represent mitochondrial function regulated by oligomycin ( figure 2b ) . dose - dependent reductions in respiration due to atp turnover ( figure s2a ) , but not proton leak ( figure s2b ) , were associated with increased mmp and ros production ( figure 2b , figure s2c and d ) . at 100 nm , the extracellular acidification rate ( ecar ) , a proxy measure of glycolytic rate , was increased with both doses ( figure 2c ) and was associated with an increase in cellular atp levels ( figure 2d ) . insulin - stimulated glucose uptake was decreased with both doses of oligomycin , and at 50 nm , basal glucose uptake was increased ( figure 2e ) . insulin action , assessed as the percentage increases in insulin - stimulated glucose uptake over basal , was ablated at both oligomycin doses ( figure 2f ) . there was also a global reduction in expression and phosphorylation of key components of the insulin signalling pathway ( figure 2 g ) . as mitochondrial ros production has been implicated as a causative factor in insulin resistance and was increased in this model , the sod mimetic mntbap was used to reduce mitochondrial ros production ( figure s2f ) . mntbap did not restore oligomycin - induced insulin resistance , but did reduce insulin - stimulated glucose uptake in the absence of oligomycin ( figure 2h ) , suggesting that increased mitochondrial ros production does not mediate insulin resistance in this model . this was further supported by evidence that antimycin a , a complex iii inhibitor that increases ros production , had no effect on glucose uptake ( figure s2 g ) at concentrations that reduced respiration by 20% and 80% respectively ( data not shown ) . these data show that mitochondrial dysfunction , in a model that replicates aspects of mitochondrial dysfunction seen in insulin resistant states in vivo , is sufficient to induce insulin resistance independent of mitochondrial ros production . as inhibition of complex i is thought to be the mechanism of action of some anti - diabetic agents , we examined the effect of the complex i inhibitor rotenone on insulin action . in 3t3l1 adipocytes , 1 nm and 5 nm rotenone reduced basal respiration by 20% and 60% respectively ( figure 2i ) . altered mitochondrial function induced by rotenone ( figure 2j ) varied from that induced by oligomycin ( figure 2b ) and did not include alterations in mmp or mitochondrial ros production ( figure s2h and i ) . however , 5 nm but not 1 nm rotenone , significantly reduced respiration associated with atp turnover ( figure s2j ) and proton leak ( figure s2k ) . while 5 nm rotenone increased ecar ( figure 2k ) , both doses of rotenone had no effect on atp concentration ( figure 2l ) or cell viability ( figure s2l ) . we next tested the effect of rotenone on insulin action in 3t3l1 adipocytes . at 5 nm , but not 1 nm , rotenone significantly reduced insulin - stimulated glucose uptake ( figure 2 m ) and insulin action ( figure 2n ) . there were no obvious impairments in insulin signalling through irs1 , akt and as160 ( figure 2o ) . these data suggest that inhibition of complex i and its associated mitochondrial dysfunction is sufficient to induce insulin resistance in 3t3l1 adipocytes . nonetheless , these data show that complex i inhibition does not have insulin sensitising effects in 3t3l1 adipocytes . the divergent effects of oligomycin and rotenone on insulin signalling and the magnitude of insulin resistance show that the exact mechanism of mitochondrial impairment is important in predicting subsequent effects on insulin action . therefore , to determine whether mitochondrial dysfunction is sufficient to induce insulin resistance in multiple cell types , we investigated these effects in fao hepatocytes . low doses of oligomycin reduced basal respiration ( figure 3a ) and altered mitochondrial function ( figure 3b ) in fao hepatocytes . at 0.5 nm , which reduced respiration by 10% ( figure 3a ) , oligomycin did not significantly alter respiration due to atp turnover ( figure s3a ) and proton leak ( figure s3b ) , but did increased mmp ( figure s3c ) and ros production ( figure s3d ) . there were no effects on ecar ( figure 3c ) or atp levels ( figure 3d ) . we tested whether oligomycin - induced mitochondrial dysfunction was sufficient to reduce insulin sensitivity in fao hepatocytes by measuring insulin suppression of glucose production ( figure 3e ) . oligomycin did not alter basal or insulin suppression of glucose production , or insulin action ( figure 3f ) . no effect on the insulin signalling pathway was detected ( figure 3 g ) . these data provide evidence that increased mitochondrial ros production does not induce insulin resistance in these cells . rather a reduction in mitochondrial ros ( figure s3f ) and alteration of the cellular redox state with mntbap completely impaired glucose production ( figure s3 g ) , suggesting a more fundamental role for ros signalling in the control of glucose production in these cells . we next assessed the effect of rotenone on respiration in fao hepatocytes ( figure 3 g ) , which altered mitochondrial function ( figure 3h ) . at 1 nm , which reduced respiration by 20% , this included decreased respiration due to atp turnover ( figure s3h ) and proton leak ( figure s3i ) but had no effect on mmp , ros production ( figure 3h , figure s3j and k ) or cell viability ( figure s3e ) . ecar was increased ( figure 3i ) and atp levels were unchanged ( figure 3j ) . interestingly , rotenone reduced both basal and insulin - stimulated glucose production ( figure 3 m ) , but had no effect on insulin action ( figure 3n ) . as rotenone increased both basal and insulin - stimulated tyrosine phosphorylation of irs1 ( figure 3o ) , we tested whether inhibiting phosphatidylinositol 3-kinase ( pi3k ) and subsequent insulin signalling with wortmannin , would eliminate the ability of rotenone to reduce glucose production . wortmannin significantly increased basal glucose production in vehicle - treated cells , but had no effect on the ability of insulin to suppress glucose production , suggesting that the canonical insulin signalling pathway is not essential for the suppressive effects of insulin on glucose production in this model . in the presence of wortmannin , rotenone was still able to decrease both basal and insulin - stimulated glucose production ( figure 3p ) , suggesting that rotenone 's actions on glucose production are not mediated through insulin signalling pathway sensitisation . our data show that mitochondrial dysfunction is not a universal initiating factor for insulin resistance . however , inducing mitochondrial dysfunction is sufficient to induce cell type - dependent insulin resistance . as rosiglitazone and phenformin exert their effects , in part , through inhibition of complex i and the mitochondrial pyruvate carrier , resulting in impaired mitochondrial respiration , we examined the acute effects of these agents on mitochondrial function and insulin sensitivity . in 3t3l1 adipocytes , rosiglitazone reduced mitochondrial respiration ( figure s4a ) and mitochondrial function ( figure 4a , figure s4b and c ) , however the nature of that dysfunction , based on multiple parameter analysis varied from that induced by oligomycin and rotenone in these same cells ( figure 2b and j ) , reinforcing the importance of measuring multiple parameters of mitochondrial function . rosiglitazone increased atp levels ( figure s4d ) independent of increases in ecar ( figure s4e ) . consistent with our data for other mitochondrial inhibitors in these cells , insulin action was decreased by rosiglitazone ( figure 4b and c ) , but this was driven by an increase in basal glucose uptake ( figure 4b ) . phenformin induced similar alterations in mitochondrial respiration ( figure s4f ) and function ( figure 4a ) , in adipocytes , albeit with greater inhibitory action on respiration associated with atp turnover and proton leak ( figures s4 g and h ) . no significant changes in atp levels ( figure s4i ) or ecar ( figure s4j ) were detected with phenformin treatment . although phenformin increased basal glucose uptake ( figure 4e ) , insulin action was also completely abrogated ( figure 4f ) . as phenformin increased mitochondrial ros production ( refer ahead to figure 5d ) , we used mntbap to counter this increase , with no effect on insulin action ( figure 4e and f ) . this shows that in this cell type , heterogeneous forms of mitochondrial dysfunction converge to induce insulin resistance , independent of increased mitochondrial ros production . these drugs also altered mitochondrial function in fao hepatocytes ( figure 4 g and k , figure s4k o , q rosiglitazone at high doses reduced both basal and insulin suppression of glucose production ( figure 4h ) and increased insulin action ( figure 4i ) . as recent evidence links the efficacy of biguanides on hepatic glucose production to reduced atp levels , we also assessed atp concentrations . however , rosiglitazone did not reduce atp levels ( figure 4j ) , nor ecar ( figure s4p ) . phenformin at higher doses also reduced basal and insulin - stimulated glucose production ( figure 4l ) , but not insulin action ( figure 4 m ) , despite reduced atp levels ( figure 4n ) and no compensatory change in ecar ( figure 4sv ) . these data show that similar to traditional mitochondrial inhibitors , these anti - diabetic agents have cell type - dependent effects on mitochondrial function and insulin action . while the exact mechanism linking the effects of rosiglitazone and phenformin on mitochondrial function and insulin action remain unclear , one proposed mechanism is that these compounds can reduce ros production under conditions of mitochondrial dysfunction , suggesting that these drugs could have altered efficacy in stressed and healthy states . while we have not found a role for ros production linking mitochondrial dysfunction and insulin resistance in our models , we nonetheless explored this possibility . we induced mitochondrial dysfunction in 3t3l1 adipocytes with glucose oxidase , which increased ros production ( figure 1f ) , and co - treated with rosiglitazone or phenformin before assessing insulin action ( figure 5a ) . glucose oxidase - induced insulin resistance was worsened with increasing doses of both rosiglitazone and phenformin ( figure 5a ) . ros production was also increased with co - treatment of glucose oxidase with rosiglitazone ( figure 5b ) and phenformin ( figure 5d ) . this was associated with a loss in mmp with rosiglitazone that was also worsened with glucose oxidase ( figure 5c ) and with 100 m phenformin and glucose oxidase ( figure 5e ) . these data suggest that these compounds do not reduce ros production under states of mitochondrial dysfunction in all cell types and that these anti - diabetic drugs can further exacerbate mitochondrial dysfunction and its associated insulin resistance under context - specific conditions . these fundamental studies on the relationship between mitochondrial function and cell - autonomous insulin action revealed that acute , physiological impairments in mitochondrial function are sufficient , but not necessary , to induce insulin resistance ( figure 6 ) . this has been partly due to the difficultly in inducing physiological impairments in mitochondrial function in animal models either through genetic or other means . in addition , interventionist human studies have found it difficult to separate mitochondrial dysfunction with other confounding variables , such as increased fatty acid availability , which is also associated with insulin resistance . however the utility of our cell - autonomous system that allows titration of mitochondrial impairment to physiological levels and analysis of insulin action under conditions of constant substrate availability , without the influence of inter - tissue crosstalk , has assisted with mechanistic dissection of this relationship . these findings support data in humans with mitochondrial impairments due to mitochondrial dna mutations , which can also manifest in insulin resistance in key insulin - sensitive tissues [ 1416 ] . numerous animal models with impaired mitochondrial function due to genetic or dietary modification of components of the electron transport chain have produced opposing conclusions on this same issue . for example , deletion of the apoptosis initiating factor ( aif ) enzyme that is a component of complex i , leads to oxidative phosphorylation defects and protection against insulin resistance in response to high fat feeding . similar findings are observed with the ablation of cytochrome c oxidase subunit vi peptide 2a ( cox6a2 ; 23 ) and iron - containing enzymes of the etc . for example , we showed that complex i inhibition decreased insulin - stimulated glucose uptake in adipocytes , but potentiated insulin suppression of glucose production in hepatocytes . these data showing that identical insults have cell type - dependent effects on mitochondrial function and insulin action highlights the complexity in this relationship ( figure 6 ) . multi - parameter measurement and representation of mitochondrial function in a multi - dimension format in the form of surface plots indeed , oligomycin and rotenone had differential effects on insulin signalling and the magnitude of insulin resistance in 3t3l1 adipocytes , despite similar impairments in respiration . these data suggest that animal models with global defects in mitochondrial function could manifest different phenotypes depending on the site of the mitochondrial impairment and on the specific insulin - sensitive tissue that dominates the whole body phenotype . the impact on whole body insulin action would therefore be difficult to identify and predict . the functional significance of these findings are that any phenotypic investigation into the role of mitochondria in the pathogenesis of insulin resistance in patients or disease models will likely have to establish the exact tissue and enzyme(s ) affected to understand the mechanisms involved . furthermore , the biological complexity in mitochondrial regulation of insulin action identified in our studies suggests that universal and reductionist theories describing this relationship may not be possible . our findings also revealed that mitochondrial ros is not a universal driver of insulin resistance in the cellular systems examined . this theory on the aetiology of insulin resistance has gained momentum following a number of recent studies showing that skeletal muscle insulin resistance can be prevented through buffering of mitochondrial ros . furthermore , buffering total cellular ros in a number of adipocyte models of insulin resistance was sufficient to reverse insulin resistance . however , the compartmentalisation of ros production and the cell type - dependent responses to ros require further consideration when interpreting these data , as ros is also required for insulin action . nonetheless , mitochondrial ros - mediated insulin resistance appears to be a valid mechanism in skeletal muscle . it is unclear why mitochondrial ros production was not associated with insulin resistance in our models , however this is an area that warrants further investigation and could involve differences in anti - oxidant capacities . such experiments could be important in determining the utility of anti - oxidant therapies in heterogeneous insulin resistant states . the mechanisms that link mitochondrial dysfunction and insulin action in adipocytes and hepatocytes are also unclear . in adipocytes , mitochondrial dysfunction - induced alterations in calcium handling could be important , as insulin - stimulated glut4 translocation and glucose uptake are calcium sensitive . notably , deletion of apoptosis - inducing factor ( aif ) , a protein typically released from the mitochondria by pro - apoptotic stimuli , protects against insulin resistance despite a reduction in mitochondrial respiration . this could suggest that protein - mediated signalling from mitochondria might impact on insulin action . in hepatocytes , a recent study has found that reduced cellular energy status can impede glucagon signalling , which opposes insulin signalling in the control of glucogeogenesis . increased amp however , we found no association between reduced atp , or any involvement for the canonical insulin signalling pathway in our mitochondrial dysfunction models that reduced insulin suppression of glucose production . the mechanisms by which mitochondrial dysfunction impacts on insulin action in hepatocytes remain to be determined . we also observed that the anti - diabetic agents phenformin and rosiglitazone showed cell type - dependent impairments in mitochondrial function and insulin action . this included enhanced suppression of glucose production and enhanced insulin action in hepatocytes , but also included induction of insulin resistance in adipocytes . the acute effects of these drugs in adipocytes might be offset by longer term remodelling of adipocyte metabolism , particularly in the case of rosiglitazone , which enhances the expression of ppar dependent genes to increase glucose disposal and lipogenesis , thereby reducing hyperglycemia in vivo . indeed , the reduced insulin action we observed in rosiglitazone treated adipocytes was primarily due to an increase in basal glucose uptake , rather than reduced insulin - stimulated glucose uptake . nonetheless , both anti - diabetic drugs altered mitochondrial function in adipocytes , which could have longer term implications in patients treated with these drugs for extended periods . indeed , these findings could explain inconsistencies in efficacy associated with these drugs and their reduced efficacy over time . the finding that both drugs exacerbated insulin resistance in the context of existing mitochondrial dysfunction also highlights that personalised medicine approaches that identify any mitochondrial defects could be used to develop improved treatment strategies for patients . in conclusion , our findings show that acute , physiological impairments in mitochondrial function are sufficient , but not necessary , for the development of cell type - dependent insulin resistance and this is independent of mitochondrial ros production . the translational implications from these findings are that therapies for insulin resistance that involve modulation of mitochondrial function should be tissue specific and their mechanism of action on mitochondria well characterised to ensure optimal therapeutic outcomes . the complexity in the relationship between mitochondrial function and insulin action revealed by these studies provides insights into opposing conclusions on this issue and questions reductionist theories that attempt to describe this relationship .

the contribution of mitochondrial dysfunction to insulin resistance is a contentious issue in metabolic research . recent evidence implicates mitochondrial dysfunction as contributing to multiple forms of insulin resistance . however , some models of mitochondrial dysfunction fail to induce insulin resistance , suggesting greater complexity describes mitochondrial regulation of insulin action . we report that mitochondrial dysfunction is not necessary for cellular models of insulin resistance . however , impairment of mitochondrial function is sufficient for insulin resistance in a cell type - dependent manner , with impaired mitochondrial function inducing insulin resistance in adipocytes , but having no effect , or insulin sensitising effects in hepatocytes . the mechanism of mitochondrial impairment was important in determining the impact on insulin action , but was independent of mitochondrial ros production . these data can account for opposing findings on this issue and highlight the complexity of mitochondrial regulation of cell type - specific insulin action , which is not described by current reductionist paradigms .

Introduction Materials and methods Results Discussion

type 2 diabetes ( t2d ) involves insulin resistance in skeletal muscle , the liver and adipose tissue . one of the most contentious issues in metabolic research is the role of mitochondrial dysfunction in the development of insulin resistance . the term mitochondrial dysfunction can describe impairments in numerous mitochondrial function indices , including respiration , atp production , membrane potential , proton leak and reactive oxygen species ( ros ) production . impaired mitochondrial function has been observed in skeletal muscle [ 36 ] , the liver [ 79 ] and adipose tissue [ 1012 ] of t2d patients and animal models of t2d . this could suggest a role for impaired mitochondrial function in the development of insulin resistance . this is supported by observations of insulin resistance in humans with mitochondrial dna mutations that result in impaired mitochondrial function [ 1416 ] . mitochondrial dysfunction has been proposed to induce insulin resistance through ectopic lipid accumulation secondary to reduced -oxidation , which impairs insulin signalling . more recently , production of ros has emerged as a direct link between mitochondrial dysfunction and insulin resistance driven by numerous insults such as saturated fatty acids , inflammatory cytokines and glucocorticoids . however , numerous animal models with impaired mitochondrial function have either unchanged or increased insulin sensitivity [ 2123 ] , questioning both the causality of this relationship and whether mitochondrial dysfunction is necessary and/or sufficient for insulin resistance . further adding to controversy on this issue , anti - diabetic agents such as the biguanide and thiazolidinedione family of compounds , which enhance insulin action primarily in the liver and adipose tissue respectively , have been reported to inhibit complex i of the electron transport chain and/or the mitochondrial pyruvate carrier ( mpc ) , which impairs mitochondrial function [ 2426 ] . however , given that most of our knowledge regarding the role of mitochondria in the regulation of insulin action has been generated from studies of skeletal muscle , coupled with the fact that the primary tissues of action of these anti - diabetic drugs are not skeletal muscle , could raise the possibility that there are cell / tissue type - specific responses in this relationship that are not yet fully understood . indeed , studies of either insulin resistant humans or animal models of mitochondrial dysfunction have not been able to mechanistically dissect this relationship with any certainty . this is due to reasons such as the non - physiological nature of gene ablation , the markedly different mitochondrial respiratory rates and metabolic function of tissues involved in whole body insulin action , the complexity in controlling substrate flux to individual insulin - sensitive tissues and inter - tissue cross - talk in vivo . while these factors are important for the development of the whole body metabolic phenotype in insulin resistance , they are nonetheless confounding variables when examining the fundamental link between mitochondrial dysfunction and insulin action . furthermore , it is unknown how alterations in many of the interdependent indices of mitochondrial function impact on cellular insulin action . as in vivo studies have been unable to dissect these mechanisms , fundamental studies in cellular systems that define the biological complexity in the relationship between mitochondrial function and insulin action in multiple cell types are required before our understanding of the physiological role of mitochondria in the development of insulin resistance can advance . determine whether mitochondrial dysfunction is necessary and/or sufficient for cellular insulin resistance ; 2 . establish whether specific mitochondrial enzyme impairment is important for this response in a cell type - dependent manner ; and 3 . assess whether ros production is a universal link between impaired mitochondrial function and insulin resistance . we used 3t3l1 adipocytes and fao hepatoma cells , as models of adipocytes and hepatocytes , respectively , to address these aims , assessing glucose uptake and suppression of glucose production as measures of insulin action . these cell lines have been used extensively to study insulin action and these cell types are characterised by impaired mitochondrial function in insulin resistant states . however , few studies have utilised these cell types to mechanistically examine the role of mitochondria in insulin action . for 3t3l1 models of insulin resistance , cells were treated with 25 mu / ml glucose oxidase ( g.o . treatment doses for oligomycin ( sigma aldrich ) and rotenone ( sigma aldrich ) models of mitochondrial dysfunction as well as antimycin a ( sigma aldrich ) and fccp ( carbonyl cyanide 4-(trifluoromethoxy ) phenylhydrazone ; sigma aldrich ) in both 3t3l1 and fao cells are as stated in figures 2 and 3 , figures s2 and s3 . calculations of respiratory parameters of mitochondrial function were performed as previously described and included subtraction non - mitochondrial respiration from all mitochondrial respiration parameters . fao hepatocytes in 48-well plates were treated for 24 h with mitochondrial inhibitors or insulin sensitising agents in gp media in the presence or absence of 0.1 nm insulin . in vivo studies have not been able to determine whether mitochondrial dysfunction is necessary for insulin resistance with any certainty . to establish whether mitochondrial dysfunction is necessary for insulin resistance , we assessed mitochondrial function indices in diverse cellular models of insulin resistance in 3t3l1 adipocytes . cellular insulin resistance in these models was not attributed to a common defect in insulin signalling at the level of irs1 , akt or as160 ( figure 1b ) . as impaired mitochondrial respiration has been linked to the development of insulin resistance , we measured multiple mitochondrial respiration parameters including basal respiration , respiration due to atp turnover and respiration due to proton leak . glucose oxidase decreased respiration due to atp turnover ( figure 1d ) and increased mitochondrial ros production ( figure 1f ) . as tnf had no effect on any of the parameters measured and there was no consistent mitochondrial perturbation across all insults , these data suggest that a common defect in mitochondrial function , and mitochondrial dysfunction more generally , is not necessary for all forms of cellular insulin resistance . we sought to clarify these findings by investigating whether physiological impairment of mitochondrial function was sufficient to induce insulin resistance . three - variable ( respiration , membrane potential and ros production ) surface plots were generated to represent mitochondrial function regulated by oligomycin ( figure 2b ) . dose - dependent reductions in respiration due to atp turnover ( figure s2a ) , but not proton leak ( figure s2b ) , were associated with increased mmp and ros production ( figure 2b , figure s2c and d ) . insulin action , assessed as the percentage increases in insulin - stimulated glucose uptake over basal , was ablated at both oligomycin doses ( figure 2f ) . as mitochondrial ros production has been implicated as a causative factor in insulin resistance and was increased in this model , the sod mimetic mntbap was used to reduce mitochondrial ros production ( figure s2f ) . mntbap did not restore oligomycin - induced insulin resistance , but did reduce insulin - stimulated glucose uptake in the absence of oligomycin ( figure 2h ) , suggesting that increased mitochondrial ros production does not mediate insulin resistance in this model . this was further supported by evidence that antimycin a , a complex iii inhibitor that increases ros production , had no effect on glucose uptake ( figure s2 g ) at concentrations that reduced respiration by 20% and 80% respectively ( data not shown ) . these data show that mitochondrial dysfunction , in a model that replicates aspects of mitochondrial dysfunction seen in insulin resistant states in vivo , is sufficient to induce insulin resistance independent of mitochondrial ros production . as inhibition of complex i is thought to be the mechanism of action of some anti - diabetic agents , we examined the effect of the complex i inhibitor rotenone on insulin action . altered mitochondrial function induced by rotenone ( figure 2j ) varied from that induced by oligomycin ( figure 2b ) and did not include alterations in mmp or mitochondrial ros production ( figure s2h and i ) . we next tested the effect of rotenone on insulin action in 3t3l1 adipocytes . at 5 nm , but not 1 nm , rotenone significantly reduced insulin - stimulated glucose uptake ( figure 2 m ) and insulin action ( figure 2n ) . these data suggest that inhibition of complex i and its associated mitochondrial dysfunction is sufficient to induce insulin resistance in 3t3l1 adipocytes . nonetheless , these data show that complex i inhibition does not have insulin sensitising effects in 3t3l1 adipocytes . the divergent effects of oligomycin and rotenone on insulin signalling and the magnitude of insulin resistance show that the exact mechanism of mitochondrial impairment is important in predicting subsequent effects on insulin action . therefore , to determine whether mitochondrial dysfunction is sufficient to induce insulin resistance in multiple cell types , we investigated these effects in fao hepatocytes . at 0.5 nm , which reduced respiration by 10% ( figure 3a ) , oligomycin did not significantly alter respiration due to atp turnover ( figure s3a ) and proton leak ( figure s3b ) , but did increased mmp ( figure s3c ) and ros production ( figure s3d ) . oligomycin did not alter basal or insulin suppression of glucose production , or insulin action ( figure 3f ) . these data provide evidence that increased mitochondrial ros production does not induce insulin resistance in these cells . rather a reduction in mitochondrial ros ( figure s3f ) and alteration of the cellular redox state with mntbap completely impaired glucose production ( figure s3 g ) , suggesting a more fundamental role for ros signalling in the control of glucose production in these cells . we next assessed the effect of rotenone on respiration in fao hepatocytes ( figure 3 g ) , which altered mitochondrial function ( figure 3h ) . at 1 nm , which reduced respiration by 20% , this included decreased respiration due to atp turnover ( figure s3h ) and proton leak ( figure s3i ) but had no effect on mmp , ros production ( figure 3h , figure s3j and k ) or cell viability ( figure s3e ) . interestingly , rotenone reduced both basal and insulin - stimulated glucose production ( figure 3 m ) , but had no effect on insulin action ( figure 3n ) . wortmannin significantly increased basal glucose production in vehicle - treated cells , but had no effect on the ability of insulin to suppress glucose production , suggesting that the canonical insulin signalling pathway is not essential for the suppressive effects of insulin on glucose production in this model . our data show that mitochondrial dysfunction is not a universal initiating factor for insulin resistance . however , inducing mitochondrial dysfunction is sufficient to induce cell type - dependent insulin resistance . as rosiglitazone and phenformin exert their effects , in part , through inhibition of complex i and the mitochondrial pyruvate carrier , resulting in impaired mitochondrial respiration , we examined the acute effects of these agents on mitochondrial function and insulin sensitivity . in 3t3l1 adipocytes , rosiglitazone reduced mitochondrial respiration ( figure s4a ) and mitochondrial function ( figure 4a , figure s4b and c ) , however the nature of that dysfunction , based on multiple parameter analysis varied from that induced by oligomycin and rotenone in these same cells ( figure 2b and j ) , reinforcing the importance of measuring multiple parameters of mitochondrial function . consistent with our data for other mitochondrial inhibitors in these cells , insulin action was decreased by rosiglitazone ( figure 4b and c ) , but this was driven by an increase in basal glucose uptake ( figure 4b ) . phenformin induced similar alterations in mitochondrial respiration ( figure s4f ) and function ( figure 4a ) , in adipocytes , albeit with greater inhibitory action on respiration associated with atp turnover and proton leak ( figures s4 g and h ) . as phenformin increased mitochondrial ros production ( refer ahead to figure 5d ) , we used mntbap to counter this increase , with no effect on insulin action ( figure 4e and f ) . this shows that in this cell type , heterogeneous forms of mitochondrial dysfunction converge to induce insulin resistance , independent of increased mitochondrial ros production . these drugs also altered mitochondrial function in fao hepatocytes ( figure 4 g and k , figure s4k o , q rosiglitazone at high doses reduced both basal and insulin suppression of glucose production ( figure 4h ) and increased insulin action ( figure 4i ) . phenformin at higher doses also reduced basal and insulin - stimulated glucose production ( figure 4l ) , but not insulin action ( figure 4 m ) , despite reduced atp levels ( figure 4n ) and no compensatory change in ecar ( figure 4sv ) . these data show that similar to traditional mitochondrial inhibitors , these anti - diabetic agents have cell type - dependent effects on mitochondrial function and insulin action . while the exact mechanism linking the effects of rosiglitazone and phenformin on mitochondrial function and insulin action remain unclear , one proposed mechanism is that these compounds can reduce ros production under conditions of mitochondrial dysfunction , suggesting that these drugs could have altered efficacy in stressed and healthy states . while we have not found a role for ros production linking mitochondrial dysfunction and insulin resistance in our models , we nonetheless explored this possibility . we induced mitochondrial dysfunction in 3t3l1 adipocytes with glucose oxidase , which increased ros production ( figure 1f ) , and co - treated with rosiglitazone or phenformin before assessing insulin action ( figure 5a ) . these data suggest that these compounds do not reduce ros production under states of mitochondrial dysfunction in all cell types and that these anti - diabetic drugs can further exacerbate mitochondrial dysfunction and its associated insulin resistance under context - specific conditions . these fundamental studies on the relationship between mitochondrial function and cell - autonomous insulin action revealed that acute , physiological impairments in mitochondrial function are sufficient , but not necessary , to induce insulin resistance ( figure 6 ) . in addition , interventionist human studies have found it difficult to separate mitochondrial dysfunction with other confounding variables , such as increased fatty acid availability , which is also associated with insulin resistance . however the utility of our cell - autonomous system that allows titration of mitochondrial impairment to physiological levels and analysis of insulin action under conditions of constant substrate availability , without the influence of inter - tissue crosstalk , has assisted with mechanistic dissection of this relationship . these findings support data in humans with mitochondrial impairments due to mitochondrial dna mutations , which can also manifest in insulin resistance in key insulin - sensitive tissues [ 1416 ] . numerous animal models with impaired mitochondrial function due to genetic or dietary modification of components of the electron transport chain have produced opposing conclusions on this same issue . for example , deletion of the apoptosis initiating factor ( aif ) enzyme that is a component of complex i , leads to oxidative phosphorylation defects and protection against insulin resistance in response to high fat feeding . for example , we showed that complex i inhibition decreased insulin - stimulated glucose uptake in adipocytes , but potentiated insulin suppression of glucose production in hepatocytes . these data showing that identical insults have cell type - dependent effects on mitochondrial function and insulin action highlights the complexity in this relationship ( figure 6 ) . multi - parameter measurement and representation of mitochondrial function in a multi - dimension format in the form of surface plots indeed , oligomycin and rotenone had differential effects on insulin signalling and the magnitude of insulin resistance in 3t3l1 adipocytes , despite similar impairments in respiration . these data suggest that animal models with global defects in mitochondrial function could manifest different phenotypes depending on the site of the mitochondrial impairment and on the specific insulin - sensitive tissue that dominates the whole body phenotype . the impact on whole body insulin action would therefore be difficult to identify and predict . the functional significance of these findings are that any phenotypic investigation into the role of mitochondria in the pathogenesis of insulin resistance in patients or disease models will likely have to establish the exact tissue and enzyme(s ) affected to understand the mechanisms involved . furthermore , the biological complexity in mitochondrial regulation of insulin action identified in our studies suggests that universal and reductionist theories describing this relationship may not be possible . our findings also revealed that mitochondrial ros is not a universal driver of insulin resistance in the cellular systems examined . this theory on the aetiology of insulin resistance has gained momentum following a number of recent studies showing that skeletal muscle insulin resistance can be prevented through buffering of mitochondrial ros . furthermore , buffering total cellular ros in a number of adipocyte models of insulin resistance was sufficient to reverse insulin resistance . however , the compartmentalisation of ros production and the cell type - dependent responses to ros require further consideration when interpreting these data , as ros is also required for insulin action . nonetheless , mitochondrial ros - mediated insulin resistance appears to be a valid mechanism in skeletal muscle . it is unclear why mitochondrial ros production was not associated with insulin resistance in our models , however this is an area that warrants further investigation and could involve differences in anti - oxidant capacities . such experiments could be important in determining the utility of anti - oxidant therapies in heterogeneous insulin resistant states . the mechanisms that link mitochondrial dysfunction and insulin action in adipocytes and hepatocytes are also unclear . in adipocytes , mitochondrial dysfunction - induced alterations in calcium handling could be important , as insulin - stimulated glut4 translocation and glucose uptake are calcium sensitive . this could suggest that protein - mediated signalling from mitochondria might impact on insulin action . in hepatocytes , a recent study has found that reduced cellular energy status can impede glucagon signalling , which opposes insulin signalling in the control of glucogeogenesis . increased amp however , we found no association between reduced atp , or any involvement for the canonical insulin signalling pathway in our mitochondrial dysfunction models that reduced insulin suppression of glucose production . the mechanisms by which mitochondrial dysfunction impacts on insulin action in hepatocytes remain to be determined . we also observed that the anti - diabetic agents phenformin and rosiglitazone showed cell type - dependent impairments in mitochondrial function and insulin action . this included enhanced suppression of glucose production and enhanced insulin action in hepatocytes , but also included induction of insulin resistance in adipocytes . the acute effects of these drugs in adipocytes might be offset by longer term remodelling of adipocyte metabolism , particularly in the case of rosiglitazone , which enhances the expression of ppar dependent genes to increase glucose disposal and lipogenesis , thereby reducing hyperglycemia in vivo . indeed , the reduced insulin action we observed in rosiglitazone treated adipocytes was primarily due to an increase in basal glucose uptake , rather than reduced insulin - stimulated glucose uptake . nonetheless , both anti - diabetic drugs altered mitochondrial function in adipocytes , which could have longer term implications in patients treated with these drugs for extended periods . the finding that both drugs exacerbated insulin resistance in the context of existing mitochondrial dysfunction also highlights that personalised medicine approaches that identify any mitochondrial defects could be used to develop improved treatment strategies for patients . in conclusion , our findings show that acute , physiological impairments in mitochondrial function are sufficient , but not necessary , for the development of cell type - dependent insulin resistance and this is independent of mitochondrial ros production . the translational implications from these findings are that therapies for insulin resistance that involve modulation of mitochondrial function should be tissue specific and their mechanism of action on mitochondria well characterised to ensure optimal therapeutic outcomes . the complexity in the relationship between mitochondrial function and insulin action revealed by these studies provides insights into opposing conclusions on this issue and questions reductionist theories that attempt to describe this relationship .

while global sales of nanoproducts are expected to reach 450 billion within the next year ( hanssen et al . 2008 ) , scientists and regulators are still left with many questions as to whether nanomaterials ( nm ) pose a risk to human health or the environment . the risk assessment framework developed for chemicals remains the predominant decision support tool , underpinning regulations that cover nm or providing information to make the case for regulatory amendment ( rocks et al . 2008 ) . this framework is well known to many , comprising hazard identification , hazard assessment and risk characterization ( including considerations of exposure ) ( rocks et al . associated uncertainty in the process can be managed , for example by the application of safety factors ( european commission 2003 ; owen et al . while it is recognised that methodologies underpinning the conceptual risk assessment frameworks ( e.g. eu technical guidance document ( tgd ) , organisation for economic co - operation and development ( oecd ) test guidelines ) may not be entirely fit for purpose ( e.g. crane et al . 2008 ) , the general consensus has been that in principle the chemical risk assessment framework is sound to use for nm with some modifications ( european commission 2008b ; oecd 2008a ; scenihr 2007 , 2009 ) . whereas , in principle , risk assessment is a sound approach , in practice , it is not an easy task to complete as there are some very significant challenges to first overcome ( e.g. maynard et al . it is now acknowledged that its completion will quite possibly take decades even for those nm that are currently on the market , let alone those that are emerging . similar challenges have been faced for other chemicals ( e.g. endocrine - disrupting chemicals , sumpter and johnson 2008 ) . however , nm may represent an extreme case due to their diversity ( e.g. particle sizes , functionalities ) and range of applications ( owen and handy 2007 ; rcep 2008 ; linkov et al . while the question of identifying the most appropriate mechanism(s ) to support timely decisions ( e.g. interventions ) in the face of little or no data is far from new ( collingridge 1980 ) , it may be more challenging for nm and , at the same time , of utmost importance given the ubiquitous nature of nano - embedded consumer products and applications ( project on emerging nanotechnologies 2008 ) . some fundamental questions concerning whether nm risk research efforts have been directed in the most effective fields for timely decisions regarding environment , health and safety risks include : how do research efforts compare between the fields dedicated to using traditional risk assessment frameworks and alternative or complementary frameworks or tools for nm ? are programs that feed into chemical - based risk assessment made at the expense of research into tools that can support near - term decisions ? what are the alternatives to traditional risk assessment currently available and what are their strengths and weaknesses ? this analysis aims to address these critical questions , in addition to providing a snapshot of publications , projects and funding schemes made in these fields . we then propose some recommendations for future nm risk research schemes to help ensure the responsible innovation of nm as well as ultimately protect human and environmental health . scientists , international organizations and regulatory agencies have been actively working towards developing risk assessment for nm in recent years ( e.g. nni 2008b ; epa 2009 ; oecd 2009d ; scenihr 2007 , 2009 ) . in general , it has not yet been possible to complete individual nm risk assessments ( scenihr 2009 ) due to extensive uncertainties and knowledge gaps within nearly all aspects of the risk assessment process ( rcep 2008 ; grieger et al . , there is a distinct lack of quantitative exposure and hazard data for both humans and environmental species for many nm ( crane et al . in addition , a number of key methodological concerns have been raised , including uncertainty in applying standardized test methods developed for chemicals to nm ( e.g. to quantify hazards including sample preparation for ( eco)toxicity studies , characterisation of nm in test systems , dosimetry and reporting metrics ) ( crane et al . ; shatkin 2008 ) . furthermore , there are also difficulties in detecting and quantifying nm in complex environmental matrices ( owen and handy 2007 ; hassellov et al . 2008 ) . based upon a reflection of these challenges and in response to calls for more research ( e.g. maynard et al . 2006 ) , increased efforts regarding these have been underway . for example , scientists and organisations have been actively generating and analysing data for effects and exposure assessments as well as reviewing risk assessment methodologies for their appropriateness for nm . academics and scientists in government and regulatory agencies all play a role in these developments , as well as industry , for example under chemicals legislation in relation to nm ( e.g. registration , evaluation , authorisation and restriction of chemical substances , reach ) ( european commission 2008b ) . in the eu , the european commission s scientific committee on emerging and newly identified health risks ( scenihr ) has recently reviewed the available data for risk assessment of nm ( scenihr 2009 ) . they conclude that the risk assessment framework seems applicable to nm although they acknowledge and describe many of its limitations , stating that the risk assessment process is still under development . others such as the european food safety authority have recently published similar conclusions , stating that although there are some major ( e.g. methodological ) challenges , current risk assessment methodologies are expected to be applicable for nm in food and feed applications in principle , and assessments should be undertaken on a case - by - case basis ( european food safety authority 2009 ) . the challenges for risk assessment posed by nm are reflected by a significant program of work at the organisation for economic co - operation and development s working party on ( manufactured ) nm ( wpmn ) . an array of projects are being directed , including the development of a nm risk research database ( oecd 2009d ) , analysis of test methods ( which underpin chemicals risk assessment ) as applied to nm and a sponsorship program that will acquire risk assessment data for 14 nm of current relevance ( oecd 2008b ) . further study has also been undertaken to prioritize research needs to close the known knowledge gaps as quickly as possible , supporting the performance of risk assessments often in the context of current regulatory testing requirements ( defra 2006 , 2007 ; maynard et al . ; scenihr 2009 ) . on the whole , a consensus is beginning to emerge : risk assessment frameworks for chemicals should be appropriate for nm , but they most likely need some methodological modifications . exactly what modifications are needed is not consistently made clear , and how long it will take to make these modifications is also not often stated . despite these serious technical challenges that risk assessors face , most knowledge gaps are expected to be reduced in due course because of their epistemic nature ( rcep 2008 ; grieger et al . it is anticipated that eventually standardized testing methodologies and analytical tools will be developed alongside full nm characterization and dose response data ( iso 2008a , b ; oecd 2008a , b ) . history has shown many examples in which regulatory test methods were developed and applied ( baun and hansen 2008 ; oecd 2009a , b ) in response to new knowledge ( e.g. endocrine - disrupting chemicals , sumpter and johnson 2008 ) , and later test methods were developed ( oecd 2009a , b ) . in fact , a recent review of the applicability of oecd test guidelines for nm ( oecd 2009e ) shows this process in action : some test guidelines for nm physical chemical properties appear to be adequate for nm ( e.g. tg 102 , 109 , 113 , 116 ) , some may be applicable in some cases or for some nm ( e.g. tg 101 , 105 , 106 , etc . ) , while other guidelines are considered inadequate ( e.g. tg 103 , 114 ) . however , how long will this process take especially given the diversity of nm and applications ? a recent analysis estimates that testing existing nanoparticles in the usa alone will cost between $ 249 million and $ 1.18 billion and take 3453 years for completion ( choi et al . earlier analyses also estimated , at least , a decade for the acquisition of some critical knowledge within the field ( maynard et al . given this , some have suggested that more rapidly deployable decision - support frameworks and/or tools need to be applied now ( hansen 2009 ; linkov et al . in addition , others have proposed the need for risk governance1 mechanisms that can be enacted far earlier in the nano - innovation process , reducing the time needed to acquire critical risk characterisation data ( lee and jose 2008 ; owen et al . , the precautionary principle2 has been invoked to support decisions in the absence of full scientific certainty . for instance , the environment agency in the uk recommended that unbound carbon nanotubes are treated as hazardous waste ( environment agency 2008 ) . others , however , have concluded that , for the most part , there is still insufficient evidence to invoke the precautionary principle ( aitken et al . 2009 ; stebbing 2009 ) , and , hence , this issue may quite possibly take many years to resolve . in order to gauge whether current nm risk research efforts are responding to the challenges outlined above , we have undertaken a snapshot review of research efforts , mainly in terms of peer - reviewed journal publications , research projects and public funding within the fields of traditional risk assessment paradigms , complementary tools to risk assessment and risk governance mechanisms as they apply to nm . this is not intended to be a complete analysis of research publications and projects within the field of environment , health and safety aspects ( ehs ) of nm ( see e.g. aguar and murcia nicols 2008 ; scenihr 2009 for more complete overviews ) ; instead , we aim to provide a synopsis of the general direction and distribution of efforts to date . we searched peer - reviewed journal articles within different nano - risk - related fields using the isi web of knowledge article database ( http://apps.isiknowledge.com ) and the international council on nanotechnology ( icon ) database ( quick and advanced searches , http://icon.rice.edu/research.cfm ) which specifically focuses on ehs issues of nm . in addition , the oecd research project database ( http://webnet.oecd.org/nanomaterials/pagelet/front/default.aspx ? ) which also specifically focuses on projects within health and environmental safety of nm was also searched . see fig . 1 for more details on the search methodology , including search terms used.fig . 1comparison of number of journal article publications and research projects within different nano - risk topics and environment , health and safety ( ehs ) issues of nanomaterials ( nm ) . the isi web of knowledge ( http://apps.isiknowledge.com ) and icon virtual journal ( http://icon.rice.edu/research.cfm ) which specifically focuses on ehs issues of nm ( using both quick and advanced search options ) were used to search for scientific journal publications . the oecd nm risk research project database ( http://webnet.oecd.org/nanomaterials/pagelet/front/default.aspx ? ) was used to search for completed or on - going research projects , shown on the secondary axis . searches were made within all years in these databases , and were accessed and used on 30 june 2009 . management and monitoring. since the isi web of knowledge database is not confined to nm risk research , as in the case of the icon and oecd databases , the previously cited search terms were used together with nanoparticle comparison of number of journal article publications and research projects within different nano - risk topics and environment , health and safety ( ehs ) issues of nanomaterials ( nm ) . the isi web of knowledge ( http://apps.isiknowledge.com ) and icon virtual journal ( http://icon.rice.edu/research.cfm ) which specifically focuses on ehs issues of nm ( using both quick and advanced search options ) were used to search for scientific journal publications . the oecd nm risk research project database ( http://webnet.oecd.org/nanomaterials/pagelet/front/default.aspx ? ) was used to search for completed or on - going research projects , shown on the secondary axis . searches were made within all years in these databases , and were accessed and used on 30 june 2009 . monitoring. since the isi web of knowledge database is not confined to nm risk research , as in the case of the icon and oecd databases , the previously cited search terms were used together with nanotechnology , nanoparticle in terms of the number of published research articles and research projects within different nano - risk fields , the majority ( 60.1% ) of these research efforts were within the topics of toxicity , there were also relatively high numbers of research articles found in topics of management and monitoring of nanotechnology , -material or -particle using the isi web of knowledge , although the number of articles found within these topics is significantly less when using the icon database which specifically focuses on ehs aspects of nm . in comparison , only 6.8 and 1.6% of research efforts were in the areas of risk governance and decision making , respectively . similar patterns are also seen in the distribution of research projects completed or underway within nm ehs issues . according to the oecd project database for health and environmental safety research of nm , there were 153 projects listed under topics of exposure and 99 projects within toxicity or ecotoxicity , compared to 45 within risk assessment , 37 within monitoring , 16 within management and 6 within risk management. only two projects are within the field of risk governance and three in the field of decision making ( fig . 1 ) . it is evident , not unsurprisingly , that much of the ongoing research fits within a chemical - based risk assessment paradigm . very little research is directly addressing the key issues of near - term decision support highlighted above and by others ( rcep 2008 ; brown 2009 ; linkov et al . of the nearly 200 projects funded either through the eu framework programmes ( totally 32 million ) , eu member states , candidate countries or countries associated to sixth framework programme ( fp6 ) or seventh framework programme ( fp7 ) ( totally 47 million ) , only a few projects focus on risk governance or decision making ( aguar and murcia nicols 2008 ) . the great majority of projects have been dedicated towards improving technical knowledge or developing new test protocols and equipment . similar patterns are seen in us public funding schemes through the national nanotechnology initiative ( nni 2008a , b ) . funding schemes outlined by the eu s current fp7 programme do not appear to deviate much from previous research priorities ( aguar and murcia nicols 2008 ; european commission 2009 ) . the recent fp7 call , ( theme 4 nanosciences , nanotechnologies , materials and new production technologies nmp ) , published 29 july 2009 , is aimed largely at gaining more technical knowledge for risk assessment ( european commission 2009 ) . very few references are made on improved governance or decision making of nanotechnologies , with no specific calls within these areas . these are largely subsumed within wider goals , such as improved social acceptance , sustainable development and the development of reference materials . this is not surprising given the overarching strategy outlined by the eu nanotechnologies action plan ( european commission 2005 ) which is yet to acknowledge the key issues raised in this article and by others ( e.g. rcep 2008 ) . this analysis has shown that despite the recognized serious challenges that nm present for fulfilling traditional chemical - based risk assessment frameworks and the time this will likely take , the large majority of decision support research is directed to fit ultimately within this framework . decision makers , therefore , may not be well equipped to make decisions concerning nm under conditions of extensive uncertainty in relation to environmental and human health protection in the near term . it is clear , in our view , that there is a need for a program of research and knowledge transfer specifically aimed at supporting near- and medium - term decision making , in real time and at the same pace as nano - innovation itself ( guston and sarewitz 2002 ) , see box 1 . furthermore , since there are already a number of nm on the market with varying degrees of potential for exposure ( limbach et al . 2008 ; rcep 2008 ; scenihr 2009 ) , we also recommend the use of environment and health surveillance as an early warning system to act as a safety net around such a decision support program , to which it may also serve to inform ( box 1 ) . we have highlighted these areas that might be worthy of consideration in such a research program in box 1 and provide further justification for these in the following section.box 1 in response to the challenges of potential health and environmental risk assessments of nanomaterials ( nm ) and making decisions regarding these potential risks , as highlighted in this analysis , we propose some key areas for future research to ensure the responsible emergence of nanotechnologiesrecommendationrationaleimplementationadaptive and more responsive risk governance frameworksrapid nanotechnologies innovation out - paces regulatory governance based on traditional risk assessmentencourage and support research on risk governance which specifically focuses on timely yet informed decision making in light of uncertainty and rapid nano - innovationlengthy , post - innovation risk investigationsintegrate responsible innovation early ( upstream ) in innovation processalternative , complementary tools to risk assessmentearly approximate risk evaluationsimmediate , rigorous testing of already - developed tools to fully evaluate their functionality and limitationsrelative comparison of nm risks for decision supportcontinue development of new tools including those which may complement risk assessmenthealth and environmental surveillanceearly warning systemuse of biomonitoring , such as : potential safety net filter - feeders in aquatic hot spots monitor health of occupational workers direct toxicity assessment of effluents in response to the challenges of potential health and environmental risk assessments of nanomaterials ( nm ) and making decisions regarding these potential risks , as highlighted in this analysis , we propose some key areas for future research to ensure the responsible emergence of nanotechnologies if the development of regulation based on quantitative risk assessment is an inherently slow governance process , then one answer might be to undertake research that leads to one or more governance frameworks that are more responsive and adaptive . this is a view that has been articulated by a number of authors , including rcep ( 2008 ) , brown ( 2009 ) and owen et al . there is a foundation for this study , including a number of alternative risk governance frameworks that have been proposed in recent years , such as those suggested by the international risk governance council ( irgc 2007 ) , environmental defense and dupont ( nano risk framework , environmental defense and dupont 2007 ) and smarten ( strategic management and assessment of risks and toxicity of engineered nanomaterials ) ( metcalfe et al . 2009 ) . these typically not only combine some of the traditional parameters of risk assessment ( hazard identification , exposure assessment , etc . ) and risk management ( in the case of the nano risk framework ) , but may also incorporate wider aspects ( e.g. social , economic and cultural aspects , expert analysis ) . for example , real - time technology assessment attempts to integrate nano - science and innovation with social science and policy at early stages of research and development rather than dealing with decision - making issues after innovation has already taken a place in society ( guston and sarewitz 2002 ) . this has the potential to prevent lengthy post - nm production debates by addressing key issues early in the innovation process , given that the inclusive steps involved are coordinated and well guided to minimize delay in the process . while real - time technology assessment mainly aims to assist decision - making processes by incorporating other ( societal ) factors , it is not yet clear if more timely decisions will ultimately be aided by this process . for example , the european commission s code of conduct for nanosciences and nanotechnologies research was established in 2007 ( european commission 2008a ) , providing guidance to steer the responsible development of nanotechnology at an early stage . this includes specific recommendations aimed towards enhancing communication of the risks , benefits and uncertainties in addition to the development of scientific tools and knowledge , albeit described , in general , rather than in specific terms . in short , while there are a number of potential avenues to pursue , a comprehensive programme of research in this area is currently lacking . therefore , we recommend the continued development of research on risk governance frameworks that specifically aims to strengthen the timeliness of decision making processes in the face of rapid nanotechnologies innovation . in addition to the risk governance frameworks and approaches described above , a wide variety of tools have either been developed or deployed for approximate risk evaluations or decision making about ( some ) nm risks using available information . these include the precautionary matrix to identify potentially dangerous applications within production ( hck et al . 2008 ) and a categorisation framework to identify different exposure potentials based on the location of the nano - structured material in a product ( hansen et al . multi - criteria decision analysis ( mcda ) and smaa tri models have also been proposed , which compare and rank alternatives in nm risk decision making ( linkov et al . furthermore , expert elicitation has been put forth to help compensate for large data gaps ( morgan 2005 ) , as well as adaptive management to help create flexible and iterative processes ( davis 2007 ; linkov et al . in general , these tools attempt to provide either an early attempt to approximate or compare some nm risks for decision support , even if it is , for example ranking the risks of different nanoparticles . while these tools and models may be valuable in many aspects , especially in terms of thinking outside the box in regard to early estimates and comparisons of nm risks and better decision - making strategies , one main limitation is that many ( if not most ) of these tools and models have not been thoroughly tested and applied to many nm , and , therefore , their robustness and applicability is not yet clear . therefore , we recommend that the developed frameworks and tools , some of which have been described here , be immediately and rigorously tested to fully evaluate their functionality and limitations . this should be complementary to the continued development and amendment of alternative governance frameworks and tools . since decisions under uncertainty and especially ignorance will always be prone to varying degrees of error , some have suggested the need for corrigibility ( collingridge 1980 ) . in other words , under uncertain circumstances , it is important to ensure that such decisions can be reversed at minimum cost ( economic or otherwise ) . hence , environmental and health surveillance may have the potential to serve as an important safety net or early warning system ( owen et al . 2009 ) to any decision support system for nm risks , whether based on prospective risk assessment or an alternate framework . in particular , the use of biomonitoring may be particularly useful given the difficulties in detecting and measuring nanoparticles in complex environmental matrices ( e.g. hassellov et al . this might include evaluating the toxicity of nm - containing environmental discharges ( e.g. whole effluent assessment or direct toxicity assessment , owen et al . 2009b ) or the use of filter feeding organisms in aquatic environments where there is an anticipated high degree of exposure to nm ( baun et al . biomonitoring may also encompass the monitoring and surveillance of occupational workers health in the light of potentially hazardous effects of nm ( nasterlack et al . in fact , the national institute of occupational health and safety ( niosh ) recently recommended hazard surveillance as a foundation to implement safety measures ( niosh 2009 ) , and the oecd has recommended the inclusion of health monitoring , surveillance and biological monitoring for occupational workers ( oecd 2009c ) . if the development of regulation based on quantitative risk assessment is an inherently slow governance process , then one answer might be to undertake research that leads to one or more governance frameworks that are more responsive and adaptive . this is a view that has been articulated by a number of authors , including rcep ( 2008 ) , brown ( 2009 ) and owen et al . there is a foundation for this study , including a number of alternative risk governance frameworks that have been proposed in recent years , such as those suggested by the international risk governance council ( irgc 2007 ) , environmental defense and dupont ( nano risk framework , environmental defense and dupont 2007 ) and smarten ( strategic management and assessment of risks and toxicity of engineered nanomaterials ) ( metcalfe et al . these typically not only combine some of the traditional parameters of risk assessment ( hazard identification , exposure assessment , etc . ) and risk management ( in the case of the nano risk framework ) , but may also incorporate wider aspects ( e.g. social , economic and cultural aspects , expert analysis ) . for example , real - time technology assessment attempts to integrate nano - science and innovation with social science and policy at early stages of research and development rather than dealing with decision - making issues after innovation has already taken a place in society ( guston and sarewitz 2002 ) . this has the potential to prevent lengthy post - nm production debates by addressing key issues early in the innovation process , given that the inclusive steps involved are coordinated and well guided to minimize delay in the process . while real - time technology assessment mainly aims to assist decision - making processes by incorporating other ( societal ) factors , it is not yet clear if more timely decisions will ultimately be aided by this process . for example , the european commission s code of conduct for nanosciences and nanotechnologies research was established in 2007 ( european commission 2008a ) , providing guidance to steer the responsible development of nanotechnology at an early stage . this includes specific recommendations aimed towards enhancing communication of the risks , benefits and uncertainties in addition to the development of scientific tools and knowledge , albeit described , in general , rather than in specific terms . in short , while there are a number of potential avenues to pursue , a comprehensive programme of research in this area is currently lacking . therefore , we recommend the continued development of research on risk governance frameworks that specifically aims to strengthen the timeliness of decision making processes in the face of rapid nanotechnologies innovation . in addition to the risk governance frameworks and approaches described above , a wide variety of tools have either been developed or deployed for approximate risk evaluations or decision making about ( some ) nm risks using available information . these include the precautionary matrix to identify potentially dangerous applications within production ( hck et al . 2008 ) and a categorisation framework to identify different exposure potentials based on the location of the nano - structured material in a product ( hansen et al . multi - criteria decision analysis ( mcda ) and smaa tri models have also been proposed , which compare and rank alternatives in nm risk decision making ( linkov et al . 2007 ; furthermore , expert elicitation has been put forth to help compensate for large data gaps ( morgan 2005 ) , as well as adaptive management to help create flexible and iterative processes ( davis 2007 ; linkov et al . 2007 ) . in general , these tools attempt to provide either an early attempt to approximate or compare some nm risks for decision support , even if it is , for example ranking the risks of different nanoparticles . while these tools and models may be valuable in many aspects , especially in terms of thinking outside the box in regard to early estimates and comparisons of nm risks and better decision - making strategies , one main limitation is that many ( if not most ) of these tools and models have not been thoroughly tested and applied to many nm , and , therefore , their robustness and applicability is not yet clear . therefore , we recommend that the developed frameworks and tools , some of which have been described here , be immediately and rigorously tested to fully evaluate their functionality and limitations . this should be complementary to the continued development and amendment of alternative governance frameworks and tools . since decisions under uncertainty and especially ignorance will always be prone to varying degrees of error , some have suggested the need for corrigibility ( collingridge 1980 ) . in other words , under uncertain circumstances , it is important to ensure that such decisions can be reversed at minimum cost ( economic or otherwise ) . hence , environmental and health surveillance may have the potential to serve as an important safety net or early warning system ( owen et al . 2009b ; saunders and mohammed 2009 ; schulte et al . 2009 ) to any decision support system for nm risks , whether based on prospective risk assessment or an alternate framework . in particular , the use of biomonitoring may be particularly useful given the difficulties in detecting and measuring nanoparticles in complex environmental matrices ( e.g. hassellov et al . this might include evaluating the toxicity of nm - containing environmental discharges ( e.g. whole effluent assessment or direct toxicity assessment , owen et al . 2009b ) or the use of filter feeding organisms in aquatic environments where there is an anticipated high degree of exposure to nm ( baun et al . biomonitoring may also encompass the monitoring and surveillance of occupational workers health in the light of potentially hazardous effects of nm ( nasterlack et al . in fact , the national institute of occupational health and safety ( niosh ) recently recommended hazard surveillance as a foundation to implement safety measures ( niosh 2009 ) , and the oecd has recommended the inclusion of health monitoring , surveillance and biological monitoring for occupational workers ( oecd 2009c ) . although the traditional chemical - based risk assessment framework is a powerful approach , its use for estimating the health and environmental risks of nm in the near - term may be limited due to the time and costs needed to generate meaningful results . this is due to the extensive risk uncertainties and complex types and numbers of nm encountered in current production . given these limitations , decision makers may not currently be well equipped if decisions are to be based mainly upon results generated from quantitative risk assessment . we see this as the central issue for the responsible emergence of nanotechnologies . despite the above , research efforts thus far have been mainly put towards acquiring knowledge that will most likely be fed into chemical - based risk assessment procedures . these efforts include a predominance of research publications and projects within the fields of ( eco)toxicology and human and environmental exposure of nm , while research in broader issues such as decision making and risk governance are comparatively minimal . research supporting quantitative risk characterization of nm is indeed important , and we do not advocate abandonment of this at any cost . however , as scientists and policy makers , we must face the reality that knowledge gaps are not expected to be closed in the near future and may take decades to close in many cases . in order to ensure the protection of the environment and human health , this suggests the need for a research programme as significant as that which feeds into quantitative risk assessment and which addresses the fundamental issue of timely , yet , informed decision making regarding potential nm risks . although a few alternative risk frameworks have been proposed , it is not clear whether they will address this key concern . furthermore , although some complementary governance approaches and decision support tools have been suggested , their comprehensive evaluation and development has yet to be undertaken . in conclusion , we suggest the need for an international research programme that specifically addresses critical issues of risk governance and timely decision making as these relate to nm specifically , and emerging technologies more generally . this programme might include research into more efficient and anticipatory risk governance mechanisms as well as a systematic evaluation of available complementary tools to risk assessment and , where necessary , further tools under development . finally , the development of environmental and health surveillance is needed to act as a safety net and an early warning system while these issues are being addressed . we recommend that active research within these areas should be among the essential steps taken in the near future to ensure the responsible emergence of nanotechnologies , and ultimately the protection of human and environmental health .

chemical - based risk assessment underpins the current approach to responsible development of nanomaterials ( nm ) . it is now recognised , however , that this process may take decades , leaving decision makers with little support in the near term . despite this , current and near future research efforts are largely directed at establishing ( eco)toxicological and exposure data for nm , and comparatively little research has been undertaken on tools or approaches that may facilitate near - term decisions , some of which we briefly outline in this analysis . we propose a reprioritisation of nm risk research efforts to redress this imbalance , including the development of more adaptive risk governance frameworks , alternative / complementary tools to risk assessment , and health and environment surveillance .

Introduction Risk assessment as a decision support tool: challenges posed by nanomaterials Current and near-future research effort distributions Redressing the imbalance: a path forward and recommendations Adaptive and more responsive risk governance frameworks Alternative, complementary tools to risk assessment The need for surveillance Conclusions

2008 ) , scientists and regulators are still left with many questions as to whether nanomaterials ( nm ) pose a risk to human health or the environment . the risk assessment framework developed for chemicals remains the predominant decision support tool , underpinning regulations that cover nm or providing information to make the case for regulatory amendment ( rocks et al . while it is recognised that methodologies underpinning the conceptual risk assessment frameworks ( e.g. 2008 ) , the general consensus has been that in principle the chemical risk assessment framework is sound to use for nm with some modifications ( european commission 2008b ; oecd 2008a ; scenihr 2007 , 2009 ) . whereas , in principle , risk assessment is a sound approach , in practice , it is not an easy task to complete as there are some very significant challenges to first overcome ( e.g. it is now acknowledged that its completion will quite possibly take decades even for those nm that are currently on the market , let alone those that are emerging . interventions ) in the face of little or no data is far from new ( collingridge 1980 ) , it may be more challenging for nm and , at the same time , of utmost importance given the ubiquitous nature of nano - embedded consumer products and applications ( project on emerging nanotechnologies 2008 ) . some fundamental questions concerning whether nm risk research efforts have been directed in the most effective fields for timely decisions regarding environment , health and safety risks include : how do research efforts compare between the fields dedicated to using traditional risk assessment frameworks and alternative or complementary frameworks or tools for nm ? are programs that feed into chemical - based risk assessment made at the expense of research into tools that can support near - term decisions ? we then propose some recommendations for future nm risk research schemes to help ensure the responsible innovation of nm as well as ultimately protect human and environmental health . scientists , international organizations and regulatory agencies have been actively working towards developing risk assessment for nm in recent years ( e.g. in general , it has not yet been possible to complete individual nm risk assessments ( scenihr 2009 ) due to extensive uncertainties and knowledge gaps within nearly all aspects of the risk assessment process ( rcep 2008 ; grieger et al . for example , scientists and organisations have been actively generating and analysing data for effects and exposure assessments as well as reviewing risk assessment methodologies for their appropriateness for nm . in the eu , the european commission s scientific committee on emerging and newly identified health risks ( scenihr ) has recently reviewed the available data for risk assessment of nm ( scenihr 2009 ) . they conclude that the risk assessment framework seems applicable to nm although they acknowledge and describe many of its limitations , stating that the risk assessment process is still under development . methodological ) challenges , current risk assessment methodologies are expected to be applicable for nm in food and feed applications in principle , and assessments should be undertaken on a case - by - case basis ( european food safety authority 2009 ) . the challenges for risk assessment posed by nm are reflected by a significant program of work at the organisation for economic co - operation and development s working party on ( manufactured ) nm ( wpmn ) . an array of projects are being directed , including the development of a nm risk research database ( oecd 2009d ) , analysis of test methods ( which underpin chemicals risk assessment ) as applied to nm and a sponsorship program that will acquire risk assessment data for 14 nm of current relevance ( oecd 2008b ) . further study has also been undertaken to prioritize research needs to close the known knowledge gaps as quickly as possible , supporting the performance of risk assessments often in the context of current regulatory testing requirements ( defra 2006 , 2007 ; maynard et al . on the whole , a consensus is beginning to emerge : risk assessment frameworks for chemicals should be appropriate for nm , but they most likely need some methodological modifications . exactly what modifications are needed is not consistently made clear , and how long it will take to make these modifications is also not often stated . in fact , a recent review of the applicability of oecd test guidelines for nm ( oecd 2009e ) shows this process in action : some test guidelines for nm physical chemical properties appear to be adequate for nm ( e.g. however , how long will this process take especially given the diversity of nm and applications ? a recent analysis estimates that testing existing nanoparticles in the usa alone will cost between $ 249 million and $ 1.18 billion and take 3453 years for completion ( choi et al . given this , some have suggested that more rapidly deployable decision - support frameworks and/or tools need to be applied now ( hansen 2009 ; linkov et al . , the precautionary principle2 has been invoked to support decisions in the absence of full scientific certainty . others , however , have concluded that , for the most part , there is still insufficient evidence to invoke the precautionary principle ( aitken et al . in order to gauge whether current nm risk research efforts are responding to the challenges outlined above , we have undertaken a snapshot review of research efforts , mainly in terms of peer - reviewed journal publications , research projects and public funding within the fields of traditional risk assessment paradigms , complementary tools to risk assessment and risk governance mechanisms as they apply to nm . this is not intended to be a complete analysis of research publications and projects within the field of environment , health and safety aspects ( ehs ) of nm ( see e.g. which also specifically focuses on projects within health and environmental safety of nm was also searched . 1comparison of number of journal article publications and research projects within different nano - risk topics and environment , health and safety ( ehs ) issues of nanomaterials ( nm ) . the oecd nm risk research project database ( http://webnet.oecd.org/nanomaterials/pagelet/front/default.aspx ? ) searches were made within all years in these databases , and were accessed and used on 30 june 2009 . since the isi web of knowledge database is not confined to nm risk research , as in the case of the icon and oecd databases , the previously cited search terms were used together with nanoparticle comparison of number of journal article publications and research projects within different nano - risk topics and environment , health and safety ( ehs ) issues of nanomaterials ( nm ) . the isi web of knowledge ( http://apps.isiknowledge.com ) and icon virtual journal ( http://icon.rice.edu/research.cfm ) which specifically focuses on ehs issues of nm ( using both quick and advanced search options ) were used to search for scientific journal publications . the oecd nm risk research project database ( http://webnet.oecd.org/nanomaterials/pagelet/front/default.aspx ? ) since the isi web of knowledge database is not confined to nm risk research , as in the case of the icon and oecd databases , the previously cited search terms were used together with nanotechnology , nanoparticle in terms of the number of published research articles and research projects within different nano - risk fields , the majority ( 60.1% ) of these research efforts were within the topics of toxicity , there were also relatively high numbers of research articles found in topics of management and monitoring of nanotechnology , -material or -particle using the isi web of knowledge , although the number of articles found within these topics is significantly less when using the icon database which specifically focuses on ehs aspects of nm . in comparison , only 6.8 and 1.6% of research efforts were in the areas of risk governance and decision making , respectively . according to the oecd project database for health and environmental safety research of nm , there were 153 projects listed under topics of exposure and 99 projects within toxicity or ecotoxicity , compared to 45 within risk assessment , 37 within monitoring , 16 within management and 6 within risk management. only two projects are within the field of risk governance and three in the field of decision making ( fig . it is evident , not unsurprisingly , that much of the ongoing research fits within a chemical - based risk assessment paradigm . very little research is directly addressing the key issues of near - term decision support highlighted above and by others ( rcep 2008 ; brown 2009 ; linkov et al . these are largely subsumed within wider goals , such as improved social acceptance , sustainable development and the development of reference materials . this is not surprising given the overarching strategy outlined by the eu nanotechnologies action plan ( european commission 2005 ) which is yet to acknowledge the key issues raised in this article and by others ( e.g. this analysis has shown that despite the recognized serious challenges that nm present for fulfilling traditional chemical - based risk assessment frameworks and the time this will likely take , the large majority of decision support research is directed to fit ultimately within this framework . decision makers , therefore , may not be well equipped to make decisions concerning nm under conditions of extensive uncertainty in relation to environmental and human health protection in the near term . it is clear , in our view , that there is a need for a program of research and knowledge transfer specifically aimed at supporting near- and medium - term decision making , in real time and at the same pace as nano - innovation itself ( guston and sarewitz 2002 ) , see box 1 . we have highlighted these areas that might be worthy of consideration in such a research program in box 1 and provide further justification for these in the following section.box 1 in response to the challenges of potential health and environmental risk assessments of nanomaterials ( nm ) and making decisions regarding these potential risks , as highlighted in this analysis , we propose some key areas for future research to ensure the responsible emergence of nanotechnologiesrecommendationrationaleimplementationadaptive and more responsive risk governance frameworksrapid nanotechnologies innovation out - paces regulatory governance based on traditional risk assessmentencourage and support research on risk governance which specifically focuses on timely yet informed decision making in light of uncertainty and rapid nano - innovationlengthy , post - innovation risk investigationsintegrate responsible innovation early ( upstream ) in innovation processalternative , complementary tools to risk assessmentearly approximate risk evaluationsimmediate , rigorous testing of already - developed tools to fully evaluate their functionality and limitationsrelative comparison of nm risks for decision supportcontinue development of new tools including those which may complement risk assessmenthealth and environmental surveillanceearly warning systemuse of biomonitoring , such as : potential safety net filter - feeders in aquatic hot spots monitor health of occupational workers direct toxicity assessment of effluents in response to the challenges of potential health and environmental risk assessments of nanomaterials ( nm ) and making decisions regarding these potential risks , as highlighted in this analysis , we propose some key areas for future research to ensure the responsible emergence of nanotechnologies if the development of regulation based on quantitative risk assessment is an inherently slow governance process , then one answer might be to undertake research that leads to one or more governance frameworks that are more responsive and adaptive . this is a view that has been articulated by a number of authors , including rcep ( 2008 ) , brown ( 2009 ) and owen et al . there is a foundation for this study , including a number of alternative risk governance frameworks that have been proposed in recent years , such as those suggested by the international risk governance council ( irgc 2007 ) , environmental defense and dupont ( nano risk framework , environmental defense and dupont 2007 ) and smarten ( strategic management and assessment of risks and toxicity of engineered nanomaterials ) ( metcalfe et al . these typically not only combine some of the traditional parameters of risk assessment ( hazard identification , exposure assessment , etc . ) this has the potential to prevent lengthy post - nm production debates by addressing key issues early in the innovation process , given that the inclusive steps involved are coordinated and well guided to minimize delay in the process . while real - time technology assessment mainly aims to assist decision - making processes by incorporating other ( societal ) factors , it is not yet clear if more timely decisions will ultimately be aided by this process . for example , the european commission s code of conduct for nanosciences and nanotechnologies research was established in 2007 ( european commission 2008a ) , providing guidance to steer the responsible development of nanotechnology at an early stage . this includes specific recommendations aimed towards enhancing communication of the risks , benefits and uncertainties in addition to the development of scientific tools and knowledge , albeit described , in general , rather than in specific terms . therefore , we recommend the continued development of research on risk governance frameworks that specifically aims to strengthen the timeliness of decision making processes in the face of rapid nanotechnologies innovation . in addition to the risk governance frameworks and approaches described above , a wide variety of tools have either been developed or deployed for approximate risk evaluations or decision making about ( some ) nm risks using available information . multi - criteria decision analysis ( mcda ) and smaa tri models have also been proposed , which compare and rank alternatives in nm risk decision making ( linkov et al . while these tools and models may be valuable in many aspects , especially in terms of thinking outside the box in regard to early estimates and comparisons of nm risks and better decision - making strategies , one main limitation is that many ( if not most ) of these tools and models have not been thoroughly tested and applied to many nm , and , therefore , their robustness and applicability is not yet clear . therefore , we recommend that the developed frameworks and tools , some of which have been described here , be immediately and rigorously tested to fully evaluate their functionality and limitations . in other words , under uncertain circumstances , it is important to ensure that such decisions can be reversed at minimum cost ( economic or otherwise ) . 2009 ) to any decision support system for nm risks , whether based on prospective risk assessment or an alternate framework . this might include evaluating the toxicity of nm - containing environmental discharges ( e.g. biomonitoring may also encompass the monitoring and surveillance of occupational workers health in the light of potentially hazardous effects of nm ( nasterlack et al . in fact , the national institute of occupational health and safety ( niosh ) recently recommended hazard surveillance as a foundation to implement safety measures ( niosh 2009 ) , and the oecd has recommended the inclusion of health monitoring , surveillance and biological monitoring for occupational workers ( oecd 2009c ) . if the development of regulation based on quantitative risk assessment is an inherently slow governance process , then one answer might be to undertake research that leads to one or more governance frameworks that are more responsive and adaptive . this is a view that has been articulated by a number of authors , including rcep ( 2008 ) , brown ( 2009 ) and owen et al . there is a foundation for this study , including a number of alternative risk governance frameworks that have been proposed in recent years , such as those suggested by the international risk governance council ( irgc 2007 ) , environmental defense and dupont ( nano risk framework , environmental defense and dupont 2007 ) and smarten ( strategic management and assessment of risks and toxicity of engineered nanomaterials ) ( metcalfe et al . these typically not only combine some of the traditional parameters of risk assessment ( hazard identification , exposure assessment , etc . ) and risk management ( in the case of the nano risk framework ) , but may also incorporate wider aspects ( e.g. while real - time technology assessment mainly aims to assist decision - making processes by incorporating other ( societal ) factors , it is not yet clear if more timely decisions will ultimately be aided by this process . for example , the european commission s code of conduct for nanosciences and nanotechnologies research was established in 2007 ( european commission 2008a ) , providing guidance to steer the responsible development of nanotechnology at an early stage . this includes specific recommendations aimed towards enhancing communication of the risks , benefits and uncertainties in addition to the development of scientific tools and knowledge , albeit described , in general , rather than in specific terms . in short , while there are a number of potential avenues to pursue , a comprehensive programme of research in this area is currently lacking . therefore , we recommend the continued development of research on risk governance frameworks that specifically aims to strengthen the timeliness of decision making processes in the face of rapid nanotechnologies innovation . in addition to the risk governance frameworks and approaches described above , a wide variety of tools have either been developed or deployed for approximate risk evaluations or decision making about ( some ) nm risks using available information . 2007 ; furthermore , expert elicitation has been put forth to help compensate for large data gaps ( morgan 2005 ) , as well as adaptive management to help create flexible and iterative processes ( davis 2007 ; linkov et al . in general , these tools attempt to provide either an early attempt to approximate or compare some nm risks for decision support , even if it is , for example ranking the risks of different nanoparticles . while these tools and models may be valuable in many aspects , especially in terms of thinking outside the box in regard to early estimates and comparisons of nm risks and better decision - making strategies , one main limitation is that many ( if not most ) of these tools and models have not been thoroughly tested and applied to many nm , and , therefore , their robustness and applicability is not yet clear . therefore , we recommend that the developed frameworks and tools , some of which have been described here , be immediately and rigorously tested to fully evaluate their functionality and limitations . in other words , under uncertain circumstances , it is important to ensure that such decisions can be reversed at minimum cost ( economic or otherwise ) . 2009 ) to any decision support system for nm risks , whether based on prospective risk assessment or an alternate framework . this might include evaluating the toxicity of nm - containing environmental discharges ( e.g. biomonitoring may also encompass the monitoring and surveillance of occupational workers health in the light of potentially hazardous effects of nm ( nasterlack et al . in fact , the national institute of occupational health and safety ( niosh ) recently recommended hazard surveillance as a foundation to implement safety measures ( niosh 2009 ) , and the oecd has recommended the inclusion of health monitoring , surveillance and biological monitoring for occupational workers ( oecd 2009c ) . although the traditional chemical - based risk assessment framework is a powerful approach , its use for estimating the health and environmental risks of nm in the near - term may be limited due to the time and costs needed to generate meaningful results . given these limitations , decision makers may not currently be well equipped if decisions are to be based mainly upon results generated from quantitative risk assessment . despite the above , research efforts thus far have been mainly put towards acquiring knowledge that will most likely be fed into chemical - based risk assessment procedures . these efforts include a predominance of research publications and projects within the fields of ( eco)toxicology and human and environmental exposure of nm , while research in broader issues such as decision making and risk governance are comparatively minimal . research supporting quantitative risk characterization of nm is indeed important , and we do not advocate abandonment of this at any cost . however , as scientists and policy makers , we must face the reality that knowledge gaps are not expected to be closed in the near future and may take decades to close in many cases . in conclusion , we suggest the need for an international research programme that specifically addresses critical issues of risk governance and timely decision making as these relate to nm specifically , and emerging technologies more generally . this programme might include research into more efficient and anticipatory risk governance mechanisms as well as a systematic evaluation of available complementary tools to risk assessment and , where necessary , further tools under development . finally , the development of environmental and health surveillance is needed to act as a safety net and an early warning system while these issues are being addressed . we recommend that active research within these areas should be among the essential steps taken in the near future to ensure the responsible emergence of nanotechnologies , and ultimately the protection of human and environmental health .

mutations affecting nuclear genomic dna repair lead to somatic stem cell ( ssc ) dysfunction and premature aging : mouse models with genomic dna repair defects show progeroid phenotypes ( barlow et al . , 1996 ; de boer et al . , 2002 ; gu et al . , 1997 ) mtdna integrity is also required for proper stem cell function : mtdna mutator mice , which accumulate random mtdna mutations in all tissues , including sscs , develop progeria , with gray hair , alopecia , osteoporosis , general wasting , anemia , reduced fertility , and shortened lifespan ( ameur et al . , 2011 ; kujoth et al . , 2005 ; trifunovic et al . , these mice have a progressive dysfunction of hematopoietic , neural , and intestinal progenitor cells , starting early during embryogenesis ( ahlqvist et al . , 2012 ; chen et al . , 2009 ; fox et al , 2012 ; norddahl et al . , 2011 ) ; however , the first progeric symptom of these mice , anemia , manifests only after 6 months of age . the embryonal ssc defect can be rescued by treatment with n - acetyl - l - cysteine ( nac ) ( ahlqvist et al . , 2012 ) , suggesting a redox component in the pathology . these data show that hematopoietic and neural progenitors are sensitive to mtdna mutagenesis and dysfunction , but the mechanisms remain unclear . somatic stem / progenitor cell metabolism mostly relies on glycolytic activity , and mitochondrial respiration typically gradually increases upon differentiation ( suda et al . , 2011 ) . however , mitochondria are present and active in stem cells even though their contribution to the cellular atp demand is minor ( zhang et al . , 2011 ) . this downregulation of mitochondrial atp production may leave the mitochondria under conditions of high proton - motive force and a reduced respiratory chain ( rc ) , which favors mitochondrial reactive oxygen species ( ros ) production ( chouchani et al . , 2014 ; murphy , 2009 ) . ros are potent signaling molecules ; for example , they are known to drive hpcs to proliferate and to contribute to erythroid differentiation ( suda et al . , 2011 ) . the consequences of primary mitochondrial dysfunction for ssc and progenitor compartments are , however , poorly understood . in mtdna mutator mice , random amino acid changes accumulate in rc complexes , including complexes ci and ciii the major producers of mitochondrial ros . changes in ros levels have been found in some tissues in aged mutators ( logan et al . , 2014 ) , but little signs of oxidative damage were apparent ( kolesar et al . , 2014 ; trifunovic et al . , 2005 ) , indirectly suggesting that modified ros signaling , not oxidative stress , mediate ssc dysfunction and progeria in mutator mice . we report here that mtdna mutagenesis increases mitochondrial h2o2 in pluripotent stem cells ( pscs ) in vitro , resulting in modification of reprogramming , pluripotency , and stem cell homeostasis . we show that both induced pscs ( ipscs ) and sscs are especially sensitive to ros and also to antioxidants nac and mitochondria - targeted ubiquinone ( mitoq ) , which both improved stem cell function . however , mitoq also showed dose - dependent toxicity specific for neural stem cells ( nscs ) and ipscs . these data indicate that long - term treatment trials in animal models , with special focus on the stem cell compartment , are warranted when assessing safety of new antioxidants . mouse embryonic fibroblast ( mef ) cultures were established from e13.514.5 mutator and wt embryos , subsets of which were supplemented with nac or mitoq during the whole embryogenesis and post - harvest in vitro culture . the different genotypes showed no difference in proliferation , clonality , viability , or transfection efficiency , and the antioxidant treatments had no effect on these ( figure s1 ) . however , when reprogramming the mefs to pluripotency , 10-fold more colonies arose from the wts than from mutator mefs ( figure 1a ) . the decreased reprogramming efficiency of mutators was significantly improved by the two antioxidants with different mechanisms of action : nac increases cellular glutathione and consequently redox - buffer capacity of the cell , whereas mitoq , a modified ubiquinone , is targeted to accumulate within mitochondria ( kelso et al . , 2001 ) . nac increased the number of mutator colonies 4-fold , and the effect of mitoq was 10-fold , the latter rescuing the efficiency to wt level . neither of the antioxidants had a significant effect on the reprogramming of wt mefs ( figure 1a ) . culture and reprogramming in 4% oxygen improved reprogramming efficiency similarly for both genotypes ( figure 1b ) . to directly assess whether mtdna mutagenesis affected mitochondrial ros production in ipscs , we utilized a mitochondria - targeted probe mitob : arylboronic acid conjugated to triphenylphosphonium ion ( tpp ) ( cochem et al . , 2012 ) . the lipophilic tpp mediates rapid uptake of mitob into cells and accumulation in mitochondria , where mitob reacts with h2o2 and forms a phenol - derivative mitop . mitop / mitob ratio can be measured by ratiometric mass spectrometry and reflects the mitochondrial h2o2 level ( cochem et al . , 2012 ) . mutator ipscs showed an increase in the mitop / mitob ratio , consistent with an increase in mitochondrial h2o2 ( figure 1c ) . neither mtdna mutagenesis nor antioxidant treatment affected expression of the pluripotency markers in the established ipsc lines ( figures s1f and s1 g ) . the tet - inducible - promoter - driven reprogramming transgenes were silenced in all the clones analyzed ( figure s1h ) , and early passage ipsc clones exhibited normal karyotypes ( figure s1i ) . upon suspension culture and withdrawal of lif ( leukemia inhibitory factor ) , all tested clones produced embryoid bodies ( wt : n = 6 ; mut : n = 6 ) , and when injected into nude mice , all tested clones ( wt : n = 3 ; mut : n = 6 ) generated teratomas with cell types from all three germ layers present in them ( figure s1j ) . these results imply that mtdna mutagenesis increases mitochondrial h2o2 and that mitochondrial ros inhibit nuclear reprogramming , which can be rescued by antioxidants . clonal capacity , i.e. , ability of a single stem cell to generate a new stem cell clone , is considered a measure of stemness . this self - renewal defect was partially rescued by nac ( 100 m ) and low concentration of mitoq ( 10 nm ) . nac treatment had no significant effect on wt cells ( figures 1d and 1e ) . however , 100 nm mitoq the concentration successfully used during mef reprogramming instead of improving mutators , had a detrimental effect on wt clonality ( figures 1d and 1e ) , whereas nac even in 10-fold concentration ( 1 mm ) improved self - renewal of mutator ipscs and showed no adverse effects on wts ( figures 1d and 1e ) . these results indicate that mitoq has a narrow therapeutic window and can be harmful to stem cells at higher concentrations . the self - renewal ability was reflected in the embryonic stem cell marker ssea1 ( stage - specific embryonic antigen-1 ) expression : a significantly larger population of ssea1-negative cells existed in mutator ipscs . mitoq treatment dose dependently increased the proportion of ssea1-negative cells , as a possible sign of impaired stemness of the ipsc ( figures 1f and 1 g ) . our results show that mtdna mutagenesis reduces ipsc clonal ability , a measure of stemness . mitochondria - targeted mitoq also effectively rescues the defect at low concentrations but is deleterious for ipsc self - renewal with concentrations that improved fibroblast reprogramming . mutator mefs showed no difference to wt growth ( figures s1a and s1b ) , but the mutator ipscs manifested a severe growth defect , with doubling times around 24 hr for mutators and 16 hr for wt cells ( figures 2a and 2b ) . also , mutator embryoid bodies ( ebs ) grew slowly ( figure 2c ) . the proportion of proliferating cells was similar between mutator and wt cultures when analyzed by brdu incorporation and facs ( figure 2d ) , and apoptosis was not increased ( annexin v and propidium iodide staining ; facs analysis ; figure 2e ) . these results indicate that mtdna mutagenesis delays stem cell growth by a redox - independent manner without inducing cell death . the adverse effect of mitoq on ipscs at a relatively low concentration range prompted us to investigate the effect of mitoq in vivo to somatic progenitor cells . we extracted nscs from the brain and hematopoietic progenitors ( hpcs ) from the liver of e14.515.5 embryos treated either with mitoq ( 0.75 mg / ml ) or as control an equivalent dose of lipophilic decyl - tpp ( dtpp ) without ubiquinone ( 0.48 mg / ml ) . these cell types previously showed either an abnormal differentiation pattern ( hpcs ) or a self - renewal defect ( nscs ) in mutators , which was rescued by nac ( ahlqvist et al . , 2012 ) . mitoq , but not dtpp , rescued the abnormal proportions of embryonal hpcs in mutator embryos , replicating our previous results with nac ( figures 3a and 3b ) . however , neurospheres extracted from the same mitoq - treated embryos showed abnormal morphology ( figure 3c ) and an increase in apoptosis ( figure 3d ) . consequently , we were not able to establish stable neurosphere cultures from the mitoq - treated embryos . concentration - dependent toxicity was also evident when previously untreated neurosphere cultures were subjected to mitoq in vitro : 10 nm mitoq maintained normal self - renewal and viability , whereas already 50 nm mitoq induced apoptosis ( figures 3c and 3d ) and reduced the proportion of self - renewing cells ( figure 3e ) . nac treatment was not toxic to nscs even at a high concentration ( figures 3c3e ) . neuronal cells differentiated from ncss ( mixed cultures of tuj-1 neurons and gfap astrocytes with increasing mtdna copy number indicating mitochondrial maturation ; figure s2 ) tolerated higher concentrations of mitoq ( figure 3f ) . however , mitoq was still toxic to immature neuronal cultures at 250 nm a concentration that did not induce cell death in mefs or ipscs ( figures 3f , 3 g , and 1h ) . mefs tolerated antioxidants extremely well , as neither 1 m mitoq nor 2 mm nac induced apoptosis in mefs ( figure 3 g ) . these results show that antioxidants have cell - type - specific effects and suggest that mitochondria - targeted mitoq can be toxic to specific cell types with nscs being especially vulnerable . in western blot analysis , mutator ipscs showed a severe decrease of complex iv ( cytochrome c oxidase [ cox ] ) protein amount ( figure 4a ) , and complexes i and iii ( ci and ciii ) were moderately decreased , indicating a combined rc defect ( figures 4a and 4b ) . the antioxidant treatments increased these protein amounts about 2-fold , rescuing the amounts of ci ( nac - treated ) and ciii ( mitoq - treated ) and inducing cox to 20% of wt mean ( figure 4b ) . a histochemical cox activity assay indicated low activity in mutators compared to wt cells ( figure 4c ) . these results showed that antioxidants can improve rc protein amounts in stem cells with mtdna mutations , implying active ros regulation of rc protein levels . we further analyzed the cellular metabolism , both cell respiration and glycolysis , by seahorse xf analyzer . mutator ipscs showed low cellular respiration ( figure 4d ) , and when kept in ample glucose , they used their maximal glycolytic capacity ( figure 4e ) , whereas wt cells utilized both respiration and glycolysis ( figures 4d and 4e ) . when cell respiration ( oxygen consumption rate [ ocr ] ) was calculated against glycolytic activity ( extracellular acidification rate [ ecar ] ) , mutators showed a strong shift toward glycolytic metabolism ( figure 4f ) . both antioxidant treatments increased respiration in mutator ipscs and normalized the ocr / ecar ratio ( figures 4f and 4 g ) but had no significant effect on wt cells . in mutator mefs , glycolytic activity was also slightly increased , although basal respiration remained at wt level ( figures s3a s3c ) . the strong dependence of the mutator cells on glycolytic metabolism was further supported by their complete dependence on glucose : a clear reduction in growth was evident in mutators when reducing glucose concentration to one - third of the original ( 4.5 g / l ) , and complete withdrawal of glucose almost completely arrested their growth ( figure s3d ) . wt cells showed a growth defect only upon complete withdrawal of glucose ( figure s3d ) . mutator growth was also inhibited by non - fermentable carbon source , galactose , which well supported wt cell growth ( figure s3d ) . both wt and mutator cells responded to glucose addition by decreasing respiration and increasing glycolytic activity , but mutators induced anaerobic glycolysis more than wt cells ( figures s3e and s3f ) . these data show that the combined rc defect in mutator ipscs is partially ros dependent and rescued by antioxidants . the metabolism of mutator ipscs strongly favors glycolysis , whereas the antioxidant treatments switch it to the more - typical ipsc mode , using both respiration and glycolysis . these data point to a role for ros in stem cell metabolic shift , requiring a delicate balance of oxidative phosphorylation and anaerobic glycolytic activity . to clarify whether mtdna mutation loads and profiles could contribute to the observed ipsc findings , we used next - generation sequencing and sequenced the whole mitochondrial genome of the parental mefs and ipscs of early and late passages . we further differentiated the ipscs to fibroblast - like cells ( secondary fibroblasts ) and analyzed their mutation frequencies . three to six clones were sequenced per group , and the average coverage per group was 11,250 ( table s1 ; ncbi sra database ; project srp056999 ) . both mutator and wt mtdna showed similar sequencing coverage throughout mtdna ( figure 5a ) , indicating absence of large - scale mtdna deletions in mutator cells . mutation frequencies were calculated per every nucleotide position , and positions showing variation in > 1% of all mtdna copies were considered variable ( figure 5b ) . mutators showed an increase in variable mtdna sites in all cell types , and this became increasingly pronounced the longer the cells were kept in culture ( figure 2b ) , consistent with constant mutagenesis . the mutation loads in the fibroblasts were partially due to maternal inheritance from the heterozygote females , similar between mutator and wt cells , because littermates were used as wt controls . to minimize maternal contribution , we maintain mutators through male mice and use only one generation of young heterozygous females to produce the study groups . mtdna mutation load increased during reprogramming in both mutators and wts , whereas , during ipsc culture , variable sites decreased in wt and also in mutator cells ( figure 5b ) , despite continuous mutagenesis . upon differentiation of ipscs to secondary fibroblasts , variable sites in mutators increased again , evident already after 2 weeks of differentiation ( figure 5b ) . to identify whether the selection during ipsc culture was random , we analyzed how different types of mutations behaved . in wt cells , mutations affecting the rc protein - coding genes showed significant negative selection and rrna gene variants showed a similar tendency , whereas mutations affecting trna genes were not selected against ( figure 5c ) . in mutator cells , similar tendencies were seen , but the pattern did not reach significance , likely due to the strong background from the constant random generation of new mutations . these data demonstrate that ipscs show strong functional selection against mtdna mutations in genes encoding the rc complex subunits , with little selection pressure directed toward trna mutations . ros is a collective term for highly reactive molecules generated upon incomplete reduction of oxygen . they are mostly known for causing oxidative damage but are increasingly recognized to also function as important intracellular signaling molecules ( hamanaka and chandel , 2010 ; suda et al . , 2011 ) . this signaling has been suggested to operate through covalent modifications in the target proteins or through specific sensors able to detect and directly respond to intracellular changes through redox - based mechanisms ( dautraux and toledano , 2007 ; nathan , 2003 ) . the physiological ros signals , not associated with oxidative damage , are especially important for stem cell functions , promoting progenitor cell proliferation ( le belle et al . , 2011 ; suda et al . , mtdna mutator mice manifest a severe early onset ssc dysfunction , explaining the progeroid phenotype of these mice ( ahlqvist et al . , 2012 ; chen et al . , 2009 two different antioxidants , nac and mitoq , efficiently rescued the reprogramming and self - renewal defects in mutator pscs , indicating that reprogramming to stem cells , as well as stem cells themselves , are sensitive to antioxidants . nac increases cellular glutathione pool and enhances processing of h2o2 in the cytosol , whereas mitoq acts upstream , probably by sequestering or preventing superoxide production within mitochondria , before it forms h2o2 . vitamin c , a cofactor with both antioxidant and pro - oxidant properties , was found to improve reprogramming efficiency ( esteban et al . , 2010 ) , raising the question whether ros contribute to reprogramming . however , vitamin c has a multitude of different functions , with a potential role in histone demethylation as well as dna repair ( mandl et al . , 2009 ) , and therefore , the role of ros in reprogramming remained open ( qi et al . , 2015)partially also due to lack of reliable methods to detect subtle changes in ros levels . we demonstrate here , utilizing a mitochondria - targeted h2o2 reactive probe and ratiometric mass spectrometry ( cochem et al . , 2011 , 2012 ) , that mtdna mutagenesis induces a subtle increase in mitochondrial h2o2 , the rescue of which by mitoq completely restores the reprogramming defect , whereas nac rescued the defect partially . these antioxidants did not , however , improve wt reprogramming efficiency , suggesting that downregulation of mitochondrial respiration together with endogenous cellular ros scavengers were sufficient to minimize h2o2 production upon reprogramming . the antioxidants did not ameliorate the growth defect of the mutator ipscs or ebs , which was similar to that previously reported in mutator nscs ( ahlqvist et al . , 2012 ) . this growth defect may be contributed by various anabolic functions of mitochondria beyond atp production , but not by ros signaling , indicating separate signaling for growth and stemness in ipscs . our results indicate that the importance of ros signaling in stem cells makes these cells also sensitive to antioxidants , with potential dose - dependent toxicity . mitoq significantly impaired self - renewal of wt ipscs at concentrations that were well tolerated by fibroblasts or differentiated neuronal cells , as well as the cells undergoing reprogramming , suggesting that the cellular metabolism of the latter is still partially fibroblast - like . in mouse embryos in vivo , doses improving mutator hpc phenotypes severely compromised nsc viability , raising the question of mitoq safety for nscs . mitoq comprises ubiquinone , the antioxidant moiety of coenzyme - q10 , covalently linked to lipophilic tpp cation , which enables mitoq to cross membranes efficiently and to accumulate several - hundred - fold within mitochondria in response to the organelle s membrane potential ( murphy and smith , 2007 ) . different tissues have been reported to accumulate mitoq with different efficiencies ( rodriguez - cuenca et al . , 2010 ) , making dose control for a specific cell type , or its mitochondria , difficult . a range of literature reports mitoq treatment effects in many kinds of pathologies , in rodent models and also in human trials , with no reported in vivo toxicity in rodents following 4- or 5-month administration or in humans following 1 year s administration ( smith and murphy , 2010 ) . for example , mitoq delayed progression of cardiac damage and various types of neurodegenerative symptoms , including als and alzheimer s disease , in animal models ( manczak et al . , 2010 ; miquel et al . , 2014 ; yancey et al . , however , a human trial for parkinson s disease found no effect of mitoq for progression of the disease ( snow et al . , 2010 ) . in contrast , high concentrations of nac did not show toxicity , which suggests to a specific feature for mitoq and potentially for other mitochondria - accumulating antioxidants . tpp alone did not induce toxicity in nscs in vivo , indicating that the high concentrations of tpp cations in mitochondria , which may lead to non - specific disruption of mitochondrial function ( murphy and smith , 2007 ) , did not explain the toxicity of mitoq to ncss . our results show that special attention should be paid to antioxidant and redox effects on the stem cell compartment , especially when developing mitochondria - targeted antioxidants . we found that mutator ipscs actively downregulated mitochondrial respiration and showed a combined rc deficiency , which did not severely compromise their highly glycolytic energy metabolism or viability in high - glucose growth conditions . the enforced glycolysis was specific for mutator ipsc , and both mitoq and nac restored the metabolism to the bivalent wt mode , typical for mouse pscs ( zhou et al . , 2012 ) . these data indicate that , even upon rc dysfunction and mtdna mutagenesis , the metabolic shift to glycolysis and rc deficiency were partially reversible and mediated by ros signaling . we propose that a glycolytic shift in stem cells exists as a protective response to maintain sensitivity to ros signals . the antioxidant rescue of rc protein amounts , restoring complex i and complex iii to wt level , suggest that the major ros producers of rc may be actively degraded as a response to mitochondrial mutagenesis . complex i deficiency is a common finding in several mitochondrial disorders ( kirby et al . , 1999 ) , and recent evidence suggests that ci deficiency in disease can also be a physiological response to metabolic insult . in patients with mtdna maintenance disorders , also including polg defects , the degree of ci deficiency in the brain did not correlate with the severity or even presence of the brain manifestations ( palin et al . , 2013 ) . furthermore , in ipsc - derived neurons carrying a pathological mitochondrial trna mutation , ci was enriched in autophagosomes prior to development of ci deficiency ( hmlinen et al . , 2013 ) . the current findings support this degradation to be active , mediated by ros , and partially rescued by antioxidant treatment . the reprogramming process itself can contribute to mutation rates : nuclear genomic mutations arise during reprogramming and are selected against during subsequent culture ( hussein et al . , ipscs have also been reported to carry mtdna mutations ( prigione et al . , 2011 ) . whether these arise during reprogramming or represent changes that were present at low frequency in the parental cells and whether they are selected against during ipsc culture is unknown . we found that both mutator and wt cells showed an increase in mtdna mutation load during reprogramming , suggesting that , similarly to nuclear dna , also mtdna mutations arise during the reprogramming process . however , whereas the mtdna mutation loads were consistently higher in mutator cells than in wt cells , no specific mutations were present at high enough frequencies to account for the different characteristics between wt and mutator cells . the high proliferation rate of pscs makes them particularly prone to generate mitochondrial mutations by replication errors . however , during in vitro culture , the wt ipscs were able to eliminate most of their mtdna mutations and the mutation loads did not increase during mutator ipsc culture either . this was unexpected , as the exonuclease - defective mtdna polymerase should frequently generate new mutations . such selection has been described to occur in oocyte development of mutator mice , with rapid elimination of especially the non - synonymous changes in the protein - coding mitochondrial genes in the so - called mtdna bottleneck ( stewart et al . , 2008 ) . indeed , ipscs showed a similar preference to select against protein - coding gene mutations and to a lesser extent also against rrna gene variants . consistently , previous reports of human ipscs generated from patients with mtdna mutations showed that both mitochondrial trna and protein - coding gene mutations survived through reprogramming , but only trna mutations maintained stable heteroplasmy , whereas rc protein - coding gene mutation was purged upon ipsc culture ( folmes et al . these results suggest that the purifying selection of germ cells could be a general feature of stem cells . in conclusion , we demonstrate that mtdna mutations can reduce reprogramming efficiency of somatic cells and lead to stemness defects via ros - mediated signaling . antioxidants can cure these defects , and mitochondrial - targeted mitoq was very effective in rescue but also showed toxic effects . our data indicate that a therapeutic dose may vary considerably between cell types : a dose that may rescue pathology in one tissue may severely challenge function in another . our data implicate the need of dose - effect studies of antioxidants on ssc pools to establish their safety as nutritional supplements or therapeutic agents especially in the case of antioxidants accumulating into mitochondria . mice with a knockin inactivating mutation ( d257a ) in the exonuclease domain of dna polymerase gamma ( kujoth et al . , 2005 ) were used . for antioxidant supplementation , the mice were given either nac ( 1 mg / ml ; sigma - aldrich ) , mitoq ( 0.75 mg / ml ; provided by m.p.m . ) or lipophilic dtpp without ubiquinone bound to cyclodextrin ( 0.48 mg / ml ; equivalent to 0.75 mg / ml mitoq ) in the drinking water of the females throughout pregnancy . fibroblasts were extracted from treated and non - treated e13.514.5 embryos using standard protocol . for in vitro culture , the medium was supplemented with either 100 m nac or 10250 nm mitoq throughout all experiments unless otherwise stated . to analyze growth of the fibroblasts , growth curves were generated using cell - iq analyzer software ( cm technologies ) . to assess the transfection efficiency , a piggybac transposon - based gfp plasmid was electroporated together with the pcyl43 transposase using the same setting as for the reprogramming plasmids . stably transfected cells were analyzed by facs for the proportion of gfp - positive cells 1 week later . for the clonality assays , the cells were plated on clonal dilutions and the number of colonies was counted 2 weeks later . the transfections were done using neon electroporator ( invitrogen ; one pulse ; 30 ms ; 1,300 v ) with 5 g of dna and 5 10 cells per electroporation . the culture media was supplemented with 1 g / ml of doxycycline on the next day . for reprogramming in different oxygen tensions , the electroporations were split on two separate plates : one cultured in 4% o2 ( biospherix exvivo chamber ) whereas the other in normal room air of around 20% o2 . to assess the reprogramming efficiency , plates were stained on day 14 for alkaline phosphatase ( ap ) activity ( vector red alkaline phosphate substrate kit ) . for clonality assays , cells were plated on day 0 on clonal dilutions and stained on day 7 . the number of ap - positive clones was counted using imagej 1.44o software ( nih ) . the screening for transgene silencing was done by lacz staining ( woltjen et al . , 2009 , 2011 ) , and karyotyping was done commercially ( chrombios ) . the ipsc growth curves were done by plating 2 10 cells per well and counting the cell number on 4 consecutive days . dna was purified from cells by phenol - chloroform extraction , and total rna was purified using trizol reagent ( invitrogen ) . quantitative rt - pcr was performed using the cfx96 platform ( bio - rad ) and dynamo flash sybrgreen qpcr kit ( thermo scientific ) . the ipscs were trypsinized to single cells , and drops of media with 200 cells / drop were hung on petri dish covers for 3 days , after which the ebs were cultured in suspension culture for an additional week and lif was withdrawn from the eb culture medium . secondary fibroblasts were generated by withdrawing lif from the culture medium and inducing differentiation with 1 m retinoic acid for 6 days . after this , the cells were passaged and cultured for 8 additional days in fibroblast media . at this point , all cells were flat and no cells with esc - like morphology were present in the cultures . for teratoma formation , about 2 10 ipscs were injected intratesticularly into nmri nude mice ( scanbur ) . after 6 weeks , tumors were collected , fixed overnight in 4% paraformaldehyde ( pfa ) , embedded in paraffin , and stained with h&e . the experimental animal welfare committee of the district government of southern finland approved the animal experiments . primary antibodies were against ssea1 ( if : 1:250 , ab16285 , abcam ; facs : 1:100 , 90230 , millipore ) , nanog ( 1:250 ; ab80892 ; abcam ) , tuj-1 ( 1:500 ; nordic biosite ) , and gfap ( 1:1,000 ; chemicon ) . flow cytometry was run using bd accuri c6 flow cytometer and analyzed using the accuri c6 analysis software . to determine the proliferation rate , ipscs were incubated in 10 m brdu ( bd pharmingen ) for 15 min , fixed with ethanol , denatured with 1 m hcl , and permeabilized using 0.5% triton x-100 . cells were stained using monoclonal anti - brdu antibody ( 1:4 ; becton dickinson ) . cells were run using bd accuri c6 flow cytometer and analyzed using the accuri c6 analysis software . apoptosis was analyzed by annexin v - fitch apoptosis detection kit ( bd pharmingen ) according to the manufacturer s instructions . a mitochondria - targeted ratiometric mass spectrometry probe mitob was used to measure hydrogen peroxide in the mitochondrial matrix as published earlier ( cochem et al . , 2011 , 2012 ) . briefly , the cells were incubated with 5 m mitob together with 50 u / ml catalase for 6 hr , after which media was collected , snap frozen , and processed for mass spectrometry . isolation and analysis of nscs and hscs were performed as previously described ( ahlqvist et al . , 2012 ; primary antibodies were against cox1 ( 1:500 ; m404 ; mitosciences ) , ci - ndufa9 ( 1:2,000 ; ab14713 ; abcam ) , cii-70kd ( 1:10,000 ; ms204 ; mitosciences ) , ciii - uqcrc2 ( 1:2,500 ; ab14745 ; abcam ) , cv - a ( 1:1,000 ; ms507 ; mitosciences ) , b - actin ( 1:2,000 ; sc1616 ; santa cruz biotechnology ) , and tom20 ( 1:500 ; sc11415 ; santa cruz biotechnology ) . ipscs were grown on gelatin in chamber glasses and washed three times with pbs + mg + ca . the cells were incubated in preincubation medium ( 10% sucrose and 0.03% cobalt chloride in 50 mm tris - hcl [ ph 7.6 ] ) for 15 min at rt . cells were washed with sodium phosphate + sucrose , followed by incubation with cytochrome c , dab hydrochloride , and catalase at + 37c for 1 hr . cell metabolism was analyzed by seahorse xf96 extracellular flux platform using the xf cell mito stress test and xf glycolysis stress test kits ( seahorse bioscience ) . data were normalized against total protein amounts per well ( analyzed by bradford assay ) and analyzed using wave2.0 software ( seahorse bioscience ) . mtdna was amplified from the genomic dna using repli - g mitochondrial dna kit ( qiagen ) . after enrichment , the amplified mtdna samples were processed to illumina - sequencing - compatible libraries with nextera dna sample preparation kit ( illumina ) . the reaction volume in nextera tagmentation and amplification steps was 20 l , and after both steps , the libraries were purified with edgebio performa v3 96-well short plate ( edge biosystems ) . after amplification , the libraries were incubated with 4 l of edgebio sope resin and purified with edgebio performa plates . after purification , the samples with different index tags were pooled together ( 2 l each ) and concentrated with dna clean & concentrator-5 ( zymo research ) . the sequencing - ready libraries were quantitated with agilent 2100 bioanalyzer high sensitivity kit ( agilent ) . statistical analysis was performed when each group had at least three samples with unpaired t test ( graphpad prism software ) . p < 0.05 was considered significant ; p < 0.05 ; p < 0.005 ; p

summarymtdna mutagenesis in somatic stem cells leads to their dysfunction and to progeria in mouse . the mechanism was proposed to involve modification of reactive oxygen species ( ros)/redox signaling . we studied the effect of mtdna mutagenesis on reprogramming and stemness of pluripotent stem cells ( pscs ) and show that pscs select against specific mtdna mutations , mimicking germline and promoting mtdna integrity despite their glycolytic metabolism . furthermore , mtdna mutagenesis is associated with an increase in mitochondrial h2o2 , reduced psc reprogramming efficiency , and self - renewal . mitochondria - targeted ubiquinone , mitoq , and n - acetyl - l - cysteine efficiently rescued these defects , indicating that both reprogramming efficiency and stemness are modified by mitochondrial ros . the redox sensitivity , however , rendered pscs and especially neural stem cells sensitive to mitoq toxicity . our results imply that stem cell compartment warrants special attention when the safety of new antioxidants is assessed and point to an essential role for mitochondrial redox signaling in maintaining normal stem cell function .

Introduction Results Discussion Experimental Procedures Author Contributions

mutations affecting nuclear genomic dna repair lead to somatic stem cell ( ssc ) dysfunction and premature aging : mouse models with genomic dna repair defects show progeroid phenotypes ( barlow et al . , 1997 ) mtdna integrity is also required for proper stem cell function : mtdna mutator mice , which accumulate random mtdna mutations in all tissues , including sscs , develop progeria , with gray hair , alopecia , osteoporosis , general wasting , anemia , reduced fertility , and shortened lifespan ( ameur et al . the embryonal ssc defect can be rescued by treatment with n - acetyl - l - cysteine ( nac ) ( ahlqvist et al . these data show that hematopoietic and neural progenitors are sensitive to mtdna mutagenesis and dysfunction , but the mechanisms remain unclear . somatic stem / progenitor cell metabolism mostly relies on glycolytic activity , and mitochondrial respiration typically gradually increases upon differentiation ( suda et al . however , mitochondria are present and active in stem cells even though their contribution to the cellular atp demand is minor ( zhang et al . this downregulation of mitochondrial atp production may leave the mitochondria under conditions of high proton - motive force and a reduced respiratory chain ( rc ) , which favors mitochondrial reactive oxygen species ( ros ) production ( chouchani et al . the consequences of primary mitochondrial dysfunction for ssc and progenitor compartments are , however , poorly understood . in mtdna mutator mice , random amino acid changes accumulate in rc complexes , including complexes ci and ciii the major producers of mitochondrial ros . , 2005 ) , indirectly suggesting that modified ros signaling , not oxidative stress , mediate ssc dysfunction and progeria in mutator mice . we report here that mtdna mutagenesis increases mitochondrial h2o2 in pluripotent stem cells ( pscs ) in vitro , resulting in modification of reprogramming , pluripotency , and stem cell homeostasis . we show that both induced pscs ( ipscs ) and sscs are especially sensitive to ros and also to antioxidants nac and mitochondria - targeted ubiquinone ( mitoq ) , which both improved stem cell function . however , mitoq also showed dose - dependent toxicity specific for neural stem cells ( nscs ) and ipscs . these data indicate that long - term treatment trials in animal models , with special focus on the stem cell compartment , are warranted when assessing safety of new antioxidants . the different genotypes showed no difference in proliferation , clonality , viability , or transfection efficiency , and the antioxidant treatments had no effect on these ( figure s1 ) . the decreased reprogramming efficiency of mutators was significantly improved by the two antioxidants with different mechanisms of action : nac increases cellular glutathione and consequently redox - buffer capacity of the cell , whereas mitoq , a modified ubiquinone , is targeted to accumulate within mitochondria ( kelso et al . nac increased the number of mutator colonies 4-fold , and the effect of mitoq was 10-fold , the latter rescuing the efficiency to wt level . to directly assess whether mtdna mutagenesis affected mitochondrial ros production in ipscs , we utilized a mitochondria - targeted probe mitob : arylboronic acid conjugated to triphenylphosphonium ion ( tpp ) ( cochem et al . mitop / mitob ratio can be measured by ratiometric mass spectrometry and reflects the mitochondrial h2o2 level ( cochem et al . mutator ipscs showed an increase in the mitop / mitob ratio , consistent with an increase in mitochondrial h2o2 ( figure 1c ) . neither mtdna mutagenesis nor antioxidant treatment affected expression of the pluripotency markers in the established ipsc lines ( figures s1f and s1 g ) . the tet - inducible - promoter - driven reprogramming transgenes were silenced in all the clones analyzed ( figure s1h ) , and early passage ipsc clones exhibited normal karyotypes ( figure s1i ) . these results imply that mtdna mutagenesis increases mitochondrial h2o2 and that mitochondrial ros inhibit nuclear reprogramming , which can be rescued by antioxidants . this self - renewal defect was partially rescued by nac ( 100 m ) and low concentration of mitoq ( 10 nm ) . nac treatment had no significant effect on wt cells ( figures 1d and 1e ) . however , 100 nm mitoq the concentration successfully used during mef reprogramming instead of improving mutators , had a detrimental effect on wt clonality ( figures 1d and 1e ) , whereas nac even in 10-fold concentration ( 1 mm ) improved self - renewal of mutator ipscs and showed no adverse effects on wts ( figures 1d and 1e ) . the self - renewal ability was reflected in the embryonic stem cell marker ssea1 ( stage - specific embryonic antigen-1 ) expression : a significantly larger population of ssea1-negative cells existed in mutator ipscs . our results show that mtdna mutagenesis reduces ipsc clonal ability , a measure of stemness . mitochondria - targeted mitoq also effectively rescues the defect at low concentrations but is deleterious for ipsc self - renewal with concentrations that improved fibroblast reprogramming . mutator mefs showed no difference to wt growth ( figures s1a and s1b ) , but the mutator ipscs manifested a severe growth defect , with doubling times around 24 hr for mutators and 16 hr for wt cells ( figures 2a and 2b ) . the proportion of proliferating cells was similar between mutator and wt cultures when analyzed by brdu incorporation and facs ( figure 2d ) , and apoptosis was not increased ( annexin v and propidium iodide staining ; facs analysis ; figure 2e ) . these results indicate that mtdna mutagenesis delays stem cell growth by a redox - independent manner without inducing cell death . the adverse effect of mitoq on ipscs at a relatively low concentration range prompted us to investigate the effect of mitoq in vivo to somatic progenitor cells . these cell types previously showed either an abnormal differentiation pattern ( hpcs ) or a self - renewal defect ( nscs ) in mutators , which was rescued by nac ( ahlqvist et al . however , neurospheres extracted from the same mitoq - treated embryos showed abnormal morphology ( figure 3c ) and an increase in apoptosis ( figure 3d ) . concentration - dependent toxicity was also evident when previously untreated neurosphere cultures were subjected to mitoq in vitro : 10 nm mitoq maintained normal self - renewal and viability , whereas already 50 nm mitoq induced apoptosis ( figures 3c and 3d ) and reduced the proportion of self - renewing cells ( figure 3e ) . however , mitoq was still toxic to immature neuronal cultures at 250 nm a concentration that did not induce cell death in mefs or ipscs ( figures 3f , 3 g , and 1h ) . these results show that antioxidants have cell - type - specific effects and suggest that mitochondria - targeted mitoq can be toxic to specific cell types with nscs being especially vulnerable . in western blot analysis , mutator ipscs showed a severe decrease of complex iv ( cytochrome c oxidase [ cox ] ) protein amount ( figure 4a ) , and complexes i and iii ( ci and ciii ) were moderately decreased , indicating a combined rc defect ( figures 4a and 4b ) . a histochemical cox activity assay indicated low activity in mutators compared to wt cells ( figure 4c ) . these results showed that antioxidants can improve rc protein amounts in stem cells with mtdna mutations , implying active ros regulation of rc protein levels . when cell respiration ( oxygen consumption rate [ ocr ] ) was calculated against glycolytic activity ( extracellular acidification rate [ ecar ] ) , mutators showed a strong shift toward glycolytic metabolism ( figure 4f ) . the strong dependence of the mutator cells on glycolytic metabolism was further supported by their complete dependence on glucose : a clear reduction in growth was evident in mutators when reducing glucose concentration to one - third of the original ( 4.5 g / l ) , and complete withdrawal of glucose almost completely arrested their growth ( figure s3d ) . both wt and mutator cells responded to glucose addition by decreasing respiration and increasing glycolytic activity , but mutators induced anaerobic glycolysis more than wt cells ( figures s3e and s3f ) . these data point to a role for ros in stem cell metabolic shift , requiring a delicate balance of oxidative phosphorylation and anaerobic glycolytic activity . we further differentiated the ipscs to fibroblast - like cells ( secondary fibroblasts ) and analyzed their mutation frequencies . three to six clones were sequenced per group , and the average coverage per group was 11,250 ( table s1 ; ncbi sra database ; project srp056999 ) . mutators showed an increase in variable mtdna sites in all cell types , and this became increasingly pronounced the longer the cells were kept in culture ( figure 2b ) , consistent with constant mutagenesis . mtdna mutation load increased during reprogramming in both mutators and wts , whereas , during ipsc culture , variable sites decreased in wt and also in mutator cells ( figure 5b ) , despite continuous mutagenesis . in mutator cells , similar tendencies were seen , but the pattern did not reach significance , likely due to the strong background from the constant random generation of new mutations . the physiological ros signals , not associated with oxidative damage , are especially important for stem cell functions , promoting progenitor cell proliferation ( le belle et al . , mtdna mutator mice manifest a severe early onset ssc dysfunction , explaining the progeroid phenotype of these mice ( ahlqvist et al . , 2009 two different antioxidants , nac and mitoq , efficiently rescued the reprogramming and self - renewal defects in mutator pscs , indicating that reprogramming to stem cells , as well as stem cells themselves , are sensitive to antioxidants . however , vitamin c has a multitude of different functions , with a potential role in histone demethylation as well as dna repair ( mandl et al . , 2009 ) , and therefore , the role of ros in reprogramming remained open ( qi et al . we demonstrate here , utilizing a mitochondria - targeted h2o2 reactive probe and ratiometric mass spectrometry ( cochem et al . , 2011 , 2012 ) , that mtdna mutagenesis induces a subtle increase in mitochondrial h2o2 , the rescue of which by mitoq completely restores the reprogramming defect , whereas nac rescued the defect partially . these antioxidants did not , however , improve wt reprogramming efficiency , suggesting that downregulation of mitochondrial respiration together with endogenous cellular ros scavengers were sufficient to minimize h2o2 production upon reprogramming . this growth defect may be contributed by various anabolic functions of mitochondria beyond atp production , but not by ros signaling , indicating separate signaling for growth and stemness in ipscs . our results indicate that the importance of ros signaling in stem cells makes these cells also sensitive to antioxidants , with potential dose - dependent toxicity . mitoq significantly impaired self - renewal of wt ipscs at concentrations that were well tolerated by fibroblasts or differentiated neuronal cells , as well as the cells undergoing reprogramming , suggesting that the cellular metabolism of the latter is still partially fibroblast - like . mitoq comprises ubiquinone , the antioxidant moiety of coenzyme - q10 , covalently linked to lipophilic tpp cation , which enables mitoq to cross membranes efficiently and to accumulate several - hundred - fold within mitochondria in response to the organelle s membrane potential ( murphy and smith , 2007 ) . , however , a human trial for parkinson s disease found no effect of mitoq for progression of the disease ( snow et al . in contrast , high concentrations of nac did not show toxicity , which suggests to a specific feature for mitoq and potentially for other mitochondria - accumulating antioxidants . tpp alone did not induce toxicity in nscs in vivo , indicating that the high concentrations of tpp cations in mitochondria , which may lead to non - specific disruption of mitochondrial function ( murphy and smith , 2007 ) , did not explain the toxicity of mitoq to ncss . our results show that special attention should be paid to antioxidant and redox effects on the stem cell compartment , especially when developing mitochondria - targeted antioxidants . the enforced glycolysis was specific for mutator ipsc , and both mitoq and nac restored the metabolism to the bivalent wt mode , typical for mouse pscs ( zhou et al . these data indicate that , even upon rc dysfunction and mtdna mutagenesis , the metabolic shift to glycolysis and rc deficiency were partially reversible and mediated by ros signaling . we propose that a glycolytic shift in stem cells exists as a protective response to maintain sensitivity to ros signals . in patients with mtdna maintenance disorders , also including polg defects , the degree of ci deficiency in the brain did not correlate with the severity or even presence of the brain manifestations ( palin et al . furthermore , in ipsc - derived neurons carrying a pathological mitochondrial trna mutation , ci was enriched in autophagosomes prior to development of ci deficiency ( hmlinen et al . the reprogramming process itself can contribute to mutation rates : nuclear genomic mutations arise during reprogramming and are selected against during subsequent culture ( hussein et al . we found that both mutator and wt cells showed an increase in mtdna mutation load during reprogramming , suggesting that , similarly to nuclear dna , also mtdna mutations arise during the reprogramming process . however , whereas the mtdna mutation loads were consistently higher in mutator cells than in wt cells , no specific mutations were present at high enough frequencies to account for the different characteristics between wt and mutator cells . however , during in vitro culture , the wt ipscs were able to eliminate most of their mtdna mutations and the mutation loads did not increase during mutator ipsc culture either . indeed , ipscs showed a similar preference to select against protein - coding gene mutations and to a lesser extent also against rrna gene variants . consistently , previous reports of human ipscs generated from patients with mtdna mutations showed that both mitochondrial trna and protein - coding gene mutations survived through reprogramming , but only trna mutations maintained stable heteroplasmy , whereas rc protein - coding gene mutation was purged upon ipsc culture ( folmes et al . these results suggest that the purifying selection of germ cells could be a general feature of stem cells . in conclusion , we demonstrate that mtdna mutations can reduce reprogramming efficiency of somatic cells and lead to stemness defects via ros - mediated signaling . antioxidants can cure these defects , and mitochondrial - targeted mitoq was very effective in rescue but also showed toxic effects . for antioxidant supplementation , the mice were given either nac ( 1 mg / ml ; sigma - aldrich ) , mitoq ( 0.75 mg / ml ; provided by m.p.m . ) to assess the transfection efficiency , a piggybac transposon - based gfp plasmid was electroporated together with the pcyl43 transposase using the same setting as for the reprogramming plasmids . to assess the reprogramming efficiency , plates were stained on day 14 for alkaline phosphatase ( ap ) activity ( vector red alkaline phosphate substrate kit ) . , 2009 , 2011 ) , and karyotyping was done commercially ( chrombios ) . dna was purified from cells by phenol - chloroform extraction , and total rna was purified using trizol reagent ( invitrogen ) . quantitative rt - pcr was performed using the cfx96 platform ( bio - rad ) and dynamo flash sybrgreen qpcr kit ( thermo scientific ) . the ipscs were trypsinized to single cells , and drops of media with 200 cells / drop were hung on petri dish covers for 3 days , after which the ebs were cultured in suspension culture for an additional week and lif was withdrawn from the eb culture medium . after 6 weeks , tumors were collected , fixed overnight in 4% paraformaldehyde ( pfa ) , embedded in paraffin , and stained with h&e . primary antibodies were against ssea1 ( if : 1:250 , ab16285 , abcam ; facs : 1:100 , 90230 , millipore ) , nanog ( 1:250 ; ab80892 ; abcam ) , tuj-1 ( 1:500 ; nordic biosite ) , and gfap ( 1:1,000 ; chemicon ) . to determine the proliferation rate , ipscs were incubated in 10 m brdu ( bd pharmingen ) for 15 min , fixed with ethanol , denatured with 1 m hcl , and permeabilized using 0.5% triton x-100 . a mitochondria - targeted ratiometric mass spectrometry probe mitob was used to measure hydrogen peroxide in the mitochondrial matrix as published earlier ( cochem et al . , 2012 ; primary antibodies were against cox1 ( 1:500 ; m404 ; mitosciences ) , ci - ndufa9 ( 1:2,000 ; ab14713 ; abcam ) , cii-70kd ( 1:10,000 ; ms204 ; mitosciences ) , ciii - uqcrc2 ( 1:2,500 ; ab14745 ; abcam ) , cv - a ( 1:1,000 ; ms507 ; mitosciences ) , b - actin ( 1:2,000 ; sc1616 ; santa cruz biotechnology ) , and tom20 ( 1:500 ; sc11415 ; santa cruz biotechnology ) . cells were washed with sodium phosphate + sucrose , followed by incubation with cytochrome c , dab hydrochloride , and catalase at + 37c for 1 hr . data were normalized against total protein amounts per well ( analyzed by bradford assay ) and analyzed using wave2.0 software ( seahorse bioscience ) . the reaction volume in nextera tagmentation and amplification steps was 20 l , and after both steps , the libraries were purified with edgebio performa v3 96-well short plate ( edge biosystems ) .

intracranial hemorrhage ( ich ) is an infrequent , but serious , complication subsequent to acute myocardial infarction ( ami ) , which is associated both with great disability and a high risk of death.1 , 2 , 3 it has a high risk of recurrence and it is therefore important to be able to accurately assess hemorrhageassociated risk factors to reduce the risk of new and recurrent events.1 , 4 , 5 , 6 , 7 , 8 , 9 during the last decade , the inpatient treatment of myocardial infarction ( mi ) has changed drastically . in many countries , there has been a shift in approach from thrombolysis to primary percutaneous coronary intervention ( pci ) , an increase in the use of parenteral anticoagulants , and moreaggressive treatment of elderly patients . these changes have affected the shortterm occurrence of ich.1 , 3 , 5 , 7 , 8 , 10 , 11 , 12 however , data remain limited regarding any change in the postdischarge risk of ich over time and long term . furthermore , previous data originate primarily from clinical trials with patient populations who have a fairly modest risk profile.13 , 14 , 15 there are discrepancies among the predictors of ich determined by earlier studies , presumably attributable to differences in the reperfusion methods used ( fibrinolysis vs primary pci ) , in patient selection ( clinical trials vs registry or communitybased studies ) , and in the duration of followup . however , advanced age , previous ischemic or hemorrhagic stroke , hypertension , female sex , lower weight , diabetes , atrial fibrillation , and renal failure are factors that have been associated with increased risk.1 , 8 , 16 , 17 the subgroup of patients who have had a previous ischemic stroke is fairly large among unselected ami populations . whether or not a previous ischemic stroke is associated with an increased risk of ich when dual antiplatelet therapy is used is , however , not known . furthermore , conflicting data exist regarding the potential association between lipid levels and statin treatment and risk of ich.18 , 19 , 20 , 21 the present study had 3 aims . first , we investigated the incidence , time trends , and predictors of ich within 1 year of discharge after ami in a large , fairly unselected population . second , we investigated the 1year risk of ich after ami compared to a reference group . third , we studied the impact of a previous ischemic stroke on ich risk in patients who received different antithrombotic therapies . patient data were obtained from the swedish register of information and knowledge about swedish heart intensive care admissions ( rikshia ) , which has now become a part of the swedish websystem for enhancement and development of evidencebased care in heart disease evaluated according to recommended therapies ( swedeheart ) . detailed descriptions of these registries have previously been published.22 , 23 rikshia is a national registry including all patients with an acute coronary syndrome ( acs ) who were admitted to a swedish hospital coronary care unit ( ccu ) . , coverage increased to 58 hospitals , and by 2007 , 74 of 77 hospitals were participating , covering almost 100% of swedish ccu admissions ( detailed information and the complete protocol are available online at http://www.ucr.uu.se ) . all participating hospitals use standardized and identical criteria for defining ami.24 , 25 patient data are collected on case record forms that include over 100 variables , including information about patient characteristics , diagnosis , medication , and procedures that were recorded upon admission , during hospital stay , and at discharge . the validity of the data entered is evaluated annually and 94% to 7% conformity between rikshia records and patient records has been demonstrated . to identify cases in which ich occurred after ami , the rikshia database was amalgamated with the swedish national patient register ( npr ) . the npr has complete national coverage since 1987 and it contains diagnoses at discharge for all hospital stays in sweden . a full description of the registry has been published previously.26 diagnoses are recorded in the npr using codes defined by the swedish international classification of disease ( icd ) system , which was adapted from the world health organization icd classification system . ich cases were identified in the registry by searches for the following codes from the 10th revision of the icd : subarachnoid hemorrhage ( i60 ) ; intracerebral hemorrhage ( i61 ) ; and other ich ( i62 ) . the npr lacks information about the principal diagnosis for 0.5% to 0.9% of somatic carerelated hospitalizations . a diagnosis of stroke or transient ischemic attack ( tia ) in the npr was determined to be correct in 98.6% of the cases , and the proportion of stroke events identified ranges from 84.2% to 98% , as shown by validation studies.26 , 27 no published studies have specifically validated the diagnosis of ich in the npr ; however , the validity is expected to be at least as good as that of the diagnosis of ischemic stroke , because the diagnosis of ich can only be made after a positive computed tomography scan , magnetic resonance tomography scan , or at autopsy . a total of 202 366 patients were added to the rikshia registry between 1998 and 2010 . to study ich predictors and time trends after discharge , we excluded all patients who had died or who had an ich during their hospital stay , resulting in a set of 187 671 patients with an ich diagnosis and followup through december 31 , 2010 . after exclusion of patients with extreme values ( creatinine < 30 or over > 1500 mol / l , age < 18 years ) , a database of 187 386 patients remained . to compare the incidence of ich in the study group with that in a group representative of the general swedish population the reference subjects were sampled with a similar yearly distribution from 1998 to 2009 as the ami cases and were linked to the npr using the same algorithm as that used for the study population . ami patients were matched with references by both age ( exact in years ) and sex , resulting in 147 475 matched pairs . all patients were informed of their participation before data were entered in rikshia and they could request to be excluded . the national board of health and welfare and the swedish data inspection board approved the registry . data for patients with and without ich were summarized as means or percentages and were tested for differences using a t test or chisquare test , as appropriate . in order to study trends over time for each of the different variables , the study period was divided into 6 time periods preceding the analyses , as follows : 19982000 ; 20012002 ; 20032004 ; 20052006 ; 20072008 ; and 20092010 . meier for estimating survival functions was used to estimate the cumulative incidence , hereafter called cumulative incidence . for group comparisons , cumulative incidence of ich within 1year postdischarge was calculated for each subperiod , and these incidences were then used to study changes over time . the same technique was used to assess the cumulative incidence of ich in the reference population and compare incidence between the cases and references . cox proportional hazards model regression analysis was used to assess uni and multivariate predictors of risk . additional candidate variables were selected based on whether they predicted a significantly increased or decreased risk in our univariate cox analysis . collection of some variables ( ie , body mass index [ bmi ] , blood pressure , serum lipids , and kidney function ) was not compulsory during the first years of the rikshia . therefore , to obtain a fairly recent and complete data set , we did not include data before 2003 in the regression analyses . of 111 749 patients , 107 431 were included in the final multivariate model that included variables with > 90% valid cases . we used the same cox model to investigate whether a previous ischemic stroke affected risk for ich in subgroups with different antithrombotic treatment regimes . the proportionality assumption for appropriate use of cox proportional hazards regression was examined using a time varying interaction . statistical analyses were performed using spss ( version 22.0 ; ibm corp , armonk , ny ) and sas software ( version 9.4 ; sas institute inc , cary , nc ) . a p value less than 0.05 was considered significant . patient data were obtained from the swedish register of information and knowledge about swedish heart intensive care admissions ( rikshia ) , which has now become a part of the swedish websystem for enhancement and development of evidencebased care in heart disease evaluated according to recommended therapies ( swedeheart ) . detailed descriptions of these registries have previously been published.22 , 23 rikshia is a national registry including all patients with an acute coronary syndrome ( acs ) who were admitted to a swedish hospital coronary care unit ( ccu ) . , coverage increased to 58 hospitals , and by 2007 , 74 of 77 hospitals were participating , covering almost 100% of swedish ccu admissions ( detailed information and the complete protocol are available online at http://www.ucr.uu.se ) . all participating hospitals use standardized and identical criteria for defining ami.24 , 25 patient data are collected on case record forms that include over 100 variables , including information about patient characteristics , diagnosis , medication , and procedures that were recorded upon admission , during hospital stay , and at discharge . the validity of the data entered is evaluated annually and 94% to 7% conformity between rikshia records and patient records has been demonstrated . to identify cases in which ich occurred after ami , the rikshia database was amalgamated with the swedish national patient register ( npr ) . the npr has complete national coverage since 1987 and it contains diagnoses at discharge for all hospital stays in sweden . a full description of the registry has been published previously.26 diagnoses are recorded in the npr using codes defined by the swedish international classification of disease ( icd ) system , which was adapted from the world health organization icd classification system . ich cases were identified in the registry by searches for the following codes from the 10th revision of the icd : subarachnoid hemorrhage ( i60 ) ; intracerebral hemorrhage ( i61 ) ; and other ich ( i62 ) . the npr lacks information about the principal diagnosis for 0.5% to 0.9% of somatic carerelated hospitalizations . a diagnosis of stroke or transient ischemic attack ( tia ) in the npr was determined to be correct in 98.6% of the cases , and the proportion of stroke events identified ranges from 84.2% to 98% , as shown by validation studies.26 , 27 no published studies have specifically validated the diagnosis of ich in the npr ; however , the validity is expected to be at least as good as that of the diagnosis of ischemic stroke , because the diagnosis of ich can only be made after a positive computed tomography scan , magnetic resonance tomography scan , or at autopsy . a total of 202 366 patients were added to the rikshia registry between 1998 and 2010 . to study ich predictors and time trends after discharge , we excluded all patients who had died or who had an ich during their hospital stay , resulting in a set of 187 671 patients with an ich diagnosis and followup through december 31 , 2010 . after exclusion of patients with extreme values ( creatinine < 30 or over > 1500 mol / l , age < 18 years ) , a database of 187 386 patients remained . to compare the incidence of ich in the study group with that in a group representative of the general swedish population the reference subjects were sampled with a similar yearly distribution from 1998 to 2009 as the ami cases and were linked to the npr using the same algorithm as that used for the study population . ami patients were matched with references by both age ( exact in years ) and sex , resulting in 147 475 matched pairs . all patients were informed of their participation before data were entered in rikshia and they could request to be excluded . the national board of health and welfare and the swedish data inspection board approved the registry . data for patients with and without ich were summarized as means or percentages and were tested for differences using a t test or chisquare test , as appropriate . in order to study trends over time for each of the different variables , the study period was divided into 6 time periods preceding the analyses , as follows : 19982000 ; 20012002 ; 20032004 ; 20052006 ; 20072008 ; and 20092010 . meier for estimating survival functions was used to estimate the cumulative incidence , hereafter called cumulative incidence . for group comparisons , cumulative incidence of ich within 1year postdischarge was calculated for each subperiod , and these incidences were then used to study changes over time . the same technique was used to assess the cumulative incidence of ich in the reference population and compare incidence between the cases and references . cox proportional hazards model regression analysis was used to assess uni and multivariate predictors of risk . for our multivariate model , we used previously established predictors of hemorrhagic stroke risk . additional candidate variables were selected based on whether they predicted a significantly increased or decreased risk in our univariate cox analysis . collection of some variables ( ie , body mass index [ bmi ] , blood pressure , serum lipids , and kidney function ) was not compulsory during the first years of the rikshia . therefore , to obtain a fairly recent and complete data set , we did not include data before 2003 in the regression analyses . of 111 749 patients , 107 431 were included in the final multivariate model that included variables with > 90% valid cases . we used the same cox model to investigate whether a previous ischemic stroke affected risk for ich in subgroups with different antithrombotic treatment regimes . the proportionality assumption for appropriate use of cox proportional hazards regression was examined using a time varying interaction . statistical analyses were performed using spss ( version 22.0 ; ibm corp , armonk , ny ) and sas software ( version 9.4 ; sas institute inc , cary , nc ) . an overview of the different patient groups and study periods is depicted in figure 1 . ami indicates acute myocardial infarction ; dapt , dual antiplatelet therapy ; ich , intracranial hemorrhage ; rikshia , swedish register of information and knowledge about swedish heart intensive care admissions . the baseline characteristics of the 187 386 patients who were discharged after ami are shown in table 1 , grouped by subsequent occurrence of ich . a total of 590 patients suffered an ich within 1 year postdischarge ( 0.32% ; 95% confidence interval [ ci ] , 0.300.34 ) . according to kaplan meier analysis , this corresponds to a cumulative incidence of 0.35% ( 95% ci , 0.320.38 ) . mean age in the matched groups of ami patients and reference subjects was 71.9 years . the patients with an ich were significantly older ( mean of 75 vs 70 years ; p<0.001 ) , with increased history of previous hemorrhagic stroke ( 7.5% vs 1.2% ; p<0.001 ) and ischemic stroke ( 17.7% vs 7.9% ; p<0.001 ) . the patients in the ich group also had a higher prevalence of diabetes ( 24.9% vs 19.4% ; p<0.001 ) , atrial fibrillation ( 22.1% vs 12.9% ; p<0.001 ) , and previous cardiac failure ( 16.3% vs 10.7% ; p<0.001 ) . in addition , ich patients also had significantly lower weight ( 75 vs 78 kg ; p<0.001 ) . sex ratio , smoking history , and systolic blood pressure did not differ significantly between groups . baseline characteristics of 187 386 patients with ami who were discharged between 1998 and 2010 , separated according to the occurrence of ich within 1 year of discharge ace indicates angiotensin converting enzyme ; ami , acute myocardial infarction ; arb , angiotensin receptor blocker ; ich , intracranial hemorrhage ; n indicates number of cases ; nstemi , nonstelevation myocardial infarction ; stemi , stelevation myocardial infarction . the baseline characteristics of the population , divided into the 6 consecutive time periods , are shown in table s1 . table 2 shows medications being taken at discharge , separated by the 6 consecutive time periods from 1998 to 2010 . over time , there was a rapid increase in the use of p2y12 inhibitors , in most cases clopidogrel in addition to aspirin . the use of statins , angiotensin converting enzyme ( ace ) inhibitor / angiotensin receptor blocker ( arb ) , and betablockers also increased over the time periods studied . medication at discharge , stratified by time period and occurrence of ich within 1 year of discharge ace indicates angiotensin converting enzyme ; arb , angiotensin receptor blocker ; ich , intracranial hemorrhage ; n , number of cases . meier analysis , did not change significantly over time ( figure 2 ) : 0.29% from 1998 to 2000 ; 0.36% from 2001 to 2002 ; 0.39% from 2003 to 2004 ; 0.32% from 2005 to 2006 ; 0.37% from 2007 to 2008 ; and 0.37% from 2009 to 2010 ( p=0.2 ) . meier , without ich 1 year after hospital discharge after ami , stratified by time period . the 1year cumulative incidence of ich in the ami patients and matched reference subjects was 0.39% and 0.18% ( p<0.001 ) , respectively ( figure 3 ) . the corresponding incidences at 3 years were 0.92% and 0.57% ( p<0.001 ) and at 5 years were 1.56% and 0.95% , ( p<0.001 ) , respectively . meier , without ich 1 year after hospital discharge after ami , compared with a matched reference group . ami indicates acute myocardial infarction ; ich , intracranial hemorrhage . the univariate analysis of predictors is presented in table s2 . moreadvanced age ( hazard ratio [ hr ] , 1.14 ; 95% ci , 1.131.15 ; p<0.001 ) , for a 5 year change , previous ischemic stroke ( hr , 1.52 ; 95% ci , 1.112.08 ; p=0.010 ) , or hemorrhagic stroke ( hr , 3.58 ; 95% ci , 2.225.80 ; p<0.001 ) , and estimated glomerular filtration rate ( egfr ) less than 60 ml / min per 1.73 m ( hr , 1.28 ; 95% ci , 1.001.63 ; p=0.049 ) were significantly associated with an increased 1year risk of ich in the adjusted analysis , whereas female sex ( hr , 0.78 ; 95% ci , 0.620.98 ; p=0.030 ) was associated with a decreased risk . previous hypertension was also found to be of some importance ( hr , 1.31 ; 95% ci , 1.101.56 ; p=0.058 ) . predictors of ich after ami in a multivariate cox regression model , 1year followup ace indicates angiotensin converting enzyme ; ami , acute myocardial infarction ; arb , angiotensin receptor blocker ; gfr , glomerular filtration rate ; ich , intracranial hemorrhage ; stemi , st elevation myocardial infarction . none of the medications was significantly associated with a change in the risk of ich . table 4 shows the unadjusted occurrence of ich correlated to different antithrombotic treatments ( 520 events , with 70 events that occurred in patients without treatment ) , stratified by previous occurrence of ischemic stroke . in the analyses concerning the association between clinical variables and ich , the different antithrombotic treatments were analyzed separately according to the methods section . in the cox model , ischemic stroke was not found to be associated with an increased risk of ich in patients who were treated either with aspirin only ( hr , 1.06 ; 95% ci , 0.571.97 ; p=0.846 ) or with aspirin combined with clopidogrel ( hr , 1.52 ; 95% ci , 0.972.37 ; p=0.066 ) in analysis of 104 and 183 ich events in these 2 groups , respectively . when an anticoagulant was used ( either alone or in combination with antiplatelet therapy ) , a previous ischemic stroke was associated with an increased risk of ich ( hr , 2.26 ; 95% ci , 1.144.47 ; p=0.019 ) , in analysis of 47 ich events ( tables s3 through s5 ) . proportion of patients with an ich within 1 year of discharge , listed by type of antithrombotic treatment and stratified by occurrence of previous ischemic stroke dapt indicates dual antiplatelet therapy ; ich , intracranial hemorrhage ; n , number of cases . an overview of the different patient groups and study periods is depicted in figure 1 . ami indicates acute myocardial infarction ; dapt , dual antiplatelet therapy ; ich , intracranial hemorrhage ; rikshia , swedish register of information and knowledge about swedish heart intensive care admissions . the baseline characteristics of the 187 386 patients who were discharged after ami are shown in table 1 , grouped by subsequent occurrence of ich . a total of 590 patients suffered an ich within 1 year postdischarge ( 0.32% ; 95% confidence interval [ ci ] , 0.300.34 ) . according to kaplan meier analysis , this corresponds to a cumulative incidence of 0.35% ( 95% ci , 0.320.38 ) . mean age in the matched groups of ami patients and reference subjects was 71.9 years . the patients with an ich were significantly older ( mean of 75 vs 70 years ; p<0.001 ) , with increased history of previous hemorrhagic stroke ( 7.5% vs 1.2% ; p<0.001 ) and ischemic stroke ( 17.7% vs 7.9% ; p<0.001 ) . the patients in the ich group also had a higher prevalence of diabetes ( 24.9% vs 19.4% ; p<0.001 ) , atrial fibrillation ( 22.1% vs 12.9% ; p<0.001 ) , and previous cardiac failure ( 16.3% vs 10.7% ; p<0.001 ) . in addition , ich patients also had significantly lower weight ( 75 vs 78 kg ; p<0.001 ) . sex ratio , smoking history , and systolic blood pressure did not differ significantly between groups . baseline characteristics of 187 386 patients with ami who were discharged between 1998 and 2010 , separated according to the occurrence of ich within 1 year of discharge ace indicates angiotensin converting enzyme ; ami , acute myocardial infarction ; arb , angiotensin receptor blocker ; ich , intracranial hemorrhage ; n indicates number of cases ; nstemi , nonstelevation myocardial infarction ; stemi , stelevation myocardial infarction . the baseline characteristics of the population , divided into the 6 consecutive time periods , are shown in table s1 . table 2 shows medications being taken at discharge , separated by the 6 consecutive time periods from 1998 to 2010 . over time , there was a rapid increase in the use of p2y12 inhibitors , in most cases clopidogrel in addition to aspirin . the use of statins , angiotensin converting enzyme ( ace ) inhibitor / angiotensin receptor blocker ( arb ) , and betablockers also increased over the time periods studied . medication at discharge , stratified by time period and occurrence of ich within 1 year of discharge ace indicates angiotensin converting enzyme ; arb , angiotensin receptor blocker ; ich , intracranial hemorrhage ; n , number of cases . meier analysis , did not change significantly over time ( figure 2 ) : 0.29% from 1998 to 2000 ; 0.36% from 2001 to 2002 ; 0.39% from 2003 to 2004 ; 0.32% from 2005 to 2006 ; 0.37% from 2007 to 2008 ; and 0.37% from 2009 to 2010 ( p=0.2 ) . meier , without ich 1 year after hospital discharge after ami , stratified by time period . the 1year cumulative incidence of ich in the ami patients and matched reference subjects was 0.39% and 0.18% ( p<0.001 ) , respectively ( figure 3 ) . the corresponding incidences at 3 years were 0.92% and 0.57% ( p<0.001 ) and at 5 years were 1.56% and 0.95% , ( p<0.001 ) , respectively . meier , without ich 1 year after hospital discharge after ami , compared with a matched reference group . moreadvanced age ( hazard ratio [ hr ] , 1.14 ; 95% ci , 1.131.15 ; p<0.001 ) , for a 5 year change , previous ischemic stroke ( hr , 1.52 ; 95% ci , 1.112.08 ; p=0.010 ) , or hemorrhagic stroke ( hr , 3.58 ; 95% ci , 2.225.80 ; p<0.001 ) , and estimated glomerular filtration rate ( egfr ) less than 60 ml / min per 1.73 m ( hr , 1.28 ; 95% ci , 1.001.63 ; p=0.049 ) were significantly associated with an increased 1year risk of ich in the adjusted analysis , whereas female sex ( hr , 0.78 ; 95% ci , 0.620.98 ; p=0.030 ) was associated with a decreased risk . previous hypertension was also found to be of some importance ( hr , 1.31 ; 95% ci , 1.101.56 ; p=0.058 ) . predictors of ich after ami in a multivariate cox regression model , 1year followup ace indicates angiotensin converting enzyme ; ami , acute myocardial infarction ; arb , angiotensin receptor blocker ; gfr , glomerular filtration rate ; ich , intracranial hemorrhage ; stemi , st elevation myocardial infarction . none of the medications was significantly associated with a change in the risk of ich . table 4 shows the unadjusted occurrence of ich correlated to different antithrombotic treatments ( 520 events , with 70 events that occurred in patients without treatment ) , stratified by previous occurrence of ischemic stroke . in the analyses concerning the association between clinical variables and ich , the different antithrombotic treatments were analyzed separately according to the methods section . in the cox model , ischemic stroke was not found to be associated with an increased risk of ich in patients who were treated either with aspirin only ( hr , 1.06 ; 95% ci , 0.571.97 ; p=0.846 ) or with aspirin combined with clopidogrel ( hr , 1.52 ; 95% ci , 0.972.37 ; p=0.066 ) in analysis of 104 and 183 ich events in these 2 groups , respectively . when an anticoagulant was used ( either alone or in combination with antiplatelet therapy ) , a previous ischemic stroke was associated with an increased risk of ich ( hr , 2.26 ; 95% ci , 1.144.47 ; p=0.019 ) , in analysis of 47 ich events ( tables s3 through s5 ) . proportion of patients with an ich within 1 year of discharge , listed by type of antithrombotic treatment and stratified by occurrence of previous ischemic stroke dapt indicates dual antiplatelet therapy ; ich , intracranial hemorrhage ; n , number of cases . after an ami , the incidence of ich within 1 year of discharge was 0.35% ( 95% ci , 0.320.38 ) . this estimate is based on analysis of a large and fairly unselected population of 187 386 ami patients and 590 ich events , which is , by far , the largest data set published to date . the incidence of ich did not change significantly over the 13year period from 1998 to 2010 . the 1year cumulative incidence in age and sexmatched reference subjects sampled from the general swedish population , at 0.18% ( 95% ci , 0.160.20 ) , was significantly lower than the incidence in ami patients and did not change over time . our analysis confirmed that age , decreased egfr , previous ischemic stroke , and previous hemorrhagic stroke , in particular , are each associated with an increased risk of ich . none of the medications included in the analysis were associated with a significant change in ich risk , despite the large data set and despite there having been large changes in the use of different medical therapies over the time period studied . in patients treated with single or dual antiplatelet therapy , previous ischemic stroke was not associated with an increased risk of ich ; however , increased risk was observed if anticoagulant therapy was used . the use of p2y12 inhibitors , which increased from < 10% at the beginning of the study period to > 70% in the end , was not associated with increased incidence of ich . good clinical judgment when prescribing combined antiplatelet therapy may have been of importance for this result . clinical trials have also demonstrated the safety of clopidogrel with respect to ich risk . in the cure ( clopidogrel in unstable angina to prevent recurrent events ) trial , combined therapy with clopidogrel and aspirin was found not to increase the risk of lifethreatening bleeding or hemorrhagic stroke compared to aspirin alone ( 2.2% vs 1.8% and 0.1% vs 0.1% , respectively).28 similarly , in the commit ( clopidogrel and metoprolol in myocardial infarction ) trial , inclusion of clopidogrel with aspirin was shown not to increase the risk of hemorrhagic stroke at 28 days compared to aspirin alone ( 0.2% vs 0.2%).29 drugs comprising the new generation of p2y12 inhibitors , ticagrelor and prasugrel , seem to be fairly safe with respect to ich risk , based on data from the plato ( a study of platelet inhibition and patient outcomes ) and triton ( trial to assess improvement in therapeutic outcomes by optimizing platelet inhibition with prasugrel ) trials , except for prasugrel treatment in some highrisk groups.13 , 30 , 31 , 32 however , because of the small number of ich events included in these analyses , the data are not conclusive . analyses of data from large , unselected populations of acs patients are necessary for confirmation of the safety of these drugs . to investigate the impact of a previous ischemic stroke on the risk of subsequent ich in acs patients who were treated with different antithrombotic therapies , we analyzed the subgroups of patients treated with aspirin alone , with aspirin combined with clopidogrel , and with anticoagulant therapy in any combination with aspirin and/or clopidogrel . in these adjusted analyses , we found that a previous ischemic stroke was not a significant risk factor for ich in groups receiving single or dual antiplatelet therapy ( based on 104 and 183 ich events in the analyses , respectively ) . however , in the dapt group , we observed a trend toward an increased risk ( p=0.07 ) . in the group treated with anticoagulants , we found that a history of previous ischemic stroke more than doubled the risk of ich ( tables s3 through s5 ) . our observations concerning the unadjusted risk of ich associated with dual antiplatelet therapy ( table 4 ) , with or without a previous ischemic stroke , correspond well with the plato trial data from subgroup analysis of patients with a previous ischemic stroke or tia . we have not found any published adjusted analysis focused on the impact of a previous ischemic stroke among dapttreated patients . results from the match ( management of atherothrombosis with clopidogrel in highrisk patients ) trial , which included patients with a recent ischemic stroke or tia , indicate that the length of the delay between the ischemic event and the start of dapt may be an important factor . when dapt was initiated within 1 month of the ischemic event in the majority of the patients , the risk of intracranial hemorrhage increased significantly.33 furthermore , in a similar group of patients , anticoagulant treatment was found to be associated with an increase in the risk of major hemorrhage.34 future registry studies of large groups of acs patients with information about the time of onset of previous cerebrovascular events may shed further light on the relationship between the timing of antithrombotic treatment and ich risk . a study using the reach ( reduction of atherothrombosis for continued health ) registry includes analysis of data from patients with stable coronary artery disease ( cad).9 the risk of nonfatal cerebral hemorrhage was observed to be particularly high in patients with cad and a history of previous cerebrovascular disease who were receiving dapt ( hr , 5.2 ; 95% ci , 1.221.9 ) . unexpectedly , anticoagulant therapy ( with or without antiplatelet therapy ) did not result in increased risk . however , this result is based on very few cases ( 8 patients with ich were included in the dapt group ) . this study also found an increased risk of nonfatal cerebral hemorrhage in patients with cad and a history of ischemic stroke within the previous year ( adjusted hr , 3.9 ; 95% ci , 1.88.3 ; p<0.001 ) , although no increased risk if the stroke occurred more than 1 year previously ( hr , 1.0 ; 95% ci , 0.42.6 ; p=0.95 ) . again , however , this result is based on fewer than 10 events and is therefore inconclusive . we did not find any difference in the 1year ich risk between the different time periods during the course of the study . this is reassuring , considering the extensive changes in medications that we observed over the study period . there are no similar trend data to use for comparison , but our results seem robust . consistent with previous studies , a history of previous hemorrhagic stroke was associated with the highest risk for ich . a history of ischemic stroke , advanced age , and decreased renal function ( gfr < 60 ml / min ) were also significant factors , which is also consistent with findings from earlier studies.5 , 8 , 9 hypertension is an important risk factor for ich . a history of hypertension was close to reach significance ( p=0.058 ) in our study . a lack in the database of actual blood pressure values could possibly explain part of this modest association . possibly also , good blood pressure control in these postami patients weakened the importance of a previous diagnosis of hypertension . we found that female gender was associated with decreased risk of ich , in contrast to a previously report.1 one possible explanation for this discrepancy is that we did not include patients with ich during the hospital stay , which is often associated with thrombolytic treatment . furthermore , our study included a fairly unselected cohort of ami patients.17 , 35 , 36 we did not find statin treatment to be associated with any increased risk of ich . the hypothesis that the 2 are related , based on the sparcle ( stroke prevention by aggressive reduction of cholesterol levels ) trial , does not seem to be valid , at least in our ami patient population.19 , 21 the rikshia data set includes patients with ami who were hospitalized in ccus . therefore , very old patients or patients with extensive comorbidity may not be included.23 this might lead to an underestimation of the ich rate because , based on previous studies and our results , older patients are expected to have a higher risk of ich than younger patients . in our analyses , there was a low number of valid cases to include for the examination of some of the variables , especially in the early time period during which some of the variables were yet to be included in the rikshia database . this was true for heart rate , blood pressure , pglucose , and arb , early in the study . it was an issue throughout the entire period with respect to bmi , pcholesterol , and plowdensity lipoprotein . furthermore , when trying to establish independent risk factors with multivariate analysis , there is always the risk of confounding factors that are not taken into account because they were not recorded as variables in the database . concerning the risk associated with antithrombotic therapy over the long term , we had no information regarding the intended duration of therapy or the actual persistence of patients therapy . during the study period , therefore , very old patients or patients with extensive comorbidity may not be included.23 this might lead to an underestimation of the ich rate because , based on previous studies and our results , older patients are expected to have a higher risk of ich than younger patients . in our analyses , there was a low number of valid cases to include for the examination of some of the variables , especially in the early time period during which some of the variables were yet to be included in the rikshia database . this was true for heart rate , blood pressure , pglucose , and arb , early in the study . it was an issue throughout the entire period with respect to bmi , pcholesterol , and plowdensity lipoprotein . furthermore , when trying to establish independent risk factors with multivariate analysis , there is always the risk of confounding factors that are not taken into account because they were not recorded as variables in the database . concerning the risk associated with antithrombotic therapy over the long term , we had no information regarding the intended duration of therapy or the actual persistence of patients therapy . during the study period , despite the marked increase in use of dual antiplatelet treatment over the course of the study , the incidence of ich within 1 year of discharge after ami was stable , at 0.35% , which is approximately 2 times higher than the incidence measured in a matched reference population . advanced age , decreased renal function , previous ischemic stroke , and , most important , previous hemorrhagic stroke are all predictors of increased ich risk , and therefore they need to be taken into account when making decisions regarding antithrombotic therapy upon hospital discharge . a previous ischemic stroke is associated with a higher risk of ich when anticoagulant therapy , and possibly dual antiplatelet therapy , is used . further research is needed to clarify the impact on ich risk of the length of delay between onset of stroke and initiation of antithrombotic therapy . this study was supported by grants from the research and development unit at jmtland county council . the swedeheart registry is supported by the swedish society of cardiology , the swedish society of thoracic radiology , the swedish society of thoracic surgery , and the swedish heart association . the registry is financed by the government and the swedish association of local authorities and regions ( salar ) . baseline characteristics of patients with acute myocardial infarction ( ami ) during 6 consecutive time periods from 1998 to 2010 , separated by occurrence of intracranial hemorrhage ( ich ) within 1 year of discharge table s2 . univariate analyses of factors predictive of intracranial hemorrhage ( ich ) within 1 year postdischarge after acute myocardial infarction ( ami ) , 20032010 table s3 . predictors of intracranial hemorrhage ( ich ) after acute myocardial infarction ( ami ) in a multivariate cox regression model oneyear followup data were collected for patients treated with single antiplatelet therapy ( aspirin ) , 27 817 patients . predictors of intracranial hemorrhage ( ich ) after acute myocardial infarction ( ami ) in a multivariate cox regression model , with data from 1year followup of 72 504 patients treated with dual antiplatelet therapy , 20032010 table s5 . predictors of intracranial hemorrhage ( ich ) after acute myocardial infarction ( ami ) in a multivariate cox regression model , with data from 1 year followup of 7474 patients treated with anticoagulant alone or with a combination with aspirin , p2y12 , or both , 20032010 click here for additional data file .

backgroundto address the lack of knowledge regarding the longterm risk of intracranial hemorrhage ( ich ) after acute myocardial infarction ( ami ) , the aims of this study were to : ( 1 ) investigate the incidence , time trends , and predictors of ich in a large population within 1 year of discharge after ami ; ( 2 ) investigate the comparative 1year risk of ich in ami patients and a reference group ; and ( 3 ) study the impact of previous ischemic stroke on ich risk in patients treated with various antithrombotic therapies.methods and resultsdata about patients whose first ami occurred between 1998 and 2010 were collected from the swedish register of information and knowledge about swedish heartintensivecare admissions ( rikshia ) . patients with an ich after discharge were identified in the national patient register . risk was compared against a matched reference population . of 187 386 patients , 590 had an ich within 1 year . the 1year cumulative incidence ( 0.35% ) was approximately twice that of the reference group , and it did not change significantly over time . advanced age , previous ischemic or hemorrhagic stroke , and reduced glomerular filtration rate were associated with increased ich risk , whereas female sex was associated with a decreased risk . previous ischemic stroke did not increase risk of ich associated with single or dual antiplatelet therapy , but increased risk with anticoagulant therapy.conclusionthe 1year incidence of ich after ami remained stable , at 0.35% , over the study period . advanced age , decreased renal function , and previous ischemic or hemorrhagic stroke are predictive of increased ich risk .

Introduction Methods Study Design Statistical Analysis Results Patient Characteristics, Incidence, and Time Trends Predictors of Risk Discussion Limitations Conclusions Sources of Funding Disclosures Supporting information

intracranial hemorrhage ( ich ) is an infrequent , but serious , complication subsequent to acute myocardial infarction ( ami ) , which is associated both with great disability and a high risk of death.1 , 2 , 3 it has a high risk of recurrence and it is therefore important to be able to accurately assess hemorrhageassociated risk factors to reduce the risk of new and recurrent events.1 , 4 , 5 , 6 , 7 , 8 , 9 during the last decade , the inpatient treatment of myocardial infarction ( mi ) has changed drastically . however , advanced age , previous ischemic or hemorrhagic stroke , hypertension , female sex , lower weight , diabetes , atrial fibrillation , and renal failure are factors that have been associated with increased risk.1 , 8 , 16 , 17 the subgroup of patients who have had a previous ischemic stroke is fairly large among unselected ami populations . whether or not a previous ischemic stroke is associated with an increased risk of ich when dual antiplatelet therapy is used is , however , not known . first , we investigated the incidence , time trends , and predictors of ich within 1 year of discharge after ami in a large , fairly unselected population . second , we investigated the 1year risk of ich after ami compared to a reference group . third , we studied the impact of a previous ischemic stroke on ich risk in patients who received different antithrombotic therapies . patient data were obtained from the swedish register of information and knowledge about swedish heart intensive care admissions ( rikshia ) , which has now become a part of the swedish websystem for enhancement and development of evidencebased care in heart disease evaluated according to recommended therapies ( swedeheart ) . ich cases were identified in the registry by searches for the following codes from the 10th revision of the icd : subarachnoid hemorrhage ( i60 ) ; intracerebral hemorrhage ( i61 ) ; and other ich ( i62 ) . a diagnosis of stroke or transient ischemic attack ( tia ) in the npr was determined to be correct in 98.6% of the cases , and the proportion of stroke events identified ranges from 84.2% to 98% , as shown by validation studies.26 , 27 no published studies have specifically validated the diagnosis of ich in the npr ; however , the validity is expected to be at least as good as that of the diagnosis of ischemic stroke , because the diagnosis of ich can only be made after a positive computed tomography scan , magnetic resonance tomography scan , or at autopsy . to study ich predictors and time trends after discharge , we excluded all patients who had died or who had an ich during their hospital stay , resulting in a set of 187 671 patients with an ich diagnosis and followup through december 31 , 2010 . to compare the incidence of ich in the study group with that in a group representative of the general swedish population the reference subjects were sampled with a similar yearly distribution from 1998 to 2009 as the ami cases and were linked to the npr using the same algorithm as that used for the study population . patient data were obtained from the swedish register of information and knowledge about swedish heart intensive care admissions ( rikshia ) , which has now become a part of the swedish websystem for enhancement and development of evidencebased care in heart disease evaluated according to recommended therapies ( swedeheart ) . a diagnosis of stroke or transient ischemic attack ( tia ) in the npr was determined to be correct in 98.6% of the cases , and the proportion of stroke events identified ranges from 84.2% to 98% , as shown by validation studies.26 , 27 no published studies have specifically validated the diagnosis of ich in the npr ; however , the validity is expected to be at least as good as that of the diagnosis of ischemic stroke , because the diagnosis of ich can only be made after a positive computed tomography scan , magnetic resonance tomography scan , or at autopsy . to study ich predictors and time trends after discharge , we excluded all patients who had died or who had an ich during their hospital stay , resulting in a set of 187 671 patients with an ich diagnosis and followup through december 31 , 2010 . to compare the incidence of ich in the study group with that in a group representative of the general swedish population the reference subjects were sampled with a similar yearly distribution from 1998 to 2009 as the ami cases and were linked to the npr using the same algorithm as that used for the study population . ami indicates acute myocardial infarction ; dapt , dual antiplatelet therapy ; ich , intracranial hemorrhage ; rikshia , swedish register of information and knowledge about swedish heart intensive care admissions . the patients with an ich were significantly older ( mean of 75 vs 70 years ; p<0.001 ) , with increased history of previous hemorrhagic stroke ( 7.5% vs 1.2% ; p<0.001 ) and ischemic stroke ( 17.7% vs 7.9% ; p<0.001 ) . baseline characteristics of 187 386 patients with ami who were discharged between 1998 and 2010 , separated according to the occurrence of ich within 1 year of discharge ace indicates angiotensin converting enzyme ; ami , acute myocardial infarction ; arb , angiotensin receptor blocker ; ich , intracranial hemorrhage ; n indicates number of cases ; nstemi , nonstelevation myocardial infarction ; stemi , stelevation myocardial infarction . the 1year cumulative incidence of ich in the ami patients and matched reference subjects was 0.39% and 0.18% ( p<0.001 ) , respectively ( figure 3 ) . moreadvanced age ( hazard ratio [ hr ] , 1.14 ; 95% ci , 1.131.15 ; p<0.001 ) , for a 5 year change , previous ischemic stroke ( hr , 1.52 ; 95% ci , 1.112.08 ; p=0.010 ) , or hemorrhagic stroke ( hr , 3.58 ; 95% ci , 2.225.80 ; p<0.001 ) , and estimated glomerular filtration rate ( egfr ) less than 60 ml / min per 1.73 m ( hr , 1.28 ; 95% ci , 1.001.63 ; p=0.049 ) were significantly associated with an increased 1year risk of ich in the adjusted analysis , whereas female sex ( hr , 0.78 ; 95% ci , 0.620.98 ; p=0.030 ) was associated with a decreased risk . predictors of ich after ami in a multivariate cox regression model , 1year followup ace indicates angiotensin converting enzyme ; ami , acute myocardial infarction ; arb , angiotensin receptor blocker ; gfr , glomerular filtration rate ; ich , intracranial hemorrhage ; stemi , st elevation myocardial infarction . in the cox model , ischemic stroke was not found to be associated with an increased risk of ich in patients who were treated either with aspirin only ( hr , 1.06 ; 95% ci , 0.571.97 ; p=0.846 ) or with aspirin combined with clopidogrel ( hr , 1.52 ; 95% ci , 0.972.37 ; p=0.066 ) in analysis of 104 and 183 ich events in these 2 groups , respectively . when an anticoagulant was used ( either alone or in combination with antiplatelet therapy ) , a previous ischemic stroke was associated with an increased risk of ich ( hr , 2.26 ; 95% ci , 1.144.47 ; p=0.019 ) , in analysis of 47 ich events ( tables s3 through s5 ) . proportion of patients with an ich within 1 year of discharge , listed by type of antithrombotic treatment and stratified by occurrence of previous ischemic stroke dapt indicates dual antiplatelet therapy ; ich , intracranial hemorrhage ; n , number of cases . ami indicates acute myocardial infarction ; dapt , dual antiplatelet therapy ; ich , intracranial hemorrhage ; rikshia , swedish register of information and knowledge about swedish heart intensive care admissions . the patients with an ich were significantly older ( mean of 75 vs 70 years ; p<0.001 ) , with increased history of previous hemorrhagic stroke ( 7.5% vs 1.2% ; p<0.001 ) and ischemic stroke ( 17.7% vs 7.9% ; p<0.001 ) . baseline characteristics of 187 386 patients with ami who were discharged between 1998 and 2010 , separated according to the occurrence of ich within 1 year of discharge ace indicates angiotensin converting enzyme ; ami , acute myocardial infarction ; arb , angiotensin receptor blocker ; ich , intracranial hemorrhage ; n indicates number of cases ; nstemi , nonstelevation myocardial infarction ; stemi , stelevation myocardial infarction . meier analysis , did not change significantly over time ( figure 2 ) : 0.29% from 1998 to 2000 ; 0.36% from 2001 to 2002 ; 0.39% from 2003 to 2004 ; 0.32% from 2005 to 2006 ; 0.37% from 2007 to 2008 ; and 0.37% from 2009 to 2010 ( p=0.2 ) . the 1year cumulative incidence of ich in the ami patients and matched reference subjects was 0.39% and 0.18% ( p<0.001 ) , respectively ( figure 3 ) . meier , without ich 1 year after hospital discharge after ami , compared with a matched reference group . moreadvanced age ( hazard ratio [ hr ] , 1.14 ; 95% ci , 1.131.15 ; p<0.001 ) , for a 5 year change , previous ischemic stroke ( hr , 1.52 ; 95% ci , 1.112.08 ; p=0.010 ) , or hemorrhagic stroke ( hr , 3.58 ; 95% ci , 2.225.80 ; p<0.001 ) , and estimated glomerular filtration rate ( egfr ) less than 60 ml / min per 1.73 m ( hr , 1.28 ; 95% ci , 1.001.63 ; p=0.049 ) were significantly associated with an increased 1year risk of ich in the adjusted analysis , whereas female sex ( hr , 0.78 ; 95% ci , 0.620.98 ; p=0.030 ) was associated with a decreased risk . predictors of ich after ami in a multivariate cox regression model , 1year followup ace indicates angiotensin converting enzyme ; ami , acute myocardial infarction ; arb , angiotensin receptor blocker ; gfr , glomerular filtration rate ; ich , intracranial hemorrhage ; stemi , st elevation myocardial infarction . in the cox model , ischemic stroke was not found to be associated with an increased risk of ich in patients who were treated either with aspirin only ( hr , 1.06 ; 95% ci , 0.571.97 ; p=0.846 ) or with aspirin combined with clopidogrel ( hr , 1.52 ; 95% ci , 0.972.37 ; p=0.066 ) in analysis of 104 and 183 ich events in these 2 groups , respectively . when an anticoagulant was used ( either alone or in combination with antiplatelet therapy ) , a previous ischemic stroke was associated with an increased risk of ich ( hr , 2.26 ; 95% ci , 1.144.47 ; p=0.019 ) , in analysis of 47 ich events ( tables s3 through s5 ) . proportion of patients with an ich within 1 year of discharge , listed by type of antithrombotic treatment and stratified by occurrence of previous ischemic stroke dapt indicates dual antiplatelet therapy ; ich , intracranial hemorrhage ; n , number of cases . after an ami , the incidence of ich within 1 year of discharge was 0.35% ( 95% ci , 0.320.38 ) . the incidence of ich did not change significantly over the 13year period from 1998 to 2010 . the 1year cumulative incidence in age and sexmatched reference subjects sampled from the general swedish population , at 0.18% ( 95% ci , 0.160.20 ) , was significantly lower than the incidence in ami patients and did not change over time . our analysis confirmed that age , decreased egfr , previous ischemic stroke , and previous hemorrhagic stroke , in particular , are each associated with an increased risk of ich . none of the medications included in the analysis were associated with a significant change in ich risk , despite the large data set and despite there having been large changes in the use of different medical therapies over the time period studied . in patients treated with single or dual antiplatelet therapy , previous ischemic stroke was not associated with an increased risk of ich ; however , increased risk was observed if anticoagulant therapy was used . the use of p2y12 inhibitors , which increased from < 10% at the beginning of the study period to > 70% in the end , was not associated with increased incidence of ich . in the cure ( clopidogrel in unstable angina to prevent recurrent events ) trial , combined therapy with clopidogrel and aspirin was found not to increase the risk of lifethreatening bleeding or hemorrhagic stroke compared to aspirin alone ( 2.2% vs 1.8% and 0.1% vs 0.1% , respectively).28 similarly , in the commit ( clopidogrel and metoprolol in myocardial infarction ) trial , inclusion of clopidogrel with aspirin was shown not to increase the risk of hemorrhagic stroke at 28 days compared to aspirin alone ( 0.2% vs 0.2%).29 drugs comprising the new generation of p2y12 inhibitors , ticagrelor and prasugrel , seem to be fairly safe with respect to ich risk , based on data from the plato ( a study of platelet inhibition and patient outcomes ) and triton ( trial to assess improvement in therapeutic outcomes by optimizing platelet inhibition with prasugrel ) trials , except for prasugrel treatment in some highrisk groups.13 , 30 , 31 , 32 however , because of the small number of ich events included in these analyses , the data are not conclusive . to investigate the impact of a previous ischemic stroke on the risk of subsequent ich in acs patients who were treated with different antithrombotic therapies , we analyzed the subgroups of patients treated with aspirin alone , with aspirin combined with clopidogrel , and with anticoagulant therapy in any combination with aspirin and/or clopidogrel . in these adjusted analyses , we found that a previous ischemic stroke was not a significant risk factor for ich in groups receiving single or dual antiplatelet therapy ( based on 104 and 183 ich events in the analyses , respectively ) . in the group treated with anticoagulants , we found that a history of previous ischemic stroke more than doubled the risk of ich ( tables s3 through s5 ) . our observations concerning the unadjusted risk of ich associated with dual antiplatelet therapy ( table 4 ) , with or without a previous ischemic stroke , correspond well with the plato trial data from subgroup analysis of patients with a previous ischemic stroke or tia . when dapt was initiated within 1 month of the ischemic event in the majority of the patients , the risk of intracranial hemorrhage increased significantly.33 furthermore , in a similar group of patients , anticoagulant treatment was found to be associated with an increase in the risk of major hemorrhage.34 future registry studies of large groups of acs patients with information about the time of onset of previous cerebrovascular events may shed further light on the relationship between the timing of antithrombotic treatment and ich risk . this study also found an increased risk of nonfatal cerebral hemorrhage in patients with cad and a history of ischemic stroke within the previous year ( adjusted hr , 3.9 ; 95% ci , 1.88.3 ; p<0.001 ) , although no increased risk if the stroke occurred more than 1 year previously ( hr , 1.0 ; 95% ci , 0.42.6 ; p=0.95 ) . during the study period , despite the marked increase in use of dual antiplatelet treatment over the course of the study , the incidence of ich within 1 year of discharge after ami was stable , at 0.35% , which is approximately 2 times higher than the incidence measured in a matched reference population . advanced age , decreased renal function , previous ischemic stroke , and , most important , previous hemorrhagic stroke are all predictors of increased ich risk , and therefore they need to be taken into account when making decisions regarding antithrombotic therapy upon hospital discharge . a previous ischemic stroke is associated with a higher risk of ich when anticoagulant therapy , and possibly dual antiplatelet therapy , is used . baseline characteristics of patients with acute myocardial infarction ( ami ) during 6 consecutive time periods from 1998 to 2010 , separated by occurrence of intracranial hemorrhage ( ich ) within 1 year of discharge table s2 . univariate analyses of factors predictive of intracranial hemorrhage ( ich ) within 1 year postdischarge after acute myocardial infarction ( ami ) , 20032010 table s3 . predictors of intracranial hemorrhage ( ich ) after acute myocardial infarction ( ami ) in a multivariate cox regression model oneyear followup data were collected for patients treated with single antiplatelet therapy ( aspirin ) , 27 817 patients . predictors of intracranial hemorrhage ( ich ) after acute myocardial infarction ( ami ) in a multivariate cox regression model , with data from 1year followup of 72 504 patients treated with dual antiplatelet therapy , 20032010 table s5 . predictors of intracranial hemorrhage ( ich ) after acute myocardial infarction ( ami ) in a multivariate cox regression model , with data from 1 year followup of 7474 patients treated with anticoagulant alone or with a combination with aspirin , p2y12 , or both , 20032010 click here for additional data file .

chronic kidney disease ( ckd ) is associated with a high risk for cardiovascular disease and mortality . this increased risk is attributed to various traditional and non - traditional risk factors such as hypertension , diabetes , proteinuria , uric acid and acidosis . in recent years the role of disorders in calcium and phosphate metabolism has been emphasized , with studies reporting increased cardiovascular risk associations with higher serum phosphate [ 612 ] , higher calcium , higher parathormone ( pth ) , and lower vitamin d levels . most recently , focus has shifted to fibroblast growth factor 23 ( fgf23 ) , a phosphaturic hormone mainly produced by the osteocyte . fgf23 is associated with cardiovascular ( cv ) morbidity and mortality in patients with ckd [ 1618 ] , and is an independent predictor for progression of kidney failure [ 16 , 17 , 19 , 20 ] . moreover , elevated fgf23 levels have been shown to be a risk factor for cv disease and mortality in the general population [ 18 , 21 , 22 ] . it inhibits expression of the sodium - phosphate transporters in the proximal tubuli of the kidneys , thus promoting renal phosphate excretion . elevated fgf23 levels in patients with ckd may partially be the result of fgf23 resistance . fgf23 resistance can be defined as a state in which the kidney and the parathyroid glands , the primary sites of fgf23 action , do not respond optimally to fgf23 by excreting less phosphate and suppressing pth less efficiently compared to healthy persons , which drives fgf23 to increase . classical fgf23 action requires binding to its receptor , using klotho as co - factor [ 24 , 25 ] . it is believed that tissue klotho levels of the kidney decline with ckd and may thus be the mechanism behind fgf23 resistance [ 2631 ] . although fgf23 is thought to have effects also independently from klotho , it is possible that elevated fgf23 concentrations reflect renal tissue klotho deficiency , and that fgf23 resistance itself contributes to the increased relative risk regarding outcome . this indeed was suggested by dominguez et al . who used fractional excretion of phosphate ( fepi ) as a marker of fgf23 resistance , and observed that fepi modified the association of fgf23 with outcome in 872 patients with relatively mild ckd [ mean estimated glomerular filtration rate ( egfr)71 ml / min/1.73 m ] . we questioned if the same could be observed in patients with more severe ckd , a situation associated with increased cv mortality , progression of ckd and higher fgf23 concentrations . we used baseline data of patients who participated in the masterplan ( multifactorial approach and superior treatment efficacy in renal patients with the aid of nurse practitioners ) study . masterplan was a randomized controlled clinical trial ( isrctn73187232 ) performed in nine dutch hospitals in which patients with ckd ( egfr 2070 ml / min/1.73 m ) were randomized to receive either usual care by the nephrologist or intensified treatment with added nurse practitioner support . inclusion started in april 2004 and ended in december 2005 . in two centers , baseline 24 h urine was collected and samples were stored at 80 c . only patients from these centers were included in the current analysis as urinary analyses were required . stored blood samples were used at a more recent date for measuring fgf23 using sandwich enzyme - linked immunosorbent assay ( elisa ) ( immutopics san clemente , ca , usa ) measuring the c - terminal fgf23 . the intra- and inter - assay coefficients of variation of this assay are < 5 and < 16 % , respectively . urine samples were thawed , acidified , and creatinine , calcium , and phosphate were measured using standard automated techniques . egfr was calculated with the four - point modification of diet in renal disease ( mdrd ) formula . fractional excretion of phosphate was calculated as ( urine phosphate serum creatinine)/(serum phosphate urine creatinine ) . a composite end - point was defined consisting of the combination of death , cv events ( myocardial infarction , cerebrovascular accident , percutaneous transluminal coronary angioplasty or coronary artery bypass graft ) and renal failure ( defined as need for renal replacement therapy , doubling of serum creatinine or death ) . thus , fgf23 resistance can be considered if a high fgf23 exists together with a low fepi . baseline characteristics are reported as median values with interquartile range ( iqr ) for skewed data and as mean values with standard deviation ( sd ) for normally distributed data . survival analyses were performed using cox regression analyses in order to adjust for possible confounding by gender , age , systolic blood pressure , egfr , pth , proteinuria and smoking . in order to evaluate if fgf23 resistance expressed as fepi increased the effect of fgf23 on outcome , patients were categorized by fgf23 above and below the median combined with fepi above and below the mean into four categories . a product term for the categorized fgf23 and fepi was added again to cox regression . in the presence of a positive interaction , the sum of hazard ratios ( hr ) for the combination of a high fgf23 and low fepi would be higher than the theoretical calculated hr of a high fgf23 and high fepi times the ratio of the hr of a low fgf23 and low fepi divided by the hr of the reference group . a second method to calculate the presence and direction of interaction was performed by calculating the relative excess risk due to interaction ( reri ) [ 39 , 40 ] . a positive additive interaction is present if the reri is > 0 and is statistically significant . statistical analyses were performed using spss 20.0 ( ibm spss software , ibm corp , armonk , ny , usa ) and stata 11.2 ( statacorp , college station , tx , usa ) software packages . we used baseline data of patients who participated in the masterplan ( multifactorial approach and superior treatment efficacy in renal patients with the aid of nurse practitioners ) study . masterplan was a randomized controlled clinical trial ( isrctn73187232 ) performed in nine dutch hospitals in which patients with ckd ( egfr 2070 ml / min/1.73 m ) were randomized to receive either usual care by the nephrologist or intensified treatment with added nurse practitioner support . inclusion started in april 2004 and ended in december 2005 . in two centers , baseline 24 h urine was collected and samples were stored at 80 c . only patients from these centers were included in the current analysis as urinary analyses were required . stored blood samples were used at a more recent date for measuring fgf23 using sandwich enzyme - linked immunosorbent assay ( elisa ) ( immutopics san clemente , ca , usa ) measuring the c - terminal fgf23 . the intra- and inter - assay coefficients of variation of this assay are < 5 and < 16 % , respectively . urine samples were thawed , acidified , and creatinine , calcium , and phosphate were measured using standard automated techniques . egfr was calculated with the four - point modification of diet in renal disease ( mdrd ) formula . fractional excretion of phosphate was calculated as ( urine phosphate serum creatinine)/(serum phosphate urine creatinine ) . a composite end - point was defined consisting of the combination of death , cv events ( myocardial infarction , cerebrovascular accident , percutaneous transluminal coronary angioplasty or coronary artery bypass graft ) and renal failure ( defined as need for renal replacement therapy , doubling of serum creatinine or death ) . thus , fgf23 resistance can be considered if a high fgf23 exists together with a low fepi . baseline characteristics are reported as median values with interquartile range ( iqr ) for skewed data and as mean values with standard deviation ( sd ) for normally distributed data . survival analyses were performed using cox regression analyses in order to adjust for possible confounding by gender , age , systolic blood pressure , egfr , pth , proteinuria and smoking . in order to evaluate if fgf23 resistance expressed as fepi increased the effect of fgf23 on outcome , patients were categorized by fgf23 above and below the median combined with fepi above and below the mean into four categories . a product term for the categorized fgf23 and fepi was added again to cox regression . in the presence of a positive interaction , the sum of hazard ratios ( hr ) for the combination of a high fgf23 and low fepi would be higher than the theoretical calculated hr of a high fgf23 and high fepi times the ratio of the hr of a low fgf23 and low fepi divided by the hr of the reference group . a second method to calculate the presence and direction of interaction was performed by calculating the relative excess risk due to interaction ( reri ) [ 39 , 40 ] . the confidence interval ( ci ) of the reri a positive additive interaction is present if the reri is > 0 and is statistically significant . statistical analyses were performed using spss 20.0 ( ibm spss software , ibm corp , armonk , ny , usa ) and stata 11.2 ( statacorp , college station , tx , usa ) software packages . baseline characteristics of these 166 patients grouped by fgf23 and fepi are shown in table 1 . the median age was 53 ( iqr 45 , 62 ) years , most patients were caucasian and the median egfr was 36 ( iqr 28 , 44 ) ml / min/1.73 m. the median of proteinuria was 0.40 ( iqr 0.20 , 1.20 ) g / day , fepi 0.32 ( iqr 0.25 , 0.44 ) and fgf23 140 ( iqr 81236 ) ru / ml . people with higher fgf23 levels more frequently used vitamin d compounds.table 1baseline characteristics of patients grouped by fgf23 and fepi ( n = 166)variabletotal group ( n = 166)low fgf23 , high fepi ( n = 31)low fgf23 , low fepi ( n = 52)high fgf23 , high fepi ( n = 51)high fgf23 , low fepi ( n = 32 ) p valuegender , male , n ( % ) 112 ( 67)26 ( 84)28 ( 54)40 ( 78)18 ( 56)0.01age , years ( range)53 ( 4562)52 ( 4364)53 ( 4561)54 ( 5063)49 ( 4364)0.38bmi ( kg / m)26 ( 2429)26 ( 2327)26 ( 2328)27 ( 2430)26 ( 2331)0.47race ( caucasian)160304851310.22cause of ckd diabetic1414720.62 renovascular91341 glomerulonephritis41718106 interstitial298687 congenital83302 cystic kidney disease223784 unknown336999 other102251systolic bp ( mmhg)126 ( 118139)124 ( 116134)124 ( 116135)130 ( 119144)125 ( 118136)0.44diastolic bp ( mmhg)75 ( 7081)75 ( 7178)76 ( 7081)75 ( 7083)76 ( 6883)0.81egfr ( mdrd4 , ml / min/1.73 m)36 ( 2744)36 ( 2945)41 ( 3652)27 ( 2034)36 ( 2742)<0.01proteinuria ( g/24 h)0.40 ( 0.201.20)0.40 ( 0.201.20)0.30 ( 0.10 - 0.58)0.80 ( 0.301.50)0.60 ( 0.201.38)0.01total cholesterol ( mmol / l)4.99 0.985.10 ( 4.505.80)5.00 ( 4.405.50)4.80 ( 4.005.30)5.15 ( 4.536.08)0.18serum phosphate ( mmol / l)1.04 ( 0.92 - 1.20)1.02 ( 0.941.13)1.00 ( 0.891.09)1.17 ( 0.961.40)1.04 ( 0.901.20)0.03serum calcium ( mmol / l)2.35 0.152.37 ( 2.302.50)2.38 ( 2.282.44)2.34 ( 2.262.42)2.33 ( 2.222.40)0.49serum albumin ( mmol / l)40 441 ( 4043)40 ( 3842)39 ( 3742)40 ( 3741)0.10serum pth ( pmol / l)10.15 ( 6.6816.18)10.15 ( 8.1816.73)9.00 ( 4.6512.00)14.25 ( 9.0023.23)10.00 ( 6.0815.85)<0.01serum cfgf-23 ( ru / ml)140 ( 81236)78 ( 54114)81.25 ( 54.10115.00)278 ( 203439)204 ( 159289)<0.01fractional phosphate excretion0.32 ( 0.250.44)0.41 ( 0.360.46)0.24 ( 0.190.28)0.49 ( 0.400.54)0.26 ( 0.220.29)<0.01fractional calcium excretion0.00 ( 0.000.01)0.00 ( 0.000.01)0.00 ( 0.000.01)0.00 ( 0.000.01)0.00 ( 0.000.01)0.99vitamin d drugs ( n , % ) 33 ( 20)3 ( 10)5 ( 10)18 ( 35)7 ( 22)0.004diabetes35281870.01smokers27261540.02events / py96/79211/15825/25737/23623/141events/1000 py1217097157163median with interquartile ranges ( 2575 % ) for skewed datamean with standard deviation ( sd ) for normally distributed data fgf23 fibroblast growth factor 23 , fepi fractional excretion of phosphate , bmi body mass index , ckd chronic kidney disease , bp blood pressure , egfr estimated glomerular filtration rate , mdrd4 4-point modification of diet in renal disease , pth parathormone , py person years baseline characteristics of patients grouped by fgf23 and fepi ( n = 166 ) median with interquartile ranges ( 2575 % ) for skewed data mean with standard deviation ( sd ) for normally distributed data fgf23 fibroblast growth factor 23 , fepi fractional excretion of phosphate , bmi body mass index , ckd chronic kidney disease , bp blood pressure , egfr estimated glomerular filtration rate , mdrd4 4-point modification of diet in renal disease , pth parathormone , py person years univariate analysis between fgf23 and parameters of kidney function and phosphate metabolism showed a significant inverse correlation between fgf23 and egfr ( r = 0.43 , p < 0.01 ) . we observed a significant positive correlation between fgf23 and serum phosphate ( r = 0.29 , p < 0.01 ) , fepi ( r = 0.36 , p < 0.01 ) , urine phosphate / creatinine ratio ( r = 0.17 , p = 0.03 ) and pth ( r = 0.30 , p < 0.01 ) . of note , there was no significant correlation between fgf23 and serum calcium . in the multivariate analysis including sex , body mass index ( bmi ) , age , pth , egfr , fepi , serum phosphate , serum calcium , total cholesterol , systolic blood pressure , proteinuria , diabetes and smoking , only egfr and smoking were independent predictors of fgf23 . clinical characteristics of the patients divided by the combination of fepi and fgf23 are reported in table 1 . compared to patients with fgf23 concentrations below the median , the patients with higher concentrations had more proteinuria , a higher pth , a higher phosphate and a lower egfr . patients with a low fepi had lower serum phosphorus levels , a lower pth , total cholesterol and were more likely female . after a median of 4.8 years of follow up , 96 of the 166 patients ( 59 % ) reached an endpoint defined as death ( 8/166 ) , cv event ( 14/166 ) , renal replacement therapy or doubling of creatinine ( 74/166 ) . in the multivariate cox regression analysis only lnfgf23 , proteinuria and systolic blood pressure remained independent predictors of the composite outcome ( table 2 ) . moreover , the hazard ratio for lnfgf23 for outcome did not change after adjustment for fepi ( table 2 ) . meier curve for the composite outcome according to quartiles of fgf23 after adjustment for baseline covariates.table 2univariate and multivariate cox regression analysis for combined outcomeunivariatemultivariatehrci p valuehrci p valueln fgf232.171.652.84<0.012.131.542.95<0.001fepi3.390.8613.470.0820.230.041.520.13serum phosphate3.571.727.45<0.0012.130.875.190.10proteinuria1.271.151.41<0.0011.251.111.41<0.001mdrd0.970.960.990.0050.990.971.020.59age0.990.971.000.1580.990.971.000.11gender ( male)1.320.852.090.2150.670.401.110.12smoking1.751.082.840.0220.980.541.750.93systolic blood pressure1.021.001.040.0011.021.001.030.02pth1.021.001.030.0171.000.981.020.98 hr hazard ratio , ci confidence interval , for other abbreviations , see table 1 fig . 1cox curve for quartiles of fgf23 and the effect on composite outcome . adjusted for age , sex , smoking , systolic blood pressure , proteinuria , egfr , tmp / gfr , serum phosphate and pth . fgf23 , fibroblast growth factor 23 ; egfr , estimated glomerular filtration rate ; tmp / gfr , ratio of the maximum rate of tubular phosphate reabsorption to the glomerular filtration rate ; pth , parathormone univariate and multivariate cox regression analysis for combined outcome hr hazard ratio , ci confidence interval , for other abbreviations , see table 1 cox curve for quartiles of fgf23 and the effect on composite outcome . adjusted for age , sex , smoking , systolic blood pressure , proteinuria , egfr , tmp / gfr , serum phosphate and pth . fgf23 , fibroblast growth factor 23 ; egfr , estimated glomerular filtration rate ; tmp / gfr , ratio of the maximum rate of tubular phosphate reabsorption to the glomerular filtration rate ; pth , parathormone tables 3 and 4 further show the hazard ratios for the associations between fgf23 and fepi categories and outcome . cox regression , which was adjusted for age , sex , smoking , systolic blood pressure , proteinuria , egfr and pth , revealed a hazard ratio for the combination of high fgf23 and low fepi of 4.15 ( 95 % ci 1.859.30 ) on the combined outcome ( table 3 ) . in the absence of hazard ratio modification , one would have expected the joint effect of a high fgf23 and low fepi to give a hr of 8.14 [ = e ] . likewise , the reri was 0.60 ( 95 % ci 4.81 to 3.70 ) . similarly , the hazard ratio of a high fgf23 combined with low fepi on renal outcome was 3.44 , lower than expected from the sum of both individual factors , hr = 7.77 [ = e].table 3multivariate cox regression on combined outcome for categories defined by fgf23 below and above the median combined with fepi below and above the meancombined survivalparticipantseventshr95 % cilow fgf23/high fepi31111referencelow fgf23/low fepi52252.561.135.80high fgf23/high fepi51373.181.476.87high fgf23/low fepi32234.151.859.30this model included fgf23 and fepi as categorical variables and their product termthe model was adjusted for factors shown and age , sex , smoking , systolic blood pressure , proteinuria , egfr , pthhazard ratio product for the product term is 0.51 ( 95 % ci 0.191.34 ) , p = 0.17reri is 0.60 ( 95 % ci 4.81 to 3.70 ) , p = 0.79 reri , relative excess risk due to interaction , for other abbreviations , see previous tablestable 4multivariate cox regression on renal outcome for categories defined by fgf23 below and above the median combined with fepi below and above the meanrenal survivalparticipantseventshr95 % cilow fgf23/high fepi3191referencelow fgf23/low fepi52202.400.816.34high fgf23/high fepi51343.221.317.84high fgf23/low fepi32183.441.378.62this model included fgf23 and fepi as categorical variables and their product termthe model was adjusted for factors shown and age , sex , smoking , systolic blood pressure , proteinuria , egfr , and pthhazard ratio product for the product term is 0.44 ( 95 % ci 0.141.41)reri is 1.18 ( 95 % ci 8.10 to 5.73 ) , p = 0.74for all abbreviations , see previous tables multivariate cox regression on combined outcome for categories defined by fgf23 below and above the median combined with fepi below and above the mean this model included fgf23 and fepi as categorical variables and their product term the model was adjusted for factors shown and age , sex , smoking , systolic blood pressure , proteinuria , egfr , pth hazard ratio product for the product term is 0.51 ( 95 % ci 0.191.34 ) , p = 0.17 reri is 0.60 ( 95 % ci 4.81 to 3.70 ) , p = 0.79 reri , relative excess risk due to interaction , for other abbreviations , see previous tables multivariate cox regression on renal outcome for categories defined by fgf23 below and above the median combined with fepi below and above the mean this model included fgf23 and fepi as categorical variables and their product term the model was adjusted for factors shown and age , sex , smoking , systolic blood pressure , proteinuria , egfr , and pth hazard ratio product for the product term is 0.44 ( 95 % ci 0.141.41 ) reri is 1.18 ( 95 % ci 8.10 to 5.73 ) , p = 0.74 for all abbreviations , see previous tables univariate analysis between fgf23 and parameters of kidney function and phosphate metabolism showed a significant inverse correlation between fgf23 and egfr ( r = 0.43 , p < 0.01 ) . we observed a significant positive correlation between fgf23 and serum phosphate ( r = 0.29 , p < 0.01 ) , fepi ( r = 0.36 , p < 0.01 ) , urine phosphate / creatinine ratio ( r = 0.17 , p = 0.03 ) and pth ( r = 0.30 , p < 0.01 ) . of note , there was no significant correlation between fgf23 and serum calcium . in the multivariate analysis including sex , body mass index ( bmi ) , age , pth , egfr , fepi , serum phosphate , serum calcium , total cholesterol , systolic blood pressure , proteinuria , clinical characteristics of the patients divided by the combination of fepi and fgf23 are reported in table 1 . compared to patients with fgf23 concentrations below the median , the patients with higher concentrations had more proteinuria , a higher pth , a higher phosphate and a lower egfr . patients with high fgf23 were also more frequently diabetics and more likely to smoke . patients with a low fepi had lower serum phosphorus levels , a lower pth , total cholesterol and were more likely female . after a median of 4.8 years of follow up , 96 of the 166 patients ( 59 % ) reached an endpoint defined as death ( 8/166 ) , cv event ( 14/166 ) , renal replacement therapy or doubling of creatinine ( 74/166 ) . in the multivariate cox regression analysis only lnfgf23 , proteinuria and systolic blood pressure remained independent predictors of the composite outcome ( table 2 ) . moreover , the hazard ratio for lnfgf23 for outcome did not change after adjustment for fepi ( table 2 ) . meier curve for the composite outcome according to quartiles of fgf23 after adjustment for baseline covariates.table 2univariate and multivariate cox regression analysis for combined outcomeunivariatemultivariatehrci p valuehrci p valueln fgf232.171.652.84<0.012.131.542.95<0.001fepi3.390.8613.470.0820.230.041.520.13serum phosphate3.571.727.45<0.0012.130.875.190.10proteinuria1.271.151.41<0.0011.251.111.41<0.001mdrd0.970.960.990.0050.990.971.020.59age0.990.971.000.1580.990.971.000.11gender ( male)1.320.852.090.2150.670.401.110.12smoking1.751.082.840.0220.980.541.750.93systolic blood pressure1.021.001.040.0011.021.001.030.02pth1.021.001.030.0171.000.981.020.98 hr hazard ratio , ci confidence interval , for other abbreviations , see table 1 fig . 1cox curve for quartiles of fgf23 and the effect on composite outcome . adjusted for age , sex , smoking , systolic blood pressure , proteinuria , egfr , tmp / gfr , serum phosphate and pth . fgf23 , fibroblast growth factor 23 ; egfr , estimated glomerular filtration rate ; tmp / gfr , ratio of the maximum rate of tubular phosphate reabsorption to the glomerular filtration rate ; pth , parathormone univariate and multivariate cox regression analysis for combined outcome hr hazard ratio , ci confidence interval , for other abbreviations , see table 1 cox curve for quartiles of fgf23 and the effect on composite outcome . adjusted for age , sex , smoking , systolic blood pressure , proteinuria , egfr , tmp / gfr , serum phosphate and pth . fgf23 , fibroblast growth factor 23 ; egfr , estimated glomerular filtration rate ; tmp / gfr , ratio of the maximum rate of tubular phosphate reabsorption to the glomerular filtration rate ; pth , parathormone tables 3 and 4 further show the hazard ratios for the associations between fgf23 and fepi categories and outcome . cox regression , which was adjusted for age , sex , smoking , systolic blood pressure , proteinuria , egfr and pth , revealed a hazard ratio for the combination of high fgf23 and low fepi of 4.15 ( 95 % ci 1.859.30 ) on the combined outcome ( table 3 ) . in the absence of hazard ratio modification , one would have expected the joint effect of a high fgf23 and low fepi to give a hr of 8.14 [ = e ] . likewise , the reri was 0.60 ( 95 % ci 4.81 to 3.70 ) . similarly , the hazard ratio of a high fgf23 combined with low fepi on renal outcome was 3.44 , lower than expected from the sum of both individual factors , hr = 7.77 [ = e].table 3multivariate cox regression on combined outcome for categories defined by fgf23 below and above the median combined with fepi below and above the meancombined survivalparticipantseventshr95 % cilow fgf23/high fepi31111referencelow fgf23/low fepi52252.561.135.80high fgf23/high fepi51373.181.476.87high fgf23/low fepi32234.151.859.30this model included fgf23 and fepi as categorical variables and their product termthe model was adjusted for factors shown and age , sex , smoking , systolic blood pressure , proteinuria , egfr , pthhazard ratio product for the product term is 0.51 ( 95 % ci 0.191.34 ) , p = 0.17reri is 0.60 ( 95 % ci 4.81 to 3.70 ) , p = 0.79 reri , relative excess risk due to interaction , for other abbreviations , see previous tablestable 4multivariate cox regression on renal outcome for categories defined by fgf23 below and above the median combined with fepi below and above the meanrenal survivalparticipantseventshr95 % cilow fgf23/high fepi3191referencelow fgf23/low fepi52202.400.816.34high fgf23/high fepi51343.221.317.84high fgf23/low fepi32183.441.378.62this model included fgf23 and fepi as categorical variables and their product termthe model was adjusted for factors shown and age , sex , smoking , systolic blood pressure , proteinuria , egfr , and pthhazard ratio product for the product term is 0.44 ( 95 % ci 0.141.41)reri is 1.18 ( 95 % ci 8.10 to 5.73 ) , p = 0.74for all abbreviations , see previous tables multivariate cox regression on combined outcome for categories defined by fgf23 below and above the median combined with fepi below and above the mean this model included fgf23 and fepi as categorical variables and their product term the model was adjusted for factors shown and age , sex , smoking , systolic blood pressure , proteinuria , egfr , pth hazard ratio product for the product term is 0.51 ( 95 % ci 0.191.34 ) , p = 0.17 reri is 0.60 ( 95 % ci 4.81 to 3.70 ) , p = 0.79 reri , relative excess risk due to interaction , for other abbreviations , see previous tables multivariate cox regression on renal outcome for categories defined by fgf23 below and above the median combined with fepi below and above the mean this model included fgf23 and fepi as categorical variables and their product term the model was adjusted for factors shown and age , sex , smoking , systolic blood pressure , proteinuria , egfr , and pth hazard ratio product for the product term is 0.44 ( 95 % ci 0.141.41 ) reri is 1.18 ( 95 % ci 8.10 to 5.73 ) , p = 0.74 for all abbreviations , see previous tables in this study we tested the hypothesis that fgf23 resistance may contribute to the increased risk for morbidity and mortality associated with elevated fgf23 levels in patients with advanced ckd . this hypothesis was based on a recent study showing interaction between fepi and fgf23 levels on outcome in patients with mild ckd . our study showed that there was no interaction between fgf23 levels and fepi on this outcome , and thus we could not confirm this hypothesis in subjects with more advanced ckd and higher fgf23 . this suggests that in patients with advanced ckd , fgf23 itself and not fgf23 resistance determines the risk for adverse outcome . fgf23 concentrations increase during progression of ckd and several studies have shown that fgf23 is associated with mortality in hemodialysis patients as well in patients with ckd [ 16 , 17 , 4143 ] . also in our analysis fgf23 remained a predictor of outcome after adjustment of egfr . interestingly , the hr of egfr itself on the composite outcome that included progression of ckd was lost in the multivariable model . this suggests that the well - established risk of ckd may actually be accounted for by high levels of fgf23 that accompany ckd . obviously , increased fgf23 levels might merely reflect the severity of other unmeasured risk factors . cohort studies , for instance , have reported associations of fgf23 with left ventricular hypertrophy [ 44 , 45 ] , progression of kidney failure [ 16 , 19 , 20 ] , and with several cardiovascular risk factors , such as endothelial dysfunction and arterial stiffness , in the general population as well as in early ckd , in the absence of clinically evident disturbances in phosphate metabolism [ 21 , 46 , 47 ] . our findings appear to contrast with the report by dominguez et al . who concluded that the association of fgf23 with outcome was reinforced if high fgf23 was accompanied by a low fepi , suggesting that kidney fgf23 resistance modifies the association between fgf23 and outcome . studied patients that participated in the heart and soul study , which included patients with prevalent occlusive coronary artery disease and normal to slightly decreased egfr , mainly attributed to vascular disease . importantly , in the egfr range of the heart and soul study ( stage ii ckd ) , ckd is not an important contributor to overall risk . in contrast , our patients had moderate - severe ckd and often defined kidney disease whereby outcome was mainly determined by renal failure we performed interaction analyses by calculating the direction of the interaction using the low fgf23 and high fepi as reference group . dominguez documented the p - value though the direction seems opposite and used low fgf23 and low fepi as reference group . another study examined the association of a poor phosphaturic response to fgf23 , as a sign of fgf23 resistance , with abdominal aortic calcification in ckd stages 34 . in this study , fgf23 correlated well to fepi except in the group of patients with severe aortic calcification , while egfr and pth correlated well to fepi irrespective of the amount of calcification , suggesting an association between fgf23 resistance and severe aortic calcification . a shortcoming of this study however is that the ratio of fepi to fgf23 ( fepi / fgf23 ) was used as a marker of resistance where this ratio was mostly determined by the denominator fgf23 as there was no difference in fepi between groups . overall , we can not exclude the possibility that fepi values might indeed modify fgf23-related risk in patients with early - stage ckd . in patients with more severe ckd , however , the increased risk is predominantly determined by the higher fgf23 levels , and not by kidney resistance of its effects . previous studies showed that also fepi was not associated with mortality and cardiovascular events in a model adjusted for fgf23 and egfr [ 17 , 33 , 49 ] . this might be because in ckd other ( non - defined ) risk factors might outweigh the risk of tubular resistance to fgf23 , or that more advanced tubular damage inhibits phosphate reabsorption by other mechanisms than the physiological effects of fgf23 , as can be observed , for instance , in fanconi s syndrome . our results thus refute a significant contribution of fgf23-resistance to outcome in patients with ckd 3 - 4 . in addition to the arguments described above , it should , however , be borne in mind that there lacks a good representative parameter and validated measure of fgf23-resistance , in that both tmp / gfr ( ratio of the maximum rate of tubular phosphate reabsorption to the glomerular filtration rate ) and fepi might not reflect fgf23-resistance optimally , especially in the setting of more advanced ckd . our study has some limitations , all being consequences of the post hoc nature of the current analysis . the main limitation is the relatively small number of patients included . although the event rate was high , the events were mainly in the group of patients with highest fgf23 levels . especially for the interaction analysis that we performed , a larger number of patients would have been preferable . due to the small number of patients , we used a composite end - point . our study had a low power to evaluate single end - points with adjustment for competing risks . another limitation is that the study is a post hoc analysis of mostly caucasian patients with more severe ckd and results might not be directly applicable to other populations . furthermore , although serum phosphate was measured in a fasting state , fepi was not a timed specimen before blood sample collection but was calculated from 24 h urine samples . this might have led to higher fepi values , and an underestimation of fgf23 resistance because fasting phosphate concentrations are generally lower than the average daytime value . a final limitation is that we used a composite end - point , and that in larger studies outcomes may differ for individual components of the currently used composite end - points . strengths of our data are the prospectively collected data , and the inclusion primarily of patients in a stage of ckd where ckd has proven impact on clinical outcome . in conclusion , in this study in patients with a median egfr of 36 ( iqr 2744 ) ml / min/1.73 m , fepi did not modify the association of fgf23 with outcome . therefore , in advanced ckd , the role of fgf23 resistance expressed by fepi is negligible compared to the risk predicted by increased concentrations of fgf23 itself . all procedures performed in this study were in accordance with the ethical standards of the institutional and international research committee and with the 1964 helsinki declaration and its later amendments or comparable ethical standards .

backgroundcardiovascular risk is increased in patients with chronic kidney disease ( ckd ) . fibroblast growth factor 23 ( fgf23 ) has emerged as an important , independent predictor of outcome in ckd patients . high fgf23 may , however , be a reflection of renal tissue resistance to its actions , reflected by low fractional excretion of phosphate ( fepi ) . we evaluated the modifying effect of fepi on the association between fgf23 and outcome in patients with ckd stage 34.methodsan analysis was performed in a subset of 166 adult patients of two participating centers of the masterplan trial of whom urine samples at baseline were available to calculate fepi . outcome was defined as a composite of death , renal failure ( defined as need for renal replacement therapy or doubling of serum creatinine ) and cardiovascular events ( myocardial infarction , cerebrovascular accident , percutaneous transluminal coronary angioplasty or coronary artery bypass graft . patients were categorized by fgf23 and fepi . a product term was added to cox regression and reris were calculated.resultspatients had a median estimated glomerular filtration rate ( egfr ) of 36 ml / min/1.73 m2 [ interquartile range ( iqr ) 2744 ] , serum phosphate 1.04 mmol / l ( iqr 0.921.20 ) , fgf23 140 ru / ml ( iqr 81236 ) and fepi 0.32 ( iqr 0.250.44 ) . a total of 96 events occurred during 5 years of follow up . lnfgf23 was a significant , independent predictor for the composite outcome [ hazard ratio ( hr ) 2.13 , 95 % confidence interval ( ci ) 1.532.95 ] . fepi did not modify the relation between fgf23 and outcome in these patients with ckd.conclusionsour study shows that fgf23 itself , but not its renal tissue resistance as reflected by fepi , is an important risk factor for clinical events in subjects with ckd stage 34 .

Introduction Patients and methods Patients Data collection FGF23 resistance Statistical analysis Results Correlations FGF23 resistance Outcome Discussion Conclusion Ethical approval Informed consent

who used fractional excretion of phosphate ( fepi ) as a marker of fgf23 resistance , and observed that fepi modified the association of fgf23 with outcome in 872 patients with relatively mild ckd [ mean estimated glomerular filtration rate ( egfr)71 ml / min/1.73 m ] . a composite end - point was defined consisting of the combination of death , cv events ( myocardial infarction , cerebrovascular accident , percutaneous transluminal coronary angioplasty or coronary artery bypass graft ) and renal failure ( defined as need for renal replacement therapy , doubling of serum creatinine or death ) . a composite end - point was defined consisting of the combination of death , cv events ( myocardial infarction , cerebrovascular accident , percutaneous transluminal coronary angioplasty or coronary artery bypass graft ) and renal failure ( defined as need for renal replacement therapy , doubling of serum creatinine or death ) . a product term for the categorized fgf23 and fepi was added again to cox regression . the median age was 53 ( iqr 45 , 62 ) years , most patients were caucasian and the median egfr was 36 ( iqr 28 , 44 ) ml / min/1.73 m. the median of proteinuria was 0.40 ( iqr 0.20 , 1.20 ) g / day , fepi 0.32 ( iqr 0.25 , 0.44 ) and fgf23 140 ( iqr 81236 ) ru / ml . people with higher fgf23 levels more frequently used vitamin d compounds.table 1baseline characteristics of patients grouped by fgf23 and fepi ( n = 166)variabletotal group ( n = 166)low fgf23 , high fepi ( n = 31)low fgf23 , low fepi ( n = 52)high fgf23 , high fepi ( n = 51)high fgf23 , low fepi ( n = 32 ) p valuegender , male , n ( % ) 112 ( 67)26 ( 84)28 ( 54)40 ( 78)18 ( 56)0.01age , years ( range)53 ( 4562)52 ( 4364)53 ( 4561)54 ( 5063)49 ( 4364)0.38bmi ( kg / m)26 ( 2429)26 ( 2327)26 ( 2328)27 ( 2430)26 ( 2331)0.47race ( caucasian)160304851310.22cause of ckd diabetic1414720.62 renovascular91341 glomerulonephritis41718106 interstitial298687 congenital83302 cystic kidney disease223784 unknown336999 other102251systolic bp ( mmhg)126 ( 118139)124 ( 116134)124 ( 116135)130 ( 119144)125 ( 118136)0.44diastolic bp ( mmhg)75 ( 7081)75 ( 7178)76 ( 7081)75 ( 7083)76 ( 6883)0.81egfr ( mdrd4 , ml / min/1.73 m)36 ( 2744)36 ( 2945)41 ( 3652)27 ( 2034)36 ( 2742)<0.01proteinuria ( g/24 h)0.40 ( 0.201.20)0.40 ( 0.201.20)0.30 ( 0.10 - 0.58)0.80 ( 0.301.50)0.60 ( 0.201.38)0.01total cholesterol ( mmol / l)4.99 0.985.10 ( 4.505.80)5.00 ( 4.405.50)4.80 ( 4.005.30)5.15 ( 4.536.08)0.18serum phosphate ( mmol / l)1.04 ( 0.92 - 1.20)1.02 ( 0.941.13)1.00 ( 0.891.09)1.17 ( 0.961.40)1.04 ( 0.901.20)0.03serum calcium ( mmol / l)2.35 0.152.37 ( 2.302.50)2.38 ( 2.282.44)2.34 ( 2.262.42)2.33 ( 2.222.40)0.49serum albumin ( mmol / l)40 441 ( 4043)40 ( 3842)39 ( 3742)40 ( 3741)0.10serum pth ( pmol / l)10.15 ( 6.6816.18)10.15 ( 8.1816.73)9.00 ( 4.6512.00)14.25 ( 9.0023.23)10.00 ( 6.0815.85)<0.01serum cfgf-23 ( ru / ml)140 ( 81236)78 ( 54114)81.25 ( 54.10115.00)278 ( 203439)204 ( 159289)<0.01fractional phosphate excretion0.32 ( 0.250.44)0.41 ( 0.360.46)0.24 ( 0.190.28)0.49 ( 0.400.54)0.26 ( 0.220.29)<0.01fractional calcium excretion0.00 ( 0.000.01)0.00 ( 0.000.01)0.00 ( 0.000.01)0.00 ( 0.000.01)0.00 ( 0.000.01)0.99vitamin d drugs ( n , % ) 33 ( 20)3 ( 10)5 ( 10)18 ( 35)7 ( 22)0.004diabetes35281870.01smokers27261540.02events / py96/79211/15825/25737/23623/141events/1000 py1217097157163median with interquartile ranges ( 2575 % ) for skewed datamean with standard deviation ( sd ) for normally distributed data fgf23 fibroblast growth factor 23 , fepi fractional excretion of phosphate , bmi body mass index , ckd chronic kidney disease , bp blood pressure , egfr estimated glomerular filtration rate , mdrd4 4-point modification of diet in renal disease , pth parathormone , py person years baseline characteristics of patients grouped by fgf23 and fepi ( n = 166 ) median with interquartile ranges ( 2575 % ) for skewed data mean with standard deviation ( sd ) for normally distributed data fgf23 fibroblast growth factor 23 , fepi fractional excretion of phosphate , bmi body mass index , ckd chronic kidney disease , bp blood pressure , egfr estimated glomerular filtration rate , mdrd4 4-point modification of diet in renal disease , pth parathormone , py person years univariate analysis between fgf23 and parameters of kidney function and phosphate metabolism showed a significant inverse correlation between fgf23 and egfr ( r = 0.43 , p < 0.01 ) . we observed a significant positive correlation between fgf23 and serum phosphate ( r = 0.29 , p < 0.01 ) , fepi ( r = 0.36 , p < 0.01 ) , urine phosphate / creatinine ratio ( r = 0.17 , p = 0.03 ) and pth ( r = 0.30 , p < 0.01 ) . after a median of 4.8 years of follow up , 96 of the 166 patients ( 59 % ) reached an endpoint defined as death ( 8/166 ) , cv event ( 14/166 ) , renal replacement therapy or doubling of creatinine ( 74/166 ) . fgf23 , fibroblast growth factor 23 ; egfr , estimated glomerular filtration rate ; tmp / gfr , ratio of the maximum rate of tubular phosphate reabsorption to the glomerular filtration rate ; pth , parathormone univariate and multivariate cox regression analysis for combined outcome hr hazard ratio , ci confidence interval , for other abbreviations , see table 1 cox curve for quartiles of fgf23 and the effect on composite outcome . fgf23 , fibroblast growth factor 23 ; egfr , estimated glomerular filtration rate ; tmp / gfr , ratio of the maximum rate of tubular phosphate reabsorption to the glomerular filtration rate ; pth , parathormone tables 3 and 4 further show the hazard ratios for the associations between fgf23 and fepi categories and outcome . similarly , the hazard ratio of a high fgf23 combined with low fepi on renal outcome was 3.44 , lower than expected from the sum of both individual factors , hr = 7.77 [ = e].table 3multivariate cox regression on combined outcome for categories defined by fgf23 below and above the median combined with fepi below and above the meancombined survivalparticipantseventshr95 % cilow fgf23/high fepi31111referencelow fgf23/low fepi52252.561.135.80high fgf23/high fepi51373.181.476.87high fgf23/low fepi32234.151.859.30this model included fgf23 and fepi as categorical variables and their product termthe model was adjusted for factors shown and age , sex , smoking , systolic blood pressure , proteinuria , egfr , pthhazard ratio product for the product term is 0.51 ( 95 % ci 0.191.34 ) , p = 0.17reri is 0.60 ( 95 % ci 4.81 to 3.70 ) , p = 0.79 reri , relative excess risk due to interaction , for other abbreviations , see previous tablestable 4multivariate cox regression on renal outcome for categories defined by fgf23 below and above the median combined with fepi below and above the meanrenal survivalparticipantseventshr95 % cilow fgf23/high fepi3191referencelow fgf23/low fepi52202.400.816.34high fgf23/high fepi51343.221.317.84high fgf23/low fepi32183.441.378.62this model included fgf23 and fepi as categorical variables and their product termthe model was adjusted for factors shown and age , sex , smoking , systolic blood pressure , proteinuria , egfr , and pthhazard ratio product for the product term is 0.44 ( 95 % ci 0.141.41)reri is 1.18 ( 95 % ci 8.10 to 5.73 ) , p = 0.74for all abbreviations , see previous tables multivariate cox regression on combined outcome for categories defined by fgf23 below and above the median combined with fepi below and above the mean this model included fgf23 and fepi as categorical variables and their product term the model was adjusted for factors shown and age , sex , smoking , systolic blood pressure , proteinuria , egfr , pth hazard ratio product for the product term is 0.51 ( 95 % ci 0.191.34 ) , p = 0.17 reri is 0.60 ( 95 % ci 4.81 to 3.70 ) , p = 0.79 reri , relative excess risk due to interaction , for other abbreviations , see previous tables multivariate cox regression on renal outcome for categories defined by fgf23 below and above the median combined with fepi below and above the mean this model included fgf23 and fepi as categorical variables and their product term the model was adjusted for factors shown and age , sex , smoking , systolic blood pressure , proteinuria , egfr , and pth hazard ratio product for the product term is 0.44 ( 95 % ci 0.141.41 ) reri is 1.18 ( 95 % ci 8.10 to 5.73 ) , p = 0.74 for all abbreviations , see previous tables univariate analysis between fgf23 and parameters of kidney function and phosphate metabolism showed a significant inverse correlation between fgf23 and egfr ( r = 0.43 , p < 0.01 ) . we observed a significant positive correlation between fgf23 and serum phosphate ( r = 0.29 , p < 0.01 ) , fepi ( r = 0.36 , p < 0.01 ) , urine phosphate / creatinine ratio ( r = 0.17 , p = 0.03 ) and pth ( r = 0.30 , p < 0.01 ) . after a median of 4.8 years of follow up , 96 of the 166 patients ( 59 % ) reached an endpoint defined as death ( 8/166 ) , cv event ( 14/166 ) , renal replacement therapy or doubling of creatinine ( 74/166 ) . fgf23 , fibroblast growth factor 23 ; egfr , estimated glomerular filtration rate ; tmp / gfr , ratio of the maximum rate of tubular phosphate reabsorption to the glomerular filtration rate ; pth , parathormone univariate and multivariate cox regression analysis for combined outcome hr hazard ratio , ci confidence interval , for other abbreviations , see table 1 cox curve for quartiles of fgf23 and the effect on composite outcome . fgf23 , fibroblast growth factor 23 ; egfr , estimated glomerular filtration rate ; tmp / gfr , ratio of the maximum rate of tubular phosphate reabsorption to the glomerular filtration rate ; pth , parathormone tables 3 and 4 further show the hazard ratios for the associations between fgf23 and fepi categories and outcome . similarly , the hazard ratio of a high fgf23 combined with low fepi on renal outcome was 3.44 , lower than expected from the sum of both individual factors , hr = 7.77 [ = e].table 3multivariate cox regression on combined outcome for categories defined by fgf23 below and above the median combined with fepi below and above the meancombined survivalparticipantseventshr95 % cilow fgf23/high fepi31111referencelow fgf23/low fepi52252.561.135.80high fgf23/high fepi51373.181.476.87high fgf23/low fepi32234.151.859.30this model included fgf23 and fepi as categorical variables and their product termthe model was adjusted for factors shown and age , sex , smoking , systolic blood pressure , proteinuria , egfr , pthhazard ratio product for the product term is 0.51 ( 95 % ci 0.191.34 ) , p = 0.17reri is 0.60 ( 95 % ci 4.81 to 3.70 ) , p = 0.79 reri , relative excess risk due to interaction , for other abbreviations , see previous tablestable 4multivariate cox regression on renal outcome for categories defined by fgf23 below and above the median combined with fepi below and above the meanrenal survivalparticipantseventshr95 % cilow fgf23/high fepi3191referencelow fgf23/low fepi52202.400.816.34high fgf23/high fepi51343.221.317.84high fgf23/low fepi32183.441.378.62this model included fgf23 and fepi as categorical variables and their product termthe model was adjusted for factors shown and age , sex , smoking , systolic blood pressure , proteinuria , egfr , and pthhazard ratio product for the product term is 0.44 ( 95 % ci 0.141.41)reri is 1.18 ( 95 % ci 8.10 to 5.73 ) , p = 0.74for all abbreviations , see previous tables multivariate cox regression on combined outcome for categories defined by fgf23 below and above the median combined with fepi below and above the mean this model included fgf23 and fepi as categorical variables and their product term the model was adjusted for factors shown and age , sex , smoking , systolic blood pressure , proteinuria , egfr , pth hazard ratio product for the product term is 0.51 ( 95 % ci 0.191.34 ) , p = 0.17 reri is 0.60 ( 95 % ci 4.81 to 3.70 ) , p = 0.79 reri , relative excess risk due to interaction , for other abbreviations , see previous tables multivariate cox regression on renal outcome for categories defined by fgf23 below and above the median combined with fepi below and above the mean this model included fgf23 and fepi as categorical variables and their product term the model was adjusted for factors shown and age , sex , smoking , systolic blood pressure , proteinuria , egfr , and pth hazard ratio product for the product term is 0.44 ( 95 % ci 0.141.41 ) reri is 1.18 ( 95 % ci 8.10 to 5.73 ) , p = 0.74 for all abbreviations , see previous tables in this study we tested the hypothesis that fgf23 resistance may contribute to the increased risk for morbidity and mortality associated with elevated fgf23 levels in patients with advanced ckd . in addition to the arguments described above , it should , however , be borne in mind that there lacks a good representative parameter and validated measure of fgf23-resistance , in that both tmp / gfr ( ratio of the maximum rate of tubular phosphate reabsorption to the glomerular filtration rate ) and fepi might not reflect fgf23-resistance optimally , especially in the setting of more advanced ckd . in conclusion , in this study in patients with a median egfr of 36 ( iqr 2744 ) ml / min/1.73 m , fepi did not modify the association of fgf23 with outcome .

the increasing quantity of available biomedical data pose a challenge to life scientist wishing to explore a particular research problem . although emerging bioinformatics services enable structured access to increasingly complex datasets , it is often the case that the primary data is only available in the published literature , and needs to be extracted and stored in a standardized format before it can be leveraged upon ( 1 ) . this is the main motivation behind biomedical curation activities : to help the life sciences community to make sense of all the data that is accumulating ( 2 ) . although human curation offers the best guarantee of high - quality results , it suffers from severe bottlenecks that have long been recognized in the curation community . the most pressing problem is that of efficiency of the process : despite the fact that typically several databases attempt to focus on a particular type of biological data , and often collaborate at least sufficiently to prevent duplication of effort and ensure compatibility of resulting data formats , it is impossible for human curators to keep up with the growing pace of publication . nobody will ever be able to manually annotate all the macromolecular biological entities that exist on this planet , and consequently automatization is the only solution . ( 2 ) nobody will ever be able to manually annotate all the macromolecular biological entities that exist on this planet , and consequently automatization is the only solution . ( 2 ) on the other hand automated text - mining tools can not offer sufficient reliability to be employed indiscriminately without human supervision of the results that they deliver . therefore , the ideal solution is to combine the best capabilities of automated systems with human supervision by highly qualified domain experts . in this article , we describe recent experiments aimed at assessing the potential contribution of a specific curation tool ( odin ) to the curation process of a well - known database ( pharmgkb ) . in the rest of this section the pharmacogenomics knowledge base ( pharmgkb ) is a publicly available online worldwide resource ( www.pharmgkb.org ) ( 3,4 ) . the mission of pharmgkb is to collect , encode and disseminate knowledge about the impact of human genetic variations on drug responses , contributing to the drive towards personalized medicine for better therapeutics . pharmgkb is an nih - funded resource , which over the past 11 years has maintained a very high - quality manually curated knowledge base of pharmacogenomics facts , curated by a team of phd and masters level scientists . one of the many tasks of the pharmgkb curators is to review past and current literature and add any relevant pharmacogenetic or genomic articles to the pharmgkb database . the curators identify relevant journal articles , largely selected from a set of about 20 journals that are followed , which include major pharmacogenomic journals and publications published by the pgrn ( pharmacogenomics research network ) . they then read the abstract or full text if necessary , and populate the knowledge base with information about the genes , drugs and phenotypes discussed . in the past , this information was gathered in microsoft excel spreadsheets and uploaded to the database . today , the information is entered through a web - based graphical user interface ( gui ) developed in - house to fit the curators ' needs , and captures data that is far more structured than in the past , such as population characteristics of the study group described in an article , and p - values of associations found between genetic variants and drug response . curators can manually enter drug and gene terms for each article using auto - complete fields that draw on pharmgkb 's standardized vocabularies . additionally , in the past , in each article curators captured the entities discussed in the form of a list of genes , drugs and phenotypes , which did not enable users to presume binary relationships between a single gene and a single drug in a pharmgkb literature annotation . today , the relationships between entities are binary , such that for example a gene drug relationship is explicitly captured , including some degree of specification regarding the type of interaction ( is associated with , a detailed description of the types of annotations in pharmgkb has been published previously ( 5 ) . the current curator gui assists the curators in their process using basic text mining by suggesting entities found in the article , but does not pre - populate fields or highlight any information found within the article text . the pharmgkb team is currently working on developing natural language processing and machine learning methods to aid in the future in tasks such as document retrieval and information extraction . the ontogene group at the university of zurich has developed advanced solutions for several text - mining tasks based upon advanced natural language processing technologies , which have been proven to be state - of - the - art by participation in several competitive evaluations ( 69 ) . the ontogene text - mining system is based on a standard nlp pipeline , composed of efficient modules for sentence splitting , tokenization , entity recognition , syntactic chunking and dependency parsing . its entity recognition component has been shown in the recent calbc shared evaluation ( 9 ) to be highly efficient and capable of delivering competitive results for several entity categories . its relation mining component has been used in the biocreative 2009 evaluation to deliver the best results for the identification of protein protein interactions ( 7 ) . [ a more detailed description of the architecture of the ontogene text - mining system is beyond the scope of this article , for further details the interested reader is invited to consult the following publications ( 6,7 ) . specific adaptations that were carried out for the pharmgkb task are described in separate forthcoming publications ( 10,11 ) ] . the results of the ontogene text - mining system are made accessible through a curation system called odin ( ontogene document inspector ; this tool is not connected in any way with the recently introduced commercial text analytics system called odintext . ) which allows a user to dynamically inspect the results of their text - mining pipeline . a previous version of odin was used for participation in the interactive curation task ( iat ) of the biocreative iii competition ( 12 ) . however , the curators who used the system commented extremely positively on its usability for a practical curation task . more recently , the ontogene group created a version of odin that allows inspection of abstracts automatically annotated with pharmgkb entities [ the annotation is performed using the ontogene pipeline ( http://www.ontogene.org/pharmgkb/ ) ] . users can access either preprocessed documents , or enter any pubmed identifier and have the corresponding abstract processed on the fly. for the documents already in pharmgkb it is also possible to inspect the gold standard and compare the results of the system against the gold standard . the curator can inspect all entities annotated by the system , and easily modify them if needed ( removing false positives with a simple click , or adding missed terms if necessary ) . the modified documents can be sent back for reprocessing if desired , obtaining therefore modified candidate interactions . the user can also inspect the set of candidate interactions generated by the system , and act upon them just as on entities , i.e. confirm those that are correct , remove those that are incorrect . candidate interactions are presented ordered according to the score that has been assigned to them by the text - mining system , therefore the curator can choose to work with only a small set of highly ranked candidates , ignoring all the rest . odin , which is based on a client server architecture , maintains a log of the interaction with the curator , which could be used for later revision by a supervisor or for reversing some specific annotation decisions . at the end of a session the modified document and its annotations are sent back to the server , together with the log , for permanent storage , and can be accessed again at the next session , which could take place on a different remote client . additionally , the curator can choose to export the annotations to a local file in a simplified format ( e.g. comma - separated values ) . first of all , text - mining tools can provide a help in the initial triage stage in order to decide which papers should be inspected by the expert curators . text classification tools are nowadays capable of reliably processing large sets of articles in order to score them and provide a ranked list of candidate papers , which can then be used to prevent inspection of less promising articles . this process is typically based on machine learning tools that can distinguish interesting and less interesting articles on the basis of similarities with previously classified articles . during inspection of individual articles , it can be very helpful for curators to use a system capable of locating the entities of interest within the article , and disambiguate them as reliably as possible . this process is based on named entity recognition tools , which recently have made considerable progress and are now capable of recognizing several types of biomedical entities with great reliability . for example , recent results in the biocreative competition ( 13 ) have shown that several systems are capable of recognizing and disambiguating gene names with f - scores above 80% . databases that are entity - focused can immediately profit from such tools , as the curators will be able to manually filter the candidates suggested by the system at greater speed , compared with a manual extraction from the paper , which would involve ( i ) spotting the mentions in the paper , ( ii ) decide which database entities are actually intended . the next major challenge for the introduction of text - mining systems within curation workflows is the automated detection of relations , which is relevant for several databases . tools that can reliably detect entity interactions are in general much less efficient than named entity recognition tools due to the much greater complexity of the problem . in order to produce candidate interactions given that some errors are inherent in this process , generation of candidate entity pairs will inevitably result in compounding that error , leading to lower performance . contextual clues that can help to identify an interaction candidate are typically very sparse , making difficult to apply machine learning techniques . nonetheless , much progress has been achieved recently , as results in the biocreative ii ( 14 ) and ii.5 ( 15 ) competitions show , and therefore it is now appropriate to start practical experimentation through collaborations between developers of text - mining solutions and database groups as potential users . although immediate integration in the curation workflow might not be the goal , these joint experiment help both groups in deciding how to improve their activities . text - mining developers will receive feedback on the quality of their systems and gain an understanding of the specific needs of the curators [ ( 12 ) stresses the importance of understanding the biocurator 's curation workflow ] , and curation groups will gain a better understanding of the current potential of technologies , which are now still experimental , but might soon become mainstream , and thus be able to choose the optimal point for integration in their workflows . additionally , the feedback provided to text - mining developers will render future systems more usable in practical applications . the need to pair developers with curators has been recognized by the organizers of the biocreative competition . a new experimental task ( iat ) dedicated to the evaluation of interactive curation environments although the specific task chosen for the experimentation was an entity recognition task , several of the conclusions reached through analysis of participating systems are applicable to all types of interactive curation environments . addressing usability of text - mining systems is a novel aspect of this task . usability enables the users to find , interact with , share , compare and manipulate important information more effectively and efficiently ( 12 ) . although fully unsupervised extraction of information from the literature is , for some time at least , unrealistic , text - mining tools are already sufficiently reliable to be used to provide hints to the curators , in order to speed up their activities . such a help is sorely needed , as it is already clear that manual curation can not keep up with the rate of data generation ( 16 ) . curatorial work done with the assistance of a text - mining system has already been shown to be much more efficient than when done by human readers without support ( 17 ) . the authors of this study state that : for biologists , an automated system with high recall and even moderate precision confers a great advantage over skimming text by eye. examples of well - known text - mining solutions are ihop ( 18 ) and chillibot ( 19 ) . among the systems developed to support the curation process , one of they use a manually annotated corpus ( gold standard ) to simulate an assisted curation environment , where the curators are given either gold standard data or the output of an ( imperfect ) nlp pipeline . ( 20,21 ) presents a system developed for the curators of flybase , a database for drosophila genetics and molecular biology . although the document analysis is based on a conventional nlp pipeline , including the dependency parser rasp ( 23 ) , the curator 's interface has been developed in strict collaboration with the end - users . ( 24 ) discuss how well the performance of a text - mining system ( in their case tailored to identify mentions of protein mutations ) , when evaluated with conventional techniques , translates into real utility of the system for a curation task . textpresso is another well - known text - mining system that is characterized by the usage of ontological categories of biological concepts ( 17,25 ) , as well as by processing full papers . a variant of textpresso ( pharmspresso ) has been used for automatic annotation of pharmacogenomic literature for pharmgkb , but was never integrated with the manual curation process ( 26 ) . although the full ontogene pipeline can deliver reliably a ranked list of candidate interactions , which can then be used by curators as prompts for annotation of novel articles , the experiment described in this article centered upon the validation of existing relations from pharmgkb . revalidation of existing data is a common practice of several biological databases , for example ( 2 ) mentions several steps of re - annotation for swiss - prot , one of the most well - known and authoritative databases . the main aim of the experiment was therefore to evaluate the usability of the interface , rather than the capabilities of the underlying text - mining tools . as ( 12 ) points out , an evaluation task must be chosen to be feasible in a given time frame , considering both the time needed by developers to adapt the existing text - mining system to the specific needs of the applications and the time available to curators for the verification of the results . we started by considering the set of articles already curated by pharmgkb , processing them with the ontogene relation extraction system . we then automatically compared for each article the results of the relation mining system with the manually extracted interactions , and computed the common subset . in general , we would expect the text - mining system to deliver a larger set of interactions than those manually curated ( ideally covering all of them ) . in practice , since in this experiment only abstracts rather than full text were used , this was true only in 3059 articles out of 5378 . false negative , i.e. a relation that was not detected by the text - mining system . ( this could be due to several factors , for example an interaction that is mentioned only in the full text and not in the abstract will be obviously impossible for the system to detect with the current settings . another possible source of false negatives is due to the way the pharmgkb data was created , i.e. for each paper , a list of genes and drugs discussed was kept , and in some cases interactions among those entities were simply hypothesized and not actually verified . therefore , some of the interactions in the pharmgkb data are not expected to be true positives ) . if full articles had been processed , we would have expected the number of false negatives to be much lower . full articles , however , are difficult to process for several reasons , most of which have little to do with text mining , such as widely different formats , or copyright restrictions , which in some cases explicitly prohibit text - mining applications . we decided for this experiment to use only articles from the set where all interactions were found by the ontogene pipeline . the aims of the experiment were the following : ( i ) evaluate the usability of the interface for revalidation of pharmgkb relationships , ( ii ) estimate whether the ranking of interactions provided by the text - mining system correlates well with decisions taken by the curators . the previously developed odin system was adapted to the needs of pharmgkb , on the basis of a close interaction with the curators . during development the following recommendations by ( 12 ) ( adapted to the specific needs of our application ) were taken into consideration : support for interactive disambiguation of domain entities;an editable list of candidate entities or interactions;a view of the document correlated with the candidate interactions ( when an interaction is selected , the corresponding entities are highlighted);ability to sort the results according to different criteria;ability to collect event and timing information at the session level ; andability to export the results in a suitable format ( e.g. csv ) . support for interactive disambiguation of domain entities ; an editable list of candidate entities or interactions ; a view of the document correlated with the candidate interactions ( when an interaction is selected , the corresponding entities are highlighted ) ; ability to sort the results according to different criteria ; ability to collect event and timing information at the session level ; and ability to export the results in a suitable format ( e.g. csv ) . the odin system allows the user to verify and modify every single annotation provided by the system , at the entity level as well as at the interaction level ( a ) . it provides a ranked list of candidate interactions ( b ) , which additionally can be sorted by the user according to different criteria ( d ) . the interface is structured around three panels : term editing panel , document panel and results panel . the term editing panel ( not shown in the pictures in this article ) the document panel and results panel are actively connected , in that selection of items from the results panel will result in their visualization ( highlighting ) in the document panel . every action of the user is stored in a log that is stored at the server level ( e ) with timing information ( figure 2 ) . finally , the results can be exported as a csv file ( f ) and additional formats can easily be added upon request . the close interaction among curators and system developers allowed the latter to implement a number of suggestions that made the usage of the system more effective . for example , in the initial demonstration the entities participating in an interaction were represented only by the pharmgkb identifier of the participating entities . the curators pointed out that it was not immediately obvious to them which entity was referred to by the identifier without consulting the database ( that the odin system allows by simple click on the identifier , see figure 1 ) , so the reference name of the entity was added . this , however , made the table of interactions more cluttered , and the curators pointed to the fact that they might optionally want to remove some information from the table ( such as the entity identifiers ) . the developers therefore modified the interface to allow precisely this type of modification directly by the user ( i.e. selection of which fields they want to be displayed ) , see figure 3 . some of the information hidden in this way could further be displayed as unobtrusive tooltip windows on mouseover by the user , another option that was added upon suggestion by the curators . ( 1 ) stresses the importance of being able to hide fields of negligible value to the curators thereby distracting their attention unnecessarily. figure 1.inspection of pharmgkb entry associated with a given entity . log of user actions as stored on the ontogene server . modifying the presentation of the interactions . another example of the fruitfullness of the interaction between developers and curators is the addition of different types of confirmation boxes for an interaction . instead of a simple confirm / reject choice , the maintainers of the database suggested the need for a more fine - grained choice . negative interactions , i.e. interactions that are stated in the paper as not to hold under the conditions investigated . another wish was to be able to state that the abstract inspected for the experiment did not provide sufficient information to either confirm or reject the proposed relation . figures 1 , 3 , and 4 show these options as four tick boxes in the top right corner of the picture , which correspond to confirm relation , reject , needs full text , negative relation confirmed. figure 4.entities which participate in the selected interaction are highlighted in the document panel . after a preliminary test phase on a few selected sample articles , which allowed the curators to gather some familiarity with odin , and the developers to fix the remaining issues , the validation experiment could start . a set of 125 articles was selected from the 3059 articles where the ontogene pipeline could detect all of the relations originally annotated by pharmgkb . the selection was based on a randomized stratified sampling process , in order to generate a distribution of relations per article that would be roughly equivalent to the distribution in the whole set . this set was split into five sets of 25 articles each , which were then randomly assigned to pharmgkb curators . this sampling lead to the following distribution of articles per curator : 8 articles with 2 relations , 9 with 3 relations , 3 with 4 relations , 3 with 5 relations , 1 with 67 relations , 1 with 89 relations , 1 with 1020 relations . the data sets were at all stages identified only by a symbolic reference ( a , b , c , d , e ) , which was randomly assigned to the curators ( and known only to each of them ) , in order to ensure anonymity . this was done to avoid generating the impression that the result of the experiment could be used to evaluate individual performance . the full cooperation of the curators is of utmost importance to guarantee unskewed results , therefore we took care to prevent the possibility of identification . curators were then asked to inspect the articles in the assigned set with the odin system and then use it to validate the interactions . during this process all of their actions were logged using the symbolic reference ( that they had to enter into the system at the beginning of the process ) as an identifier . the resulting validation decisions are automatically saved by the system and transferred to a server , together with detailed logs of the activity . this data set forms the basis of the evaluation presented in the next section . at the end of the curation experiment we asked the curators to fill a questionnaire that was partly modeled on the questions used in the bciii iat task ( 12 ) . the feedback received through this survey is discussed at the end of the next section . as explained in the previous section , the experiments described in this article were centered on the revalidation of relations already stored in pharmgkb . in order to evaluate the correlation of the rankings provided by the text - mining system with curator 's validation decisions , we were limited to use only articles for which all pharmgkb interactions could be detected by the text - mining system . since only abstracts were used for automatic processing , only about 56% of pharmgkb curated articles ( 3059 ) could be taken into consideration . we also observed that articles that contained a single curated relation would not be particularly interesting for this experiment , since it can be presumed that the vast majority of these cases are correct , and in any case there is no ranking to evaluate . additionally , articles containing more than 20 interactions were also excluded , because there was a very limited number of them , and they would require too much time for revalidation . excluding these cases , we were left with a set of 1407 articles . out of this set , we selected by stratified random sampling five sets of 25 articles each , as described in the previous section . in the rest of this section , we describe in detail the results of our experiments through descriptive statistics computed from the logs of the interactions . confirm ( the abstract supports the interactions ) , reject ( there is no support in the abstract for the interaction ) , negative ( the abstract states that the mentioned entities do not interact ) , needs full text ( there is no sufficient information in the abstract to decide either way ) . the pie chart on the left of figure 5 shows the total distribution of these decision across all articles . however , this distribution appears to be strongly dependent on the type of the entities participating in a relationship . the distribution of such decision by relation type is shown on the left of figure 6 . the relationship drug / gene has been chosen to be the main focus of future revalidation work and the results show that this type of interaction has a relatively low rejection rate . one of the aims of the experiment was to verify how the ranking of interactions produced by the text - mining system correlates with validation decisions by the curators . the bar chart on the right of figure 5 shows a clear positive correlation at least for the best ranked cases ( ranks 15 ) . the proportion of interactions that the curators confirm as positive is greater at rank 1 and gradually decreases . at higher ranks there is no visible correlation , but this is partially due to the sparsity of data ( in general there are fewer articles that have 5 interactions , and very few that have more than 20 ) ( among the articles selected for the experiment , 30.06% have 2 interactions , 37.81% 3 interactions , 6.39% 4 interactions , 11.02% 5 , 5.90% 6 or 7 , 4.33% 8 or 9 and 4.48% 1020 . ) . after validating all relations in each document , the curators were asked to express their opinion about the quality of concept identification ( provided by the text - mining system ) for that particular document . however , since this comment was not mandatory , in about 1/4 of articles such judgments are missing . figure 7 on the left shows the totals , and on the right distributed per curator . these values represent the perceived quality of concept identifications , i.e. the subjective judgment of the curators about the correctness of the entities suggested by the system . we also measured ( through the logs ) the exact time span between opening of a document and saving it after completing the validation of its interactions . this time was then divided by the number of interactions that had to be validated in each specific document . the average time needed for the validation of each interaction varies strongly among different curators , from 15 s up to 122 s. the box - and - whisker plots in figure 8 illustrate the distribution of the mean time ( in seconds ) used for the curation of all relations of an article . the graph on the left shows these timings in relation to the curator 's subjective judgment about the quality of concept recognition in the article , the graph on the right shows the timings per curator . the bottom whisker gives the minimum mean time , the top whisker gives the maximum mean time . whiskers are shortened as usual to a length of 1.5 the box length and possible outliers are plotted separately with points . the bottom line of box is the first quantile , the upper line of box is the third quantile . figure 8.box-and-whisker plots illustrating curation time ( on the left according to the decision taken , on the right per curator ) . box - and - whisker plots illustrating curation time ( on the left according to the decision taken , on the right per curator ) . an interesting research question is to evaluate whether the quality of a text - mining solution has a correlation with the time necessary for the curation of a specific article . the left graph in figure 8 clearly shows such a correlation for our experiment : articles where concept identification was regarded by curators as bad was selected in a too small number of cases , and therefore results in this category might not be very informative . as mentioned in the previous section , at the end of the experiment the curators were asked to fill in a brief questionnaire in order to collect subjective feedback about their experience with the curation environment . below we list the questions that were asked and the feedback received.q1 do you consider the system easy and intuitive to use?not intuitive at allpartly intuitive ( 25%)mostly intuitive ( 75%)very intuitiveq2 do you consider the organization of the panels to be practical?not practical at allpartly practicalmostly practical ( 100%)very practicalq3 what aspects of the system are most appealing to you ? having the abstract to the left of the terms makes sense to me . it did help in that the entities in a proposed relationship were listed already so matched a pharmgkb term and were spelled correctly . that saves a bit of time.speeds things up because relationships are already entered just need to verify themhighlighting the genes and drugs in the abstract , especially matching gene synonyms to gene symbol.q4 what problems / limitations of the system did you notice ? i would like to see the mapping of terms that are different in the abstract versus displayed gene names or diseases in the panel . i would like if just the gene and drug would be highlighted in the sentences used by the program to define the relationship not every mention of the gene and drug in the abstract . at the moment too many objects are highlighted.whenever i checked the green checkbox , it would turn on the highlighting for that row(i did not have that experience with the other choices).i think it missed some relationships.sometimes needs to resort to full text . some gene / drug relationships may not be identified through the system but are true relationships . would be more valuable to extract the types of relationships between the concepts ( eg . metabolize , transport , inhibit , induce etc)q5 please mention any aspect of the system that did not appeal to you , or suggestions for changes . i find highlighting and underlining to be distracting and to clutter up the thing i m trying to read ; i do not find them helpful . maybe there was an option to do that and i simply did not notice it.it would be helpful to have a place to add free text note to each relationshipwould be more valuable to extract the types of relationships between the concepts ( eg . metabolize , transport , inhibit , induce , treat etc ) , also would be nice to extract genetic variations as another type of concept.q6 was the system helpful in performing the validation task?not helpful at allpartly helpfulmostly helpful ( 75%)very helpful ( 25%)q7 would you consider using a similar system for your regular curation task?noprobably notprobably yes ( 75%)yes ( 25%)q8 do you agree that a similar system could increase the efficiency of the manual curation process?noprobably notprobably yes ( 75%)yes ( 25% ) q1 do you consider the system easy and intuitive to use?not intuitive at allpartly intuitive ( 25%)mostly intuitive ( 75%)very intuitive partly intuitive ( 25% ) mostly intuitive ( 75% ) q2 do you consider the organization of the panels to be practical?not practical at allpartly practicalmostly practical ( 100%)very practical mostly practical ( 100% ) q3 what aspects of the system are most appealing to you ? having the abstract to the left of the terms makes sense to me . it did help in that the entities in a proposed relationship were listed already so matched a pharmgkb term and were spelled correctly . that saves a bit of time.speeds things up because relationships are already entered just need to verify themhighlighting the genes and drugs in the abstract , especially matching gene synonyms to gene symbol . having the abstract to the left of the terms makes sense to me . it did help in that the entities in a proposed relationship were listed already so matched a pharmgkb term and were spelled correctly . speeds things up because relationships are already entered just need to verify them highlighting the genes and drugs in the abstract , especially matching gene synonyms to gene symbol . i would like to see the mapping of terms that are different in the abstract versus displayed gene names or diseases in the panel . i would like if just the gene and drug would be highlighted in the sentences used by the program to define the relationship not every mention of the gene and drug in the abstract . at the moment too many objects are highlighted.whenever i checked the green checkbox , it would turn on the highlighting for that row(i did not have that experience with the other choices).i think it missed some relationships.sometimes needs to resort to full text . some gene / drug relationships may not be identified through the system but are true relationships . would be more valuable to extract the types of relationships between the concepts ( eg . metabolize , transport , inhibit , induce etc ) i would like to see the mapping of terms that are different in the abstract versus displayed gene names or diseases in the panel . i would like if just the gene and drug would be highlighted in the sentences used by the program to define the relationship not every mention of the gene and drug in the abstract . whenever i checked the green checkbox , it would turn on the highlighting for that row(i did not have that experience with the other choices ) . some gene / drug relationships may not be identified through the system but are true relationships . would be more valuable to extract the types of relationships between the concepts ( eg . metabolize , transport , inhibit , induce etc ) q5 please mention any aspect of the system that did not appeal to you , or suggestions for changes . i find highlighting and underlining to be distracting and to clutter up the thing i m trying to read ; i do not find them helpful . maybe there was an option to do that and i simply did not notice it.it would be helpful to have a place to add free text note to each relationshipwould be more valuable to extract the types of relationships between the concepts ( eg . metabolize , transport , inhibit , induce , treat etc ) , also would be nice to extract genetic variations as another type of concept . i find highlighting and underlining to be distracting and to clutter up the thing i m trying to read ; i do not find them helpful . maybe there was an option to do that and i simply did not notice it . it would be helpful to have a place to add free text note to each relationship would be more valuable to extract the types of relationships between the concepts ( eg . metabolize , transport , inhibit , induce , treat etc ) , also would be nice to extract genetic variations as another type of concept . q6 was the system helpful in performing the validation task?not helpful at allpartly helpfulmostly helpful ( 75%)very helpful ( 25% ) q7 would you consider using a similar system for your regular curation task?noprobably notprobably yes ( 75%)yes ( 25% ) q8 do you agree that a similar system could increase the efficiency of the manual curation process?noprobably notprobably yes ( 75%)yes ( 25% ) usability issues are crucial for the acceptance of any specific it tool by the end users . in the case of biomedical curation , it is essential that text - mining results are delivered to the curator in a transparent fashion , without the need of dealing with system technicalities , in order to prevent cognitive overload . the tool should not disrupt the rhythm , flow of thinking and mental modeling process of the users , otherwise even minor problems with the interface could turn into major disruptions ( 1 ) . ideally the user should be put in a situation where he / she can make a quick but well - motivated decision based on the information provided by the system . according to ( 1 ) the output of a text - mining system should respect the following five criteria in order to be really helpful in the curation process : relevance : a connection to the disease of interest or to synonyms , homologs of interest.valid : not likely to be a false positive ( has some statistic of significance associated with it).credible : trustworthy methods generated the evidence plus numerous lines of evidence , number of research publications , convincing public metadata.plausible : function , location / structure , interaction type , biological process , and cellular component suggest an explanatory story.manageable : enough interactions for promising insights but not so many as to be overwhelming ( roughly between 10 and 50 ) . relevance : a connection to the disease of interest or to synonyms , homologs of interest . valid : not likely to be a false positive ( has some statistic of significance associated with it ) . credible : trustworthy methods generated the evidence plus numerous lines of evidence , number of research publications , convincing public metadata . plausible : function , location / structure , interaction type , biological process , and cellular component suggest an explanatory story . manageable : enough interactions for promising insights but not so many as to be overwhelming ( roughly between 10 and 50 ) . another point mentioned by the same author is that it would be very helpful if the tool could present an interaction type : interaction type was crucial for scientists ' judgments about whether results might help construct a plausible explanatory story. this is a wish that has also been expressed by the curators in the experiment described in this article . on the basis of the final survey , it appears that the users appreciate the comfort and support that odin gives them . they consider it as helpful in several ways . according to the qualitative feedback provided in the survey , visual highlighting depends on personal preferences , and therefore users should be given the possibility to customize some additional aspects of the interface . several curators mentioned that they would like to be able to add more specific information at the level of individual relationships , for example by means of a free text comment . particularly useful would be to add to each relation an indication of its type as mentioned in the document ( inhibition , activation , etc . ) . if the system could provide hints in this direction , this would be a very helpful feature . the ontogene text - mining system is capable of extracting this kind of interaction type indicators , however this feature was not used in the experiment as the developers assumed it would not be needed . this is another example that shows the importance of close collaboration between system developers and database curators . the experiment described in this article aims primarily at verifying the usability of the odin system in the context of curation of the pharmgkb database . the initial assumption was that we could separate an evaluation of the interface from an evaluation of the underlying text - mining system by asking curators to perform a revalidation task rather than a novel extraction task . the revalidation task consists in using the odin functionalities to quickly check the correctness of interactions already stored in pharmgkb . the results of the experiment confirm the initial assumptions : ( i ) the odin system offers a comfortable environment for relation validation that can considerably speed up this particular curation task ( ii ) the positive validation decisions are strongly correlated with the rankings provided by the text - mining system . as a next step , we intend to verify the quality of the interaction mining component on novel unseen articles but using the same interface . since the curators at this stage are already familiar with the interface , we will be able to verify how the results delivered by the text - mining system can actually improve their effectiveness in annotating interactions , without being impaired by an unfamiliar interface . the ontogene group ( f.r . and s.c . ) is supported by the swiss national science foundation ( grant 100014 - 118396/1 ) and novartis pharma ag , nibr - it , text mining services , ch-4002 , basel , switzerland . the pharmgkb group ( all other authors ) is supported by national institute of health / national institute of general medical sciences ( r24 gm61374 ) and national library of medicine ( contract hhsn276201000025c ) .

the need for efficient text - mining tools that support curation of the biomedical literature is ever increasing . in this article , we describe an experiment aimed at verifying whether a text - mining tool capable of extracting meaningful relationships among domain entities can be successfully integrated into the curation workflow of a major biological database . we evaluate in particular ( i ) the usability of the system 's interface , as perceived by users , and ( ii ) the correlation of the ranking of interactions , as provided by the text - mining system , with the choices of the curators .

Introduction Related work Methods Evaluation Discussion Conclusion and future work Funding

although emerging bioinformatics services enable structured access to increasingly complex datasets , it is often the case that the primary data is only available in the published literature , and needs to be extracted and stored in a standardized format before it can be leveraged upon ( 1 ) . the most pressing problem is that of efficiency of the process : despite the fact that typically several databases attempt to focus on a particular type of biological data , and often collaborate at least sufficiently to prevent duplication of effort and ensure compatibility of resulting data formats , it is impossible for human curators to keep up with the growing pace of publication . ( 2 ) on the other hand automated text - mining tools can not offer sufficient reliability to be employed indiscriminately without human supervision of the results that they deliver . in this article , we describe recent experiments aimed at assessing the potential contribution of a specific curation tool ( odin ) to the curation process of a well - known database ( pharmgkb ) . the curators identify relevant journal articles , largely selected from a set of about 20 journals that are followed , which include major pharmacogenomic journals and publications published by the pgrn ( pharmacogenomics research network ) . today , the information is entered through a web - based graphical user interface ( gui ) developed in - house to fit the curators ' needs , and captures data that is far more structured than in the past , such as population characteristics of the study group described in an article , and p - values of associations found between genetic variants and drug response . the current curator gui assists the curators in their process using basic text mining by suggesting entities found in the article , but does not pre - populate fields or highlight any information found within the article text . the ontogene text - mining system is based on a standard nlp pipeline , composed of efficient modules for sentence splitting , tokenization , entity recognition , syntactic chunking and dependency parsing . [ a more detailed description of the architecture of the ontogene text - mining system is beyond the scope of this article , for further details the interested reader is invited to consult the following publications ( 6,7 ) . the results of the ontogene text - mining system are made accessible through a curation system called odin ( ontogene document inspector ; this tool is not connected in any way with the recently introduced commercial text analytics system called odintext . ) which allows a user to dynamically inspect the results of their text - mining pipeline . however , the curators who used the system commented extremely positively on its usability for a practical curation task . for the documents already in pharmgkb it is also possible to inspect the gold standard and compare the results of the system against the gold standard . the curator can inspect all entities annotated by the system , and easily modify them if needed ( removing false positives with a simple click , or adding missed terms if necessary ) . the user can also inspect the set of candidate interactions generated by the system , and act upon them just as on entities , i.e. candidate interactions are presented ordered according to the score that has been assigned to them by the text - mining system , therefore the curator can choose to work with only a small set of highly ranked candidates , ignoring all the rest . odin , which is based on a client server architecture , maintains a log of the interaction with the curator , which could be used for later revision by a supervisor or for reversing some specific annotation decisions . at the end of a session the modified document and its annotations are sent back to the server , together with the log , for permanent storage , and can be accessed again at the next session , which could take place on a different remote client . first of all , text - mining tools can provide a help in the initial triage stage in order to decide which papers should be inspected by the expert curators . during inspection of individual articles , it can be very helpful for curators to use a system capable of locating the entities of interest within the article , and disambiguate them as reliably as possible . databases that are entity - focused can immediately profit from such tools , as the curators will be able to manually filter the candidates suggested by the system at greater speed , compared with a manual extraction from the paper , which would involve ( i ) spotting the mentions in the paper , ( ii ) decide which database entities are actually intended . the next major challenge for the introduction of text - mining systems within curation workflows is the automated detection of relations , which is relevant for several databases . nonetheless , much progress has been achieved recently , as results in the biocreative ii ( 14 ) and ii.5 ( 15 ) competitions show , and therefore it is now appropriate to start practical experimentation through collaborations between developers of text - mining solutions and database groups as potential users . although immediate integration in the curation workflow might not be the goal , these joint experiment help both groups in deciding how to improve their activities . text - mining developers will receive feedback on the quality of their systems and gain an understanding of the specific needs of the curators [ ( 12 ) stresses the importance of understanding the biocurator 's curation workflow ] , and curation groups will gain a better understanding of the current potential of technologies , which are now still experimental , but might soon become mainstream , and thus be able to choose the optimal point for integration in their workflows . additionally , the feedback provided to text - mining developers will render future systems more usable in practical applications . the need to pair developers with curators has been recognized by the organizers of the biocreative competition . addressing usability of text - mining systems is a novel aspect of this task . although fully unsupervised extraction of information from the literature is , for some time at least , unrealistic , text - mining tools are already sufficiently reliable to be used to provide hints to the curators , in order to speed up their activities . such a help is sorely needed , as it is already clear that manual curation can not keep up with the rate of data generation ( 16 ) . curatorial work done with the assistance of a text - mining system has already been shown to be much more efficient than when done by human readers without support ( 17 ) . examples of well - known text - mining solutions are ihop ( 18 ) and chillibot ( 19 ) . although the document analysis is based on a conventional nlp pipeline , including the dependency parser rasp ( 23 ) , the curator 's interface has been developed in strict collaboration with the end - users . ( 24 ) discuss how well the performance of a text - mining system ( in their case tailored to identify mentions of protein mutations ) , when evaluated with conventional techniques , translates into real utility of the system for a curation task . textpresso is another well - known text - mining system that is characterized by the usage of ontological categories of biological concepts ( 17,25 ) , as well as by processing full papers . although the full ontogene pipeline can deliver reliably a ranked list of candidate interactions , which can then be used by curators as prompts for annotation of novel articles , the experiment described in this article centered upon the validation of existing relations from pharmgkb . the main aim of the experiment was therefore to evaluate the usability of the interface , rather than the capabilities of the underlying text - mining tools . as ( 12 ) points out , an evaluation task must be chosen to be feasible in a given time frame , considering both the time needed by developers to adapt the existing text - mining system to the specific needs of the applications and the time available to curators for the verification of the results . we then automatically compared for each article the results of the relation mining system with the manually extracted interactions , and computed the common subset . in general , we would expect the text - mining system to deliver a larger set of interactions than those manually curated ( ideally covering all of them ) . a relation that was not detected by the text - mining system . full articles , however , are difficult to process for several reasons , most of which have little to do with text mining , such as widely different formats , or copyright restrictions , which in some cases explicitly prohibit text - mining applications . the aims of the experiment were the following : ( i ) evaluate the usability of the interface for revalidation of pharmgkb relationships , ( ii ) estimate whether the ranking of interactions provided by the text - mining system correlates well with decisions taken by the curators . the previously developed odin system was adapted to the needs of pharmgkb , on the basis of a close interaction with the curators . during development the following recommendations by ( 12 ) ( adapted to the specific needs of our application ) were taken into consideration : support for interactive disambiguation of domain entities;an editable list of candidate entities or interactions;a view of the document correlated with the candidate interactions ( when an interaction is selected , the corresponding entities are highlighted);ability to sort the results according to different criteria;ability to collect event and timing information at the session level ; andability to export the results in a suitable format ( e.g. support for interactive disambiguation of domain entities ; an editable list of candidate entities or interactions ; a view of the document correlated with the candidate interactions ( when an interaction is selected , the corresponding entities are highlighted ) ; ability to sort the results according to different criteria ; ability to collect event and timing information at the session level ; and ability to export the results in a suitable format ( e.g. the odin system allows the user to verify and modify every single annotation provided by the system , at the entity level as well as at the interaction level ( a ) . it provides a ranked list of candidate interactions ( b ) , which additionally can be sorted by the user according to different criteria ( d ) . the term editing panel ( not shown in the pictures in this article ) the document panel and results panel are actively connected , in that selection of items from the results panel will result in their visualization ( highlighting ) in the document panel . the close interaction among curators and system developers allowed the latter to implement a number of suggestions that made the usage of the system more effective . for example , in the initial demonstration the entities participating in an interaction were represented only by the pharmgkb identifier of the participating entities . the curators pointed out that it was not immediately obvious to them which entity was referred to by the identifier without consulting the database ( that the odin system allows by simple click on the identifier , see figure 1 ) , so the reference name of the entity was added . this , however , made the table of interactions more cluttered , and the curators pointed to the fact that they might optionally want to remove some information from the table ( such as the entity identifiers ) . some of the information hidden in this way could further be displayed as unobtrusive tooltip windows on mouseover by the user , another option that was added upon suggestion by the curators . instead of a simple confirm / reject choice , the maintainers of the database suggested the need for a more fine - grained choice . figures 1 , 3 , and 4 show these options as four tick boxes in the top right corner of the picture , which correspond to confirm relation , reject , needs full text , negative relation confirmed. during this process all of their actions were logged using the symbolic reference ( that they had to enter into the system at the beginning of the process ) as an identifier . the resulting validation decisions are automatically saved by the system and transferred to a server , together with detailed logs of the activity . at the end of the curation experiment we asked the curators to fill a questionnaire that was partly modeled on the questions used in the bciii iat task ( 12 ) . as explained in the previous section , the experiments described in this article were centered on the revalidation of relations already stored in pharmgkb . in order to evaluate the correlation of the rankings provided by the text - mining system with curator 's validation decisions , we were limited to use only articles for which all pharmgkb interactions could be detected by the text - mining system . we also observed that articles that contained a single curated relation would not be particularly interesting for this experiment , since it can be presumed that the vast majority of these cases are correct , and in any case there is no ranking to evaluate . in the rest of this section , we describe in detail the results of our experiments through descriptive statistics computed from the logs of the interactions . one of the aims of the experiment was to verify how the ranking of interactions produced by the text - mining system correlates with validation decisions by the curators . at higher ranks there is no visible correlation , but this is partially due to the sparsity of data ( in general there are fewer articles that have 5 interactions , and very few that have more than 20 ) ( among the articles selected for the experiment , 30.06% have 2 interactions , 37.81% 3 interactions , 6.39% 4 interactions , 11.02% 5 , 5.90% 6 or 7 , 4.33% 8 or 9 and 4.48% 1020 . ) after validating all relations in each document , the curators were asked to express their opinion about the quality of concept identification ( provided by the text - mining system ) for that particular document . the subjective judgment of the curators about the correctness of the entities suggested by the system . we also measured ( through the logs ) the exact time span between opening of a document and saving it after completing the validation of its interactions . this time was then divided by the number of interactions that had to be validated in each specific document . the average time needed for the validation of each interaction varies strongly among different curators , from 15 s up to 122 s. the box - and - whisker plots in figure 8 illustrate the distribution of the mean time ( in seconds ) used for the curation of all relations of an article . an interesting research question is to evaluate whether the quality of a text - mining solution has a correlation with the time necessary for the curation of a specific article . the left graph in figure 8 clearly shows such a correlation for our experiment : articles where concept identification was regarded by curators as bad was selected in a too small number of cases , and therefore results in this category might not be very informative . as mentioned in the previous section , at the end of the experiment the curators were asked to fill in a brief questionnaire in order to collect subjective feedback about their experience with the curation environment . below we list the questions that were asked and the feedback received.q1 do you consider the system easy and intuitive to use?not intuitive at allpartly intuitive ( 25%)mostly intuitive ( 75%)very intuitiveq2 do you consider the organization of the panels to be practical?not practical at allpartly practicalmostly practical ( 100%)very practicalq3 what aspects of the system are most appealing to you ? that saves a bit of time.speeds things up because relationships are already entered just need to verify themhighlighting the genes and drugs in the abstract , especially matching gene synonyms to gene symbol.q4 what problems / limitations of the system did you notice ? i would like if just the gene and drug would be highlighted in the sentences used by the program to define the relationship not every mention of the gene and drug in the abstract . metabolize , transport , inhibit , induce etc)q5 please mention any aspect of the system that did not appeal to you , or suggestions for changes . metabolize , transport , inhibit , induce , treat etc ) , also would be nice to extract genetic variations as another type of concept.q6 was the system helpful in performing the validation task?not helpful at allpartly helpfulmostly helpful ( 75%)very helpful ( 25%)q7 would you consider using a similar system for your regular curation task?noprobably notprobably yes ( 75%)yes ( 25%)q8 do you agree that a similar system could increase the efficiency of the manual curation process?noprobably notprobably yes ( 75%)yes ( 25% ) q1 do you consider the system easy and intuitive to use?not intuitive at allpartly intuitive ( 25%)mostly intuitive ( 75%)very intuitive partly intuitive ( 25% ) mostly intuitive ( 75% ) q2 do you consider the organization of the panels to be practical?not practical at allpartly practicalmostly practical ( 100%)very practical mostly practical ( 100% ) q3 what aspects of the system are most appealing to you ? i would like if just the gene and drug would be highlighted in the sentences used by the program to define the relationship not every mention of the gene and drug in the abstract . metabolize , transport , inhibit , induce etc ) q5 please mention any aspect of the system that did not appeal to you , or suggestions for changes . q6 was the system helpful in performing the validation task?not helpful at allpartly helpfulmostly helpful ( 75%)very helpful ( 25% ) q7 would you consider using a similar system for your regular curation task?noprobably notprobably yes ( 75%)yes ( 25% ) q8 do you agree that a similar system could increase the efficiency of the manual curation process?noprobably notprobably yes ( 75%)yes ( 25% ) usability issues are crucial for the acceptance of any specific it tool by the end users . in the case of biomedical curation , it is essential that text - mining results are delivered to the curator in a transparent fashion , without the need of dealing with system technicalities , in order to prevent cognitive overload . the tool should not disrupt the rhythm , flow of thinking and mental modeling process of the users , otherwise even minor problems with the interface could turn into major disruptions ( 1 ) . ideally the user should be put in a situation where he / she can make a quick but well - motivated decision based on the information provided by the system . according to ( 1 ) the output of a text - mining system should respect the following five criteria in order to be really helpful in the curation process : relevance : a connection to the disease of interest or to synonyms , homologs of interest.valid : not likely to be a false positive ( has some statistic of significance associated with it).credible : trustworthy methods generated the evidence plus numerous lines of evidence , number of research publications , convincing public metadata.plausible : function , location / structure , interaction type , biological process , and cellular component suggest an explanatory story.manageable : enough interactions for promising insights but not so many as to be overwhelming ( roughly between 10 and 50 ) . this is a wish that has also been expressed by the curators in the experiment described in this article . according to the qualitative feedback provided in the survey , visual highlighting depends on personal preferences , and therefore users should be given the possibility to customize some additional aspects of the interface . the ontogene text - mining system is capable of extracting this kind of interaction type indicators , however this feature was not used in the experiment as the developers assumed it would not be needed . the experiment described in this article aims primarily at verifying the usability of the odin system in the context of curation of the pharmgkb database . the initial assumption was that we could separate an evaluation of the interface from an evaluation of the underlying text - mining system by asking curators to perform a revalidation task rather than a novel extraction task . the results of the experiment confirm the initial assumptions : ( i ) the odin system offers a comfortable environment for relation validation that can considerably speed up this particular curation task ( ii ) the positive validation decisions are strongly correlated with the rankings provided by the text - mining system . as a next step , we intend to verify the quality of the interaction mining component on novel unseen articles but using the same interface . since the curators at this stage are already familiar with the interface , we will be able to verify how the results delivered by the text - mining system can actually improve their effectiveness in annotating interactions , without being impaired by an unfamiliar interface .

the more - than - ten - years - long history of the large - scale laser gravitation - wave ( gw ) detectors ( the first one , tama started to operate in 1999 , and the most powerful pair , the two detectors of the ligo project , in 2001 , not to forget about the two european members of the international interferometric gw detectors network , also having a pretty long history , namely , the german - british interferometer geo 600 located near hannover , germany , and the joint european large - scale detector virgo , operating near pisa , italy ) can be considered both as a great success and a complete failure , depending on the point of view . on the one hand , virtually all technical requirements for these detectors have been met , and the planned sensitivity levels have been achieved . on the other hand , the possibility of this result had been envisaged by the community , and during the same last ten years , plans for the second - generation detectors were developed [ 143 , 64 , 4 , 169 , 6 , 96 ] . currently ( 2012 ) , both ligo detectors are shut down , and their upgrade to the advanced ligo , which should take about three years , is underway . the goal of this upgrade is to increase the detectors sensitivity by about one order of magnitude , and therefore the rate of the detectable events by three orders of magnitude , from some half per year ( by the optimistic astrophysical predictions ) of the second generation detectors to , probably , hundreds per year . this goal will be achieved , mostly , by means of quantitative improvements ( higher optical power , heavier mirrors , better seismic isolation , lower loss , both optical and mechanical ) and evolutionary changes of the interferometer configurations , most notably , by introduction of the signal recycling mirror . as a result , the second - generation detectors will be quantum noise limited . at higher gw frequencies , the main sensitivity limitation will be due to phase fluctuations of light inside the interferometer ( shot noise ) . at lower frequencies , the random force created by the amplitude fluctuations ( radiation - pressure noise ) will be the main or among the major contributors to the sum noise . it is important that these noise sources both have the same quantum origin , stemming from the fundamental quantum uncertainties of the electromagnetic field , and thus that they obey the heisenberg uncertainty principle and can not be reduced simultaneously . in particular , the shot noise can ( and will , in the second generation detectors ) be reduced by means of the optical power increase . however , as a result , the radiation - pressure noise will increase . in the naively designed measurement schemes , built on the basis of a michelson interferometer , kin to the first and the second generation gw detectors , but with sensitivity chiefly limited by quantum noise , the best strategy for reaching a maximal sensitivity at a given spectral frequency would be to make these noise source contributions ( at this frequency ) in the total noise budget equal . the corresponding sensitivity point is known as the standard quantum limit ( sql ) [ 16 , 22 ] . this limitation is by no means an absolute one , and can be evaded using more sophisticated measurement schemes . starting from the first pioneering works oriented on solid - state gw detectors [ 28 , 29 , 144 ] , many methods of overcoming the sql were proposed , including the ones suitable for practical implementation in laser - interferometer gw detectors . the primary goal of this review is to give a comprehensive introduction of these methods , as well as into the underlying theory of linear quantum measurements , such that it remains comprehensible to a broad audience . we give a classical ( that is , non - quantum ) treatment of the problem , with the goal to familiarize the reader with the main components of laser gw detectors . in section 3 we provide the necessary basics of quantum optics . in section 4 we demonstrate the main principles of linear quantum measurement theory , using simplified toy examples of the quantum optical position meters . in section 5 , we provide the full - scale quantum treatment of the standard fabry - prot - michelson topology of the modern optical gw detectors . at last , in section 6 , we consider three methods of overcoming the sql , which are viewed now as the most probable candidates for implementation in future laser gw detectors . concluding remarks are presented in section 7 . throughout the review we use the notations and conventions presented in table 1 below . table 1notations and conventions , used in this review , given in alphabetical order for both , greek ( first ) and latin ( after greek ) symbols.notation and valuecomments|coherent state of light with dimensionless complex amplitude = arctan /normalized detuning interferometer half - bandwidth \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\gamma = \sqrt { { \gamma ^2 } + { \delta ^2}}$\end{document } effective bandwidth = p 0optical pump detuning from the cavity resonance frequency 0 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\epsilon_d } = \sqrt { { 1 \over { { \eta _ d } } } - 1}$\end{document } excess quantum noise due to optical losses in the detector readout system with quantum efficiency d = t x / cspace - time - dependent argument of the field strength of a light wave , propagating in the positive direction of the x - axis d quantum efficiency of the readout system ( e.g. , of a photodetector ) squeeze angle , some short time intervaloptical wave length reduced massv = 0mechanical detuning from the resonance frequency \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\xi = \sqrt { { s \over { { s_{{\rm{sql}}}}}}}$\end{document } sql beating factor signal - 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action force of the meter g signal force h dimensionless gw signal ( a.k.a . metrics variation ) \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$h = \left [ { \begin{array}{*{20}c } { \cos { \phi _ { { \rm{lo } } } } } \\ { \sin { \phi _ { { \rm{lo } } } } } \\ \end{array } } \right]$\end{document } homodyne vector \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat{\mathcal h}$\end{document } hamiltonian of a quantum system planck s constant \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathbb i}$\end{document } identity matrix \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal i}$\end{document } optical power \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}_c}$\end{document } circulating optical power in a cavity \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}_{{\rm{arm}}}}$\end{document } circulating optical power per interferometer arm cavity \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$j = { { 4{\omega _ 0}{{\mathcal i}_c } } \over { mcl}}$\end{document } normalized circulating powerkp = p / coptical pumping wave number k rigidity , including optical rigidity \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal k } = { { 2j\gamma } \over { { \omega ^2}({\gamma ^2 } + { \omega ^2})}}$\end{document } kimble s optomechanical coupling factor \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal k}_{{\rm{sm } } } } = { { 4j\gamma } \over { { { ( { \gamma ^2 } + { \omega ^2})}^2}}}$\end{document } optomechanical coupling factor of the sagnac speed meter l cavity length m probe - body mass o general linear meter readout observable \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathbb p}[\alpha ] = \left [ { \begin{array}{*{20}c } { \cos \alpha } & { - \sin \alpha } \\ { \sin \alpha } & { \cos \alpha } \\ \end{array } } \right]$\end{document } matrix of counterclockwise rotation ( pivoting ) by angle r amplitude squeezing factor ( e)rdb = 20r log10epower squeezing factor in decibels r power reflectivity of a mirror()reflection matrix of the fabry - prot cavitys()noise power spectral density ( double - sided ) \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${s_{{\mathcal x}{\mathcal x}}}(\omega)$\end{document } measurement noise power spectral density ( double - sided ) \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${s_{{\mathcal f}{\mathcal f}}}(\omega)$\end{document } back - action noise power spectral density ( double - sided ) \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${s_{{\mathcal x}{\mathcal f}}}(\omega)$\end{document } cross - correlation power spectral density ( double - sided ) \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathbb s}_{{\rm{vac}}}}(\omega ) = { 1 \over 2}{\mathbb i}$\end{document } vacuum quantum state power spectral density matrix \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathbb s}_{{\rm{sqz}}}}(\omega)$\end{document } squeezed quantum state power spectral density matrix \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathbb s}_{{\rm{sqz}}}}[r,\theta ] = { \mathbb p}[\theta ] \;\,\left [ { \begin{array}{*{20}c } { { e^r } } & 0 \\ 0 & { { e^{- r } } } \\ \end{array } } \right]\;\,{\mathbb p } [ - \theta ] $ \end{document } squeezing matrix t power transmissivity of a mirror \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathbb t}$\end{document } transmissivity matrix of the fabry - prot cavity test - mass velocity \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal w}$\end{document } optical energyw|(x , y)wigner function of the quantum state | x test - mass position \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat x = { { \hat a + { { \hat a}^\dagger } } \over { \sqrt 2}}$\end{document } dimensionless oscillator ( mode ) displacement operator \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat y = { { \hat a - { { \hat a}^\dagger } } \over { i\sqrt 2}}$\end{document } dimensionless oscillator ( mode ) momentum operator notations and conventions , used in this review , given in alphabetical order for both , greek ( first ) and latin ( after greek ) symbols . in order to have a firm basis for understanding how quantum noise influences the sensitivity of a gw detector it would be illuminating to give a brief description of the interferometers as weak force / tiny displacement meters . it is by no means our intention to give a comprehensive survey of this ample field that is certainly worthy of a good book , which there are in abundance , but rather to provide the reader with the wherewithal for grasping the very principles of the gw interferometers operation as well as of other similar ultrasensitive optomechanical gauges . the reader interested in a more detailed description of the interferometric techniques being used in the field of gw detectors might enjoy reading this book or the comprehensive living reviews on the subject by freise and strain and by pitkin et al . . let us , for the time being , imagine that we are capable of measuring an electromagnetic ( e.g. , light ) wave - phase shift with respect to some coherent reference of the same frequency . having such a hypothetical tool , what would be the right way to use it , if one had a task to measure some tiny classical force ? it consists of a movable totally - reflective mirror with mass m and a coherent paraxial light beam , that impinges on the mirror and then gets reflected towards our hypothetical phase - sensitive device . the mirror acts as a probe for an external force g that one seeks to measure . the response of the mirror on the external force g depends upon the details of its dynamics . for definiteness , then the mechanical equation of motion gives a connection between the mirror displacement x and the external force g in the very familiar form of the harmonic oscillator equation of motion : 1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$m\ddot x + m\omega _ m^2x = g(t)\,,\ ; \rightarrow \;x(t ) = { x_0}(t ) + \int\nolimits_0^t d t\prime \,{\chi _ { xx}}(t - t\prime)g(t\prime),$$\end{document } where x0(t ) = x(0 ) cosmt + p(0)/(mm)sinmt is the free motion of the mirror defined by its initial displacement x(0 ) and momentum p(0 ) at t = 0 and 2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\chi _ { xx}}(t - t\prime ) = { { \sin { \omega _ m}(t - t\prime ) } \over { m{\omega _ m}}}\,,\quad t { \geqslant } t\prime \,,$$\end{document } is the oscillator green s function . it is easy to see that the reflected light beam carries in its phase the information about the displacement x(t ) = x(t ) x(0 ) induced by the external force g. indeed , there is a phase shift between the incident and reflected beams , that matches the additional distance the light must propagate to the new position of the mirror and back , i.e. , 3\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta \phi = { { 2{\omega _ 0}\delta x } \over c } = 4\pi { { \delta x } \over { { \lambda _ 0}}}\,,$$\end{document } with 0 = 2c/0 the incident light frequency , c the speed of light and 0 the light wavelength . here figure 1scheme of a simple weak force measurement : an external signal force g pulls the mirror from its equilibrium position x = 0 , causing displacement x . the signal displacement is measured by monitoring the phase shift of the light beam , reflected from the mirror . scheme of a simple weak force measurement : an external signal force g pulls the mirror from its equilibrium position x = 0 , causing displacement x . the signal displacement is measured by monitoring the phase shift of the light beam , reflected from the mirror . apparently , the information about the signal force g(t ) can be obtained from the measured phase shift by post - processing of the measurement data record \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\delta \tilde \phi ( t ) \propto ( 1 ) instead of x. thus , the estimate of the signal force g reads : 4\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tilde g = { { mc } \over { 2{\omega _ 0}}}\,\left [ { \delta \ddot \tilde \phi + \omega _ m^2\delta \tilde \phi } \right].$$\end{document } this kind of post - processing pursues an evident goal of getting rid of any information about the eigenmotion of the test object while keeping only the signal - induced part of the total motion . the above time - domain expression can be further simplified by transforming it into a fourier domain , since it does not depend anymore on the initial values of the mirror displacement x(0 ) and momentum p(0 ) : 5\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tilde g(\omega ) = { { mc } \over { 2{\omega _ 0}}}\left [ { \omega _ m^2 - { \omega ^2 } } \right]\delta \tilde \phi ( \omega)\,,$$\end{document } where 6\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$a(\omega ) = \int\nolimits_{- \infty}^\infty d t\,a(t){e^{i\omega t}}$$\end{document } denotes a fourier transform of an arbitrary time - domain function a(t ) . if the expected signal spectrum occupies a frequency range that is much higher than the mirror - oscillation frequency m as is the case for ground based interferometric gw detectors , the oscillator behaves as a free mass and the term proportional to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\omega _ m^2$\end{document } in the equation of motion can be omitted yielding : 7\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\tilde g^{{\rm{f.m.}}}}(\omega ) = - { { mc{\omega ^2 } } \over { 2{\omega _ 0}}}\delta { \tilde \phi _ \omega}\,.$$\end{document } above , we assumed a direct light phase measurement with a hypothetical device in order to detect a weak external force , possibly created by a gw . however , in reality , direct phase measurement are not so easy to realize at optical frequencies . at the same time , physicists know well how to measure light intensity ( amplitude ) with very high precision using different kinds of photodetectors ranging from ancient - yet - die - hard reliable photographic plates to superconductive photodetectors capable of registering individual photons . how can one transform the signal , residing in the outgoing light phase , into amplitude or intensity variation ? this question is rhetorical for physicists , for interference of light as well as the multitude of interferometers of various design and purpose have become common knowledge since a couple of centuries ago . indeed , the amplitude of the superposition of two coherent waves depends on the relative phase of these two waves , thus transforming phase variation into the variation of the light amplitude . for the detection of gws , the most popular design is the michelson interferometer [ 15 , 12 , 59 ] , which schematic view is presented in figure 2 . the light wave from a laser source gets split by a semi - transparent mirror , called a beamsplitter , into two waves with equal amplitudes , travelling towards two highly - reflective mirrors mn , e1 to get reflected off them , and then recombine at the beamsplitter . the interferometer is usually tuned in such a way as to operate at a dark fringe , which means that by default the lengths of the arms are taken so that the optical paths for light , propagating back and forth in both arms , are equal to each other , and when they recombine at the signal port , they interfere destructively , leaving the photodetector unilluminated . on the opposite , the situation changes if the end mirrors get displaced by some external force in a differential manner , i.e. , such that the difference of the arms lengths is non - zero : l = le ln 0 . let a laser send to the interferometer a monochromatic wave that , at the beamsplitter , can be written as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${e_{{\rm{laser}}}}(t ) = { e_0}\cos ( { \omega _ 0}t)\,.$$\end{document } hence , the waves reflected off the interferometer arms at the beamsplitter ( before interacting with it for the second time ) are2 : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$e_{n , e}^{{\rm{out}}}(t ) = - { { { e_0 } } \over { \sqrt 2}}\cos ( { \omega _ 0}t - 2{\omega _ 0}{l_{n , e}}/c)\,,$$\end{document } and after the beamsplitter : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { e_{{\rm{dark}}}}(t ) = { { e_n^{{\rm{out}}}(t ) - e_e^{{\rm{out}}}(t ) } \over { \sqrt 2 } } = { e_0}\sin { { { \omega _ 0}\delta l } \over c}\sin \left({{\omega _ 0}t - { \omega _ 0}[{l_n } + { l_e}]/c } \right)\,,\quad } \\ { { e_{{\rm{bright}}}}(t ) = { { e_n^{{\rm{out}}}(t ) + e_e^{{\rm{out}}}(t ) } \over { \sqrt 2 } } = - { e_0}\cos { { { \omega _ 0}\delta l } \over c}\cos \left({{\omega _ 0}t - { \omega _ 0}[{l_n } + { l_e}]/c } \right)\ , . } \\ \end{array}$$\end{document } and the intensity of the outgoing light in both ports can be found using a relation \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal i } \propto \overline { { e^2}}$\end{document } with overline meaning time - average over many oscillation periods : 8\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\mathcal i}_{{\rm{dark}}}}(\delta l/{\lambda _ 0 } ) = { { { { \mathcal i}_0 } } \over 2}\left({1 - \cos 4\pi { { \delta l } \over { { \lambda _ 0 } } } } \right)\,,\quad { \rm{and}}\quad { { \mathcal i}_{{\rm{bright}}}}(\delta l/{\lambda _ 0 } ) = { { { { \mathcal i}_0 } } \over 2}\left({1 + \cos 4\pi { { \delta l } \over { { \lambda _ 0 } } } } \right)\,.$$\end{document } apparently , for small differential displacements l 0 , the michelson interferometer tuned to operate at the dark fringe has a sensitivity to ( l/0 ) that yields extremely weak light power on the photodetector and therefore very high levels of dark current noise . in practice , the interferometer , in the majority of cases , is slightly detuned from the dark fringe condition that can be viewed as an introduction of some constant small bias l0 between the arms lengths . by this simple trick experimentalists get linear response to the signal nonstationary displacement x(t ) : 9\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { { \mathcal i}_{{\rm{dark}}}}(\delta x/{\lambda _ 0})\ , = { { { { \mathcal i}_0 } } \over 2}\left({1 - \cos 4\pi { { \delta { l_0 } + \delta x } \over { { \lambda _ 0 } } } } \right ) \simeq } \\ { 8{\pi ^2}{{\mathcal i}_0}{{\delta { l_0}\delta x } \over { \lambda _ 0 ^ 2 } } + { \mathcal o}\left({{{\delta { x^2 } } \over { \lambda _ 0 ^ 2}},\,{{\delta l_0 ^ 2 } \over { \lambda _ 0 ^ 2 } } } \right)\ , = { \rm{const . } } \times 4\pi { { \delta x } \over { { \lambda _ 0 } } } + { \mathcal o}\left({{{\delta { x^2 } } \over { \lambda _ 0 ^ 2}},\,{{\delta l_0 ^ 2 } \over { \lambda _ 0 ^ 2 } } } \right)\ , . } \\ ( 3 ) should immediately conjure up the striking similarity between the response of the michelson interferometer and the single moving mirror . the nonstationary phase difference of light beams in two interferometer arms (t ) = 4x(t)/0 is absolutely the same as in the case of a single moving mirror ( cf . it is no coincidence , though , but a manifestation of the internal symmetry that all michelson - type interferometers possess with respect to coupling between mechanical displacements of their arm mirrors and the optical modes of the outgoing fields . in the next section 2.1.3 , when the end mirrors of the interferometer arms mn , e are at rest the length of the arms l is such that the light from the laser gets reflected back entirely ( bright port ) , while at the dark port the reflected waves suffer destructive interference keeping it really dark . if , due to some reason , e.g. , because of gws , the lengths of the arms changed in such a way that their difference was l , the photodetector at the dark port should measure light intensity \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${i_{{\rm{dark}}}}(\delta l ) = { { { i_0 } } \over 2}\left({1 - { \rm{cos}}\,{\rm{4}}\pi { { \delta l } \over \lambda } } \right)$\end{document}. scheme of a michelson interferometer . when the end mirrors of the interferometer arms mn , e are at rest the length of the arms l is such that the light from the laser gets reflected back entirely ( bright port ) , while at the dark port the reflected waves suffer destructive interference keeping it really dark . if , due to some reason , e.g. , because of gws , the lengths of the arms changed in such a way that their difference was l , the photodetector at the dark port should measure light intensity \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${i_{{\rm{dark}}}}(\delta l ) = { { { i_0 } } \over 2}\left({1 - { \rm{cos}}\,{\rm{4}}\pi { { \delta l } \over \lambda } } \right)$\end{document}. let us see how a michelson interferometer interacts with the gw . for this purpose we need to understand , on a very basic level , what a gw is . following the poetic , yet precise , definition by kip thorne , gravitational waves are ripples in the curvature of spacetime that are emitted by violent astrophysical events , and that propagate out from their source with the speed of light [ 13 , 110 ] . a weak gw far away from its birthplace can be most easily understood from analyzing its action on the probe bodies motion in some region of spacetime . usually , the deformation of a circular ring of free test particles is considered ( see chapter 26 : section 26.3.2 of for more rigorous treatment ) when a gw impinges it along the z - direction , perpendicular to the plane where the test particles are located . each particle , having plane coordinates ( x , y ) with respect to the center of the ring , undergoes displacement r ( x , y ) from its position at rest , induced by gws : 10\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta x = { 1 \over 2}{h _ + } x,\,\delta y = - { 1 \over 2}{h _ + } y\,,$$\end{document } 11\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta x = { 1 \over 2}{h _ \times}y\,,\,\,\delta y = { 1 \over 2}{h _ \times}x\,.$$\end{document } here , h+ h+(t z / c ) and h h ( t z / c ) stand for two independent polarizations of a gw that creates an acceleration field resulting in the above deformations . the above expressions comprise a solution to the equation of motion for free particles in the tidal acceleration field created by a gw : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta \ddot r = \frac{1}{2}\left [ { \left ( { { { \ddot h } _ + } x + { { \ddot h } _ \times } y } \right){e_x } + \left ( { - { { \ddot h } _ + } y + { { \ddot h } _ \times } x } \right){e_y } } \right],$$\end{document } with ex = { 1 , 0 } and ey = { 0 , 1 } the unit vectors pointing in the x and y direction , respectively . for our michelson interferometer , one can consider the end mirrors to be those test particles that lie on a circular ring with beamsplitter located in its center . one can choose arms directions to coincide with the frame x and y axes , then the mirrors will have coordinates ( 0 , ln ) and ( le , 0 ) , correspondingly . for this case , it is evident from this picture and from the above formulas that an h-polarized component of the gw does not change the relative lengths of the michelson interferometer arms and thus does not contribute to its output signal ; at the same time , h+-polarized gws act on the end masses of the interferometer as a pair of tidal forces of the same value but opposite in direction : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${g_n } = - { 1 \over 2}{m_n}{\ddot h _ + } { l_n}\,,\quad { g_e } = { 1 \over 2}{m_e}{\ddot h _ + } { l_e}\,.$$\end{document } figure 3action of the gw on a michelson interferometer : ( a ) h+-polarized gw periodically stretch and squeeze the interferometer arms in the x- and y - directions , ( b ) h-polarized gw though have no impact on the interferometer , yet produce stretching and squeezing of the imaginary test particle ring , but along the directions , rotated by 45 with respect to the x and y directions of the frame . the lower pictures feature field lines of the corresponding tidal acceleration fields \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\propto { \ddot h_{+ . \times}}$\end{document}. action of the gw on a michelson interferometer : ( a ) h+-polarized gw periodically stretch and squeeze the interferometer arms in the x- and y - directions , ( b ) h-polarized gw though have no impact on the interferometer , yet produce stretching and squeezing of the imaginary test particle ring , but along the directions , rotated by 45 with respect to the x and y directions of the frame . the lower pictures feature field lines of the corresponding tidal acceleration fields \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\propto { \ddot h_{+ . \times}}$\end{document}. assuming ge = gn = g , mn = me = m , and le = ln = l , one can write down the equations of motion for the interferometer end mirrors that are now considered free ( m gw ) as : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$m\ddot x = g\,,\quad m\ddot y = - g\,,$$\end{document } and for the differential displacement of the mirrors l = le ln = x y , which , we have shown above , the michelson interferometer is sensitive to , one gets the following equation of motion : 12\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$m\delta \ddot l = 2g(t ) = m{\ddot h _ + } ( t)l$$\end{document } that is absolutely analogous to eq . ( 1 ) for a single free mirror with mass m. therefore , we have proven that a michelson interferometer has the same dynamical behavior with respect to the tidal force \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$g(t ) = m{\ddot h _ + } ( t)l/2$\end{document } created by gws , as the single movable mirror with mass m to some external generic force g(t ) . the foregoing conclusion can be understood in the following way : for gws are inherently quadruple and , when the detector s plane is orthogonal to the wave propagation direction , can only excite a differential mechanical motion of its mirrors , one can reduce a complicated dynamics of the interferometer probe masses to the dynamics of a single effective particle that is the differential motion of the mirrors in the arms . this useful observation appears to be invaluably helpful for calculation of the real complicated interferometer responses to gws and also for estimation of its optical quantum noise , that comprises the rest of this review . to proceed with the analysis of quantum noise in gw interferometers we first need to familiarize ourselves with how a light field is transformed by an interferometer and how the ability of its mirrors to move modifies the outgoing field . in the following paragraphs , we endeavor to give a step - by - step introduction to the mathematical description of light in the interferometer and the interaction with its movable mirrors . we first consider how the light wave is described and how its characteristics transform , when it propagates from one point of free space to another . yet the real light beams in the large scale interferometers have a rather complicated inhomogeneous transverse spatial structure , the approximation of a plane monochromatic wave should suffice for our purposes , since it comprises all the necessary physics and leads to right results . inquisitive readers could find abundant material on the field structure of light in real optical resonators in particular , in the introductory book and in the living review by vinet . so , consider a plane monochromatic linearly polarized light wave propagating in vacuo in the positive direction of the x - axis . this field can be fully characterized by the strength of its electric component e(t x / c ) that should be a sinusoidal function of its argument = t x / c and can be written in three equivalent ways : 13\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$e\left(\zeta \right ) = { { \mathcal e}_0}\cos \left [ { { \omega _ 0}\zeta - { \phi _ 0 } } \right ] \equiv { { \mathcal e}_c}\cos { \omega _ 0}\zeta + { { \mathcal e}_s}\sin { \omega _ 0}\zeta \equiv { { { \mathcal e}{e^{- i{\omega _ 0}\zeta } } + { { \mathcal e}^{\ast}}{e^{i{\omega _ 0}\zeta } } } \over { \sqrt 2}}\,,$$\end{document } where 0 and 0 are called amplitude and phase , c and s take names of cosine and sine quadrature amplitudes , and complex number = ||e is known as the complex amplitude of the electromagnetic wave . here , we see that our wave needs two real or one complex parameter to be fully characterized in the given location x at a given time t. the amplitude - phase description is traditional for oscillations but is not very convenient since all the transformations are nonlinear in phase . therefore , in optics , either quadrature amplitudes or complex amplitude description is applied to the analysis of wave propagation . all three descriptions are related by means of straightforward transformations : 14\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { { \mathcal e}_0 } = \sqrt { { \mathcal e}_c^2 + { \mathcal e}_s^2 } = \sqrt 2 \vert { \mathcal e}\vert \quad \quad \quad \quad } & { \tan { \phi _ 0 } = { { \mathcal e}_s}/{{\mathcal e}_c } = \arg { \mathcal e},\quad { \phi _ 0 } \in [ 0,\,2\pi ] \ , , } \\ { { { \mathcal e}_c } = { { { \mathcal e } + { { \mathcal e}^{\ast } } } \over { \sqrt 2 } } = \sqrt 2 { \rm{re}}\left [ { \mathcal e } \right ] = { { \mathcal e}_0}\cos { \phi _ 0 } } & { { { \mathcal e}_s } = { { { \mathcal e } - { { \mathcal e}^{\ast } } } \over { i\sqrt 2 } } = \sqrt 2 { \rm{im}}\left [ { \mathcal e } \right ] = { { \mathcal e}_0}\sin { \phi _ 0}\,,\quad } \\ { { \mathcal e } = { { { { \mathcal e}_c } + i{{\mathcal e}_s } } \over { \sqrt 2 } } = { { { { \mathcal e}_0 } } \over { \sqrt 2}}{e^{i{\phi _ 0}}}\quad \quad \quad \quad \quad } & { { { \mathcal e}^{\ast } } = { { { { \mathcal e}_c } - i{{\mathcal e}_s } } \over { \sqrt 2 } } = { { { { \mathcal e}_0 } } \over { \sqrt 2}}{e^{- i{\phi _ 0}}}.\quad \quad \quad \quad \quad \quad } \\ \end{array}$$\end{document } the aforesaid means that for complete understanding of how the light field transforms in the optical device , knowing the rules of transformation for only two characteristic real numbers real and imaginary parts of the complex amplitude suffice . note also that the electric field of a plane wave is , in essence , a function of a single argument = t x / c ( for a forward propagating wave ) and thus can be , without loss of generality , substituted by a time dependence of electric field in some fixed point , say with x = 0 , thus yielding e( ) e(t ) now let us elaborate the way to establish a link between the wave electric field strength values taken in two spatially separated points , x1 = 0 and x2 = l. obviously , if nothing obscures light propagation between these two points , the value of the electric field in the second point at time t is just the same as the one in the first point , but at earlier time , i.e. , at t = t l / c : e ( t ) = e ( t l / c ) . this allows us to introduce a transformation that propagates em - wave from one spatial point to another . for complex amplitude , the transformation is very simple : 15\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\mathcal e}^{(l ) } } = { e^{i{\omega _ 0}l / c}}{{\mathcal e}^{(0)}}\,.$$\end{document } basically , this transformation is just a counterclockwise rotation of a wave complex amplitude vector on a complex plane by an angle \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\phi _ l } = { \left [ { { { { w_0}l } \over c } } \right]_{{\rm{mod}}\,{\rm{2}}\pi}}$\end{document}. this fact becomes even more evident if we look at the transformation for a 2-dimensional vector of quadrature amplitudes = { c , s } , that are : 16\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\mathcal e}^{(l ) } } = \left [ { \begin{array}{*{20}c } { \cos { \phi _ l } } & { - \sin { \phi _ l } } \\ { \sin { \phi _ l } } & { \cos { \phi _ l } } \\ \end{array } } \right]\cdot\left [ { \begin{array}{*{20}c } { { \mathcal e}_c^{(0 ) } } \\ { { \mathcal e}_s^{(0 ) } } \\ \end{array } } \right ] = \mathbb { p } \left [ { { \phi _ l } } \right]{{\mathcal e}^{(0)}}\,,$$\end{document } where 17\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathbb{p}\left [ \theta \right ] = \left [ { \begin{array}{*{20}c } { \cos \theta } & { - \sin \theta } \\ { \sin \theta } & { \cos \theta } \\ \end{array } } \right]$$\end{document } stands for a standard counterclockwise rotation ( pivoting ) matrix on a 2d plane . in the special case when the propagation distance is much smaller than the light wavelength l , the above two expressions can be expanded into taylor s series in l = 2l/ 1 up to the first order : 18\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${e^{(l \ll \lambda ) } } = ( 1 + i{\phi _ l}){e^{(0)}}$$\end{document } and 19\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\mathcal e}^{(l \ll \lambda ) } } = \left [ { \begin{array}{*{20}c } 1 & { - { \phi _ l } } \\ { { \phi _ l } } & 1 \\ \end{array } } \right]\cdot\left [ { \begin{array}{*{20}c } { { \mathcal e}_c^{(0 ) } } \\ { { \mathcal e}_s^{(0 ) } } \\ \end{array } } \right ] = \left({\left [ { \begin{array}{*{20}c } { 1\,\,0 } \\ { 0\,\,1 } \\ \end{array } } \right ] + \left [ { \begin{array}{*{20}c } 0 & { - { \phi _ l } } \\ { { \phi _ l } } & 0 \\ \end{array } } \right ] } \right)\cdot\left [ { \begin{array}{*{20}c } { { \mathcal e}_c^{(0 ) } } \\ { { \mathcal e}_s^{(0 ) } } \\ \end{array } } \right ] = \left({{\mathbb{i}{+}}\delta { \mathbb{p}}\left [ { { \phi _ l } } \right ] } \right){{\mathcal e}^{\left(0 \right)}},$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathbb i}$\end{document } stands for an identity matrix and [l ] is an infinitesimal increment matrix that generate the difference between the field quadrature amplitudes vector after and before the propagation , respectively . it is worthwhile to note that the quadrature amplitudes representation is used more frequently in literature devoted to quantum noise calculation in gw interferometers than the complex amplitudes formalism and there is a historical reason for this . notwithstanding the fact that these two descriptions are absolutely equivalent , the quadrature amplitudes representation was chosen by caves and schumaker as a basis for their two - photon formalism for the description of quantum fluctuations of light [ 39 , 40 ] that became from then on the workhorse of quantum noise calculation . more details about this extremely useful technique are given in sections 3.1 and 3.2 of this review . unless otherwise specified , we predominantly keep ourselves to this formalism and give all results in terms of it . above , we have seen that a gw signal displays itself in the modulation of the phase of light , passing through the interferometer . therefore , it is illuminating to see how the modulation of the light phase and/or amplitude manifests itself in a transformation of the field complex amplitude and quadrature amplitudes . throughout this section we assume our carrier field is a monochromatic light wave with frequency 0 , amplitude 0 and initial phase 0 = 0 : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${e_{{\rm{car}}}}(t ) = { { \mathcal e}_0}\cos { \omega _ 0}t = { \rm{re}}\left [ { { { \mathcal e}_0}{e^{- i{\omega _ 0}t } } } \right]\,.$$\end{document } amplitude modulation . the modulation of light amplitude is straightforward to analyze . let us do it for pedagogical sake : imagine one managed to modulate the carrier field amplitude slow enough compared to the carrier oscillation period , i.e. , 0 , then : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${e_{{\rm{am}}}}(t ) = { { \mathcal e}_0}(1 + { \epsilon_m}\cos ( \omega t + { \phi _ m}))\cos { \omega _ 0}t\,,$$\end{document } where m 1 and m are some constants called modulation depth and relative phase , respectively . the complex amplitude of the modulated wave equals to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\mathcal e}_{{\rm{am}}}}(t ) = { { { { \mathcal e}_0 } } \over { \sqrt 2}}\left({1 + { \epsilon_m}\cos ( \omega t + { \phi _ m } ) } \right)\,,$$\end{document } and the carrier quadrature amplitudes are , apparently , transformed as follows : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\mathcal e}_{c,{\rm{am}}}}(t ) = { { \mathcal e}_0}\left({1 + { \epsilon_m}\cos ( \omega t + { \phi _ m } ) } \right)\quad { \rm{and}}\quad { { \mathcal e}_{s,{\rm{am}}}}(t ) = 0\,.$$\end{document } the fact that the amplitude modulation shows up only in the quadrature that is in phase with the carrier field sets forth why this quadrature is usually named amplitude quadrature in the literature . in our review , we shall also keep to this terminology and refer to cosine quadrature as amplitude one . illuminating also is the calculation of the modulated light spectrum , that in our simple case of single frequency modulation is straightforward : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${e_{{\rm{am}}}}(t ) = { \rm{re}}\left [ { { { \mathcal e}_0}{e^{- i{\omega _ 0}t } } + { { { { \mathcal e}_0}{\epsilon_m } } \over 2}{e^{- i{\phi _ m}}}{e^{- i({\omega _ 0 } + \omega)t } } + { { { { \mathcal e}_0}{\epsilon_m } } \over 2}{e^{i{\phi _ m}}}{e^{- i({\omega _ 0 } - \omega)t } } } \right]\,.$$\end{document } apparently , the spectrum is discrete and comprises three components , i.e. , the harmonic at carrier frequency 0 with amplitude \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${a_{w0 } } = { \varepsilon _ 0}$\end{document } and two satellites at frequencies 0 , also referred to as modulation sidebands , with ( complex ) amplitudes \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${a_{w0}}_{\pm \omega } = { \epsilon _ m}{\varepsilon _ 0}e{\mp ^{i{\phi _ m}}}/2$\end{document}. the graphical interpretation of the above considerations is given in the left panel of figure 4 . here , carrier fields as well as sidebands are represented by rotating vectors on a complex plane . the carrier field vector has length 0 and rotates clockwise with the rate 0 , while sideband components participate in two rotations at a time . the sum of these three vectors yields a complex vector , whose length oscillates with time , and its projection on the real axis represents the amplitude - modulated light field . figure 4phasor diagrams for amplitude ( left panel ) and phase ( right panel ) modulated light . carrier field is given by a brown vector rotating clockwise with the rate w0 around the origin of the complex plane frame . the lower ( w0 ) sideband vector origin rotates with the tip of the carrier vector , while its own tip also rotates with respect to its origin counterclockwise with the rate . the upper ( w0 + ) sideband vector origin rotates with the tip of the upper sideband vector , while its own tip also rotates with respect to its origin counterclockwise with the rate . modulated oscillation is a sum of these three vectors an is given by the red vector . in the case of amplitude modulation ( am ) , the modulated oscillation vector is always in phase with the carrier field while its length oscillates with the modulation frequency . the time dependence of its projection onto the real axis that gives the am - light electric field strength is drawn to the right of the corresponding phasor diagram . in the case of phase modulation ( pm ) , sideband fields have a /2 constant phase shift with respect to the carrier field ( note factor i in front of the corresponding terms in eq . ( 22 ) ; therefore its sum is always orthogonal to the carrier field vector , and the resulting modulated oscillation vector ( red arrow ) has approximately the same length as the carrier field vector but outruns or lags behind the latter periodically with the modulation frequency . the resulting oscillation of the pm light electric field strength is drawn to the right of the pm phasor diagram and is the projection of the pm oscillation vector on the real axis of the complex plane . phasor diagrams for amplitude ( left panel ) and phase ( right panel ) modulated light . carrier field is given by a brown vector rotating clockwise with the rate w0 around the origin of the complex plane frame . the lower ( w0 ) sideband vector origin rotates with the tip of the carrier vector , while its own tip also rotates with respect to its origin counterclockwise with the rate . the upper ( w0 + ) sideband vector origin rotates with the tip of the upper sideband vector , while its own tip also rotates with respect to its origin counterclockwise with the rate . modulated oscillation is a sum of these three vectors an is given by the red vector . in the case of amplitude modulation ( am ) , the modulated oscillation vector is always in phase with the carrier field while its length oscillates with the modulation frequency . the time dependence of its projection onto the real axis that gives the am - light electric field strength is drawn to the right of the corresponding phasor diagram . in the case of phase modulation ( pm ) , sideband fields have a /2 constant phase shift with respect to the carrier field ( note factor i in front of the corresponding terms in eq . ( 22 ) ; therefore its sum is always orthogonal to the carrier field vector , and the resulting modulated oscillation vector ( red arrow ) has approximately the same length as the carrier field vector but outruns or lags behind the latter periodically with the modulation frequency . the resulting oscillation of the pm light electric field strength is drawn to the right of the pm phasor diagram and is the projection of the pm oscillation vector on the real axis of the complex plane . the above can be generalized to an arbitrary periodic modulation function \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$a(t ) = \sum\nolimits_{k = 1}^\infty { { a_k}\,{\rm{cos(}}k\omega + { \phi ^k})}$\end{document } , with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${e_{{\rm{am}}}}(t ) = { \varepsilon _ 0}(1 + { \epsilon _ m}a(t))$\end{document}. then the spectrum of the modulated light consists again of a carrier harmonic at 0 and an infinite discrete set of sideband harmonics at frequencies \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\omega _ 0 } \pm k\omega ( h = \overline { 1,\infty})$\end{document } : 20\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${e_{{\rm{am}}}}(t ) = { { \mathcal e}_0}\cos { \omega _ 0}t + { { { \epsilon_m}{{\mathcal e}_0 } } \over 2}\sum\limits_{k = 1}^\infty { { a_k } } \left\{{\cos [ ( { \omega _ 0 } - k\omega)t - { \phi _ k } ] + \cos [ ( { \omega _ 0 } + k\omega)t + { \phi _ k } ] } \right\}.$$\end{document } further generalization to an arbitrary ( real ) non - periodic modulation function \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$a(t ) = \int\nolimits_{- \infty}^\infty { { { d\omega } \over { 2\pi}}a(\omega ) } { e^{- i\omega t}}$\end{document } is apparent : 21\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { e_{{\rm{am}}}}\left(t \right ) = { \rm{re}}\left [ { { { \mathcal e}_0}{e^{- i{\omega _ 0}t } } + { \epsilon_m}{{\mathcal e}_0}{e^{- i{\omega _ 0}t}}\int\nolimits_{- \infty}^\infty { { { d\omega } \over { 2\pi}}a(\omega){e^{- i\omega t } } } } \right ] = } \\ { \quad \quad \quad \quad \quad \quad \quad \quad { { \mathcal e}_0}\cos { \omega _ 0}t + { { { \epsilon_m}{{\mathcal e}_0 } } \over 2}\int\nolimits_{- \infty}^\infty { { { d\omega } \over { 2\pi } } } \left\{{a\left({\omega - { \omega _ 0 } } \right ) + a\left({\omega + { \omega _ 0 } } \right ) } \right\}{e^{- i\omega t}}. } \\ \end{array}$$\end{document } from the above expression , one readily sees the general structure of the modulated light spectrum , i.e. , the central carrier peaks at frequencies 0 and the modulation sidebands around it , whose shape retraces the modulation function spectrum a( ) shifted by the carrier frequency 0 . phase modulation . the general feature of the modulated signal that we pursued to demonstrate by this simple example is the creation of the modulation sidebands in the spectrum of the modulated light . let us now see how it goes with a phase modulation that is more related to the topic of the current review . the simplest single - frequency phase modulation is given by the expression : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${e_{{\rm{pm}}}}(t ) = { { \mathcal e}_0}\cos ( { \omega _ 0}t + { \delta _ m}\cos ( \omega t + { \phi _ m}))\,,$$\end{document } where 0 , and the phase deviation m is assumed to be much smaller than 1 . using eqs . ( 14 ) , one can write the complex amplitude of the phase - modulated light as : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\mathcal e}_{{\rm{pm}}}}(t ) = { { { { \mathcal e}_0 } } \over { \sqrt 2}}{e^{i{\delta _ m}\cos ( \omega t + { \phi _ m})}}\,,$$\end{document } and quadrature amplitudes as : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\mathcal e}_{c,{\rm{pm}}}}(t ) = { { \mathcal e}_0}\cos \left [ { { \delta _ m}\cos ( \omega t + { \phi _ m } ) } \right]\quad { \rm{and}}\quad { { \mathcal e}_{s,{\rm{pm}}}}(t ) = { { \mathcal e}_0}\sin \left [ { { \delta _ m}\cos ( \omega t + { \phi _ m } ) } \right]\,.$$\end{document } note that in the weak modulation limit ( m 1 ) , the above equations can be approximated as : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\mathcal e}_{c,{\rm{pm}}}}(t ) \simeq { { \mathcal e}_0}\quad { \rm{and}}\quad { { \mathcal e}_{s,{\rm{pm}}}}(t ) \simeq { \delta _ m}{{\mathcal e}_0}\cos ( \omega t + { \phi _ m})\,.$$\end{document } this testifies that for a weak modulation only the sine quadrature , which is /2 out - of - phase with respect to the carrier field , contains the modulation signal . it is also what we will call this quadrature throughout the rest of this review . in order to get the spectrum of the phase - modulated light it is necessary to refer to the theory of bessel functions that provides us with the following useful relation ( known as the jacobi - anger expansion ) : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${e^{i{\delta _ m}\cos ( \omega t + { \phi _ m } ) } } = \sum\limits_{k = - \infty}^\infty { { i^k } } { j_k}({\delta _ m}){e^{ik(\omega t + { \phi _ m})}}\,,$$\end{document } where jk(m ) stands for the k - th bessel function of the first kind this looks a bit intimidating , yet for m 1 these expressions simplify dramatically , since near zero bessel functions can be approximated as : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${j_0}({\delta _ m } ) \simeq 1 - { { \delta _ m^2 } \over 4 } + { \mathcal o}(\delta _ m^4)\,,\quad { j_1}({\delta _ m } ) = { { { \delta _ m } } \over 2 } + { \mathcal o}(\delta _ m^3)\,,\quad { j_k}({\delta _ m } ) = { 1 \over { k!}}{\left({{{{\delta _ m } } \over 2 } } \right)^k } + { \mathcal o}(\delta _ m^{k + 2})\;(k { \geqslant } 2)\,.$$\end{document } thus , for sufficiently small m , we can limit ourselves only to the terms of order \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\delta _ m^0$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\delta _ m^1$\end{document } , which yields : 22\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${e_{{\rm{pm}}}}(t ) \simeq { \rm{re}}\left [ { { { \mathcal e}_0}{e^{i{\omega _ 0}t } } + i{{{\delta _ m}{{\mathcal e}_0 } } \over 2}\left({{e^{i[({\omega _ 0 } + \omega)t + { \phi _ m } ] } } + { e^{i[({\omega _ 0 } - \omega)t - { \phi _ m } ] } } } \right ) } \right]\,,$$\end{document } and we again face the situation in which modulation creates a pair of sidebands around the carrier frequency . the difference from the amplitude modulation case is in the way these sidebands behave on the complex plane . the corresponding phasor diagram for phase modulated light is drawn in figure 4 . in the case of pm , sideband fields have /2 constant phase shift with respect to the carrier field ( note factor i in front of the corresponding terms in eq . ( 22 ) ) ; therefore its sum is always orthogonal to the carrier field vector , and the resulting modulated oscillation vector has approximately the same length as the carrier field vector but outruns or lags behind the latter periodically with the modulation frequency . the resulting oscillation of the pm light electric field strength is drawn to the right of the pm phasor diagram and is the projection of the pm oscillation vector on the real axis of the complex plane . let us now generalize the obtained results to an arbitrary modulation function (t ) : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${e_{{\rm{pm}}}}(t ) = { { \mathcal e}_0}\cos ( { \omega _ 0}t + { \delta _ m}\phi ( t))\,.$$\end{document } in the most general case of arbitrary modulation index m , the corresponding formulas are very cumbersome and do not give much insight . then one can approximate the phase - modulated oscillation as follows : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${e_{{\rm{pm}}}}(t ) = { \rm{re}}\left [ { { { \mathcal e}_0}{e^{- i{\omega _ 0}t}}{e^{i{\delta _ m}\phi ( t ) } } } \right ] \simeq { \rm{re}}\left [ { { { \mathcal e}_0}{e^{- i{\omega _ 0}t}}\left\{{1 + i{\delta _ m}\phi ( t ) } \right\ } } \right]\,.$$\end{document } when (t ) is a periodic function : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\phi ( t ) = \sum\nolimits_{k = 1}^\infty { { \phi _ k}\,{\rm{cos } } \ , k\omega + { \phi _ k}}$\end{document } , and in weak modulation limit m 1 , the spectrum of the pm light is apparent from the following expression : 23\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { e_{{\rm{pm}}}}(t ) \simeq { { \mathcal e}_0}\cos { \omega _ 0}t - { { { \delta _ m}{{\mathcal e}_0 } } \over 2}\sum\limits_{k = 1}^\infty { { \phi _ k } } \left\{{\sin \left [ { ( { \omega _ 0 } - k\omega)t - { \phi _ k } } \right ] + \sin \left [ { ( { \omega _ 0 } + k\omega)t + { \phi _ k } } \right ] } \right\ } } \\ { { \rm{= re}}\left [ { { { \mathcal e}_0}{e^{- i{\omega _ 0}t } } + { { i{\delta _ m}{{\mathcal e}_0 } } \over 2}\sum\limits_{k = 1}^\infty { { \phi _ k } } \left\{{{e^{- i\left [ { ( { \omega _ 0 } - k\omega)t - { \phi _ k } } \right ] } } + { e^{- i\left [ { ( { \omega _ 0 } + k\omega)t + { \phi _ k } } \right ] } } } \right\ } } \right]\,,\quad \quad } \\ \end{array}$$\end{document } while for the real non - periodic modulation function \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\phi ( t ) = \int\nolimits_{- \infty}^\infty { { { d\omega } \over { 2\pi}}\phi ( \omega ) } { e^{- i\omega t}}$\end{document } the spectrum can be obtained from the following relation : 24\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { e_{{\rm{pm}}}}(t ) \simeq { \rm{re}}\left [ { { { \mathcal e}_0}{e^{- i{\omega _ 0}t } } + i{\delta _ m}{{\mathcal e}_0}{e^{- i{\omega _ 0}t}}\int\nolimits_{- \infty}^\infty { { { d\omega } \over { 2\pi } } } \phi ( \omega){e^{- i\omega t } } } \right]\quad \quad \quad \quad \quad \quad \quad \quad \quad } \\ { = { { \mathcal e}_0}\cos { \omega _ 0}t + { { { \delta _ m}{{\mathcal e}_0 } } \over 2}\int\nolimits_{- \infty}^\infty { { { d\omega } \over { 2\pi } } } \left\{{i\phi ( \omega - { \omega _ 0 } ) - i\phi ( \omega + { \omega _ 0 } ) } \right\}{e^{- i\omega t}}\ , . } \\ \end{array}$$\end{document } and again we get the same general structure of the spectrum with carrier peaks at 0 and shifted modulation spectra i( 0 ) as sidebands around the carrier peaks . the difference with the amplitude modulation is an additional /2 phase shifts added to the sidebands . thus far we have assumed the carrier field to be perfectly monochromatic having a single spectral component at carrier frequency 0 fully characterized by a pair of classical quadrature amplitudes represented by a 2-vector . in reality , this picture is no good at all ; indeed , a real laser emits not a monochromatic light but rather some spectral line of finite width with its central frequency and intensity fluctuating . these fluctuations are usually divided into two categories : ( i ) quantum noise that is associated with the spontaneous emission of photons in the gain medium , and ( ii ) technical noise arising , e.g. , from excess noise of the pump source , from vibrations of the laser resonator , or from temperature fluctuations and so on . it is beyond the goals of this review to discuss the details of the laser noise origin and methods of its suppression , since there is an abundance of literature on the subject that a curious reader might find interesting , e.g. , the following works [ 119 , 120 , 121 , 167 , 68 , 76 ] . for our purposes , the very existence of the laser noise is important as it makes us to reconsider the way we represent the carrier field . apparently , the proper account for laser noise prescribes us to add a random time - dependent modulation of the amplitude ( for intensity fluctuations ) and phase ( for phase and frequency fluctuations ) of the carrier field ( 13 ) : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$e(t ) = ( { { \mathcal e}_0 } + { \hat e_n}(t))\cos \left [ { { \omega _ 0}t + { \phi _ 0 } + { { \hat \phi}_n}(t ) } \right]\,,$$\end{document } where we placed hats above the noise terms on purpose , to emphasize that quantum noise is a part of laser noise and its quantum nature has to be taken into account , and that the major part of this review will be devoted to the consequences these hats lead to . apparently , the corrections to the amplitude and phase of the carrier light due to the laser noise are small enough to enable us to use the weak modulation approximation as prescribed above . in this case one can introduce a more handy amplitude and phase quadrature description for the laser noise contribution in the following manner : 25\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$e(t ) = \left({{{\mathcal e}_c } + { { \hat e}_c}(t ) } \right)\cos { \omega _ 0}t + \left({{{\mathcal e}_s } + { { \hat e}_s}(t ) } \right)\sin { \omega _ 0}t\,,$$\end{document } where c , s are related to n and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat \phi _ n}$\end{document } in the same manner as prescribed by eqs . it is convenient to represent a noisy laser field in the fourier domain : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\hat e_{c , s}}(t ) = \,\int\nolimits_{- \infty}^\infty { { { d\omega } \over { 2\pi } } } { \hat e_{c , s}}(\omega){e^{- i\omega t}}\,.$$\end{document } worth noting is the fact that c , s( ) is a spectral representation of a real quantity and thus satisfies an evident equality \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat e_{c , s}^\dagger ( - \omega ) = { \hat e_{c , s}}(\omega)$\end{document } ( by we denote the hermitian conjugate that for classical functions corresponds to taking the complex conjugate of this function ) . what happens if we want to know the light field of our laser with noise at some distance l from our initial reference point x = 0 ? for the carrier field component at 0 , nothing changes and the corresponding transform is given by eq . ( 16 ) , yet for the noise component \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta { \hat e_{{\rm{noise}}}}(t ) = { \hat e_c}(t)\cos { \omega _ 0}t + { \hat e_s}(t)\sin { \omega _ 0}t$$\end{document } there is a slight modification . since the field continuity relation holds for the noise field to the same extent as for the carrier field : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta \hat e_{{\rm{noise}}}^{(l)}(t ) = \delta \hat e_{{\rm{noise}}}^{(0)}(t - l / c)\,,$$\end{document } the following modification applies : 26\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\hat e^{(l)}}(\omega ) = \left [ { \begin{array}{*{20}c } { \hat e_c^{(l)}(\omega ) } \\ { \hat e_s^{(l)}(\omega ) } \\ \end{array } } \right ] = { e^{i\omega l / c}}\left [ { \begin{array}{*{20}c } { \cos { { { \omega _ 0}l } \over c } } & { - \sin { { { \omega _ 0}l } \over c } } \\ { \sin { { { \omega _ 0}l } \over c } } & { \cos { { { \omega _ 0}l } \over c } } \\ \end{array } } \right]\cdot\left [ { \begin{array}{*{20}c } { \hat e_c^{(0)}(\omega ) } \\ { \hat e_s^{(0)}(\omega ) } \\ \end{array } } \right ] = { e^{i\omega l / c}}\mathbb{p}\left [ { { { { \omega _ 0}l } \over c } } \right]{\hat e^{(0)}}(\omega)\,.$$\end{document } therefore , for sideband field components the propagation rule shall be modified by adding a frequency - dependent phase factor e that describes an extra phase shift acquired by a sideband field relative to the carrier field because of the frequency difference = 0 . so , we are one step closer to understanding how to calculate the quantum noise of the light coming out of the gw interferometer . it is necessary to understand what happens with light when it is reflected from such optical elements as mirrors and beamsplitters . one can also refer to section 2 of the living review by freise and strain for a more detailed treatment of this topic . mirrors of the modern interferometers are rather complicated optical systems usually consisting of a dielectric slab with both surfaces covered with multilayer dielectric coatings . these coatings are thoroughly constructed in such a way as to make one surface of the mirror highly reflective , while the other one is anti - reflective . we will not touch on the aspects of coating technology in this review and would like to refer the interested reader to an abundant literature on this topic , e.g. , to the following book and reviews and articles [ 154 , 72 , 100 , 73 , 122 , 49 , 97 , 117 , 57 ] . for our purposes , assuming the reflective surface of the mirror is flat and lossless should suffice . thus , we represent a mirror by a reflective plane with ( generally speaking , complex ) coefficients of reflection r and r and transmission t and t as drawn in figure 5 . let us now see how the ingoing and outgoing light beams couple on the mirrors in the interferometer . mirrors : from the general point of view , the mirror is a linear system with 2 input and 2 output ports . the way how it transforms input signals into output ones is featured by a 2 2 matrix that is known as the transfer matrix of the mirror \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathbb m}$\end{document } : 27\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\left [ { \begin{array}{*{20}c } { e_1^{{\rm{out}}}(t ) } \\ { e_2^{{\rm{out}}}(t ) } \\ \end{array } } \right ] = { \mathbb { m}}\,\cdot\,\,\left [ { \begin{array}{*{20}c } { e_1^{{\rm{in}}}(t ) } \\ { e_2^{{\rm{in}}}(t ) } \\ \end{array } } \right ] = \left [ { \begin{array}{*{20}c } r & t \\ { t\prime } & { r\prime } \\ \end{array } } \right]\cdot\left [ { \begin{array}{*{20}c } { e_1^{{\rm{in}}}(t ) } \\ { e_2^{{\rm{in}}}(t ) } \\ \end{array } } \right].$$\end{document } since we assume no absorption in the mirror , reflection and transmission coefficients should satisfy stokes relations [ 139 , 15 ] ( see also section 12.12 of ): 28\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\vert r\vert = \vert r\prime \vert \,,\qquad \vert t\vert = \vert t\prime \vert \,,\qquad \vert r{\vert ^2 } + \vert t{\vert ^2 } = 1\,,\qquad { r^{\ast}}t\prime + r\prime { t^{\ast } } = 0\,,\qquad { r^{\ast}}t + r\prime { t\prime ^{\ast } } = 0\,,$$\end{document } that is simply a consequence of the conservation of energy . this conservation of energy yields that the optical transfer matrix \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathbb m}$\end{document } must be unitary : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathbb m}^\dagger } = { { \mathbb m}^{- 1}}$\end{document}. stokes relations leave some freedom in defining complex reflectivity and transmissivity coefficients . two of the most popular variants are given by the following matrices : 29\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathbb{m}_{{\rm{sym } } } = \left [ { \begin{array}{*{20}c } { \sqrt r } & { i\sqrt t } \\ { i\sqrt t } & { \sqrt r } \\ \end{array } } \right]\,,\quad { \rm{and}}\quad { { \mathbb{m}}_{{\rm{real } } } } = \,\left [ { \begin{array}{*{20}c } { - \sqrt r } & { \sqrt t } \\ { \sqrt t } & { \sqrt r } \\ \end{array } } \right],$$\end{document } where we rewrote transfer matrices in terms of real power reflectivity and transmissivity coefficients r = |r| and t = |t| that will find extensive use throughout the rest of this review . the transformation rule , or putting it another way , coupling relations for the quadrature amplitudes can easily be obtained from eq . therefore , one has to deal with 4-dimensional vectors comprising of quadrature amplitudes of both input and output fields , and the transformation matrix become 4 4-dimensional , which can be expressed in terms of the outer product of a 2 2 matrix \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathbb m}_{{\rm{real}}}}$\end{document } by a 2 2 identity matrix \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathbb i}$\end{document } : 30\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\left [ { \begin{array}{*{20}c } { { \mathcal e}_{1c}^{{\rm{out } } } } \\ { { \mathcal e}_{1s}^{{\rm{out } } } } \\ { { \mathcal e}_{2c}^{{\rm{out } } } } \\ { { \mathcal e}_{2s}^{{\rm{out } } } } \\ \end{array } } \right ] = \left [ { \begin{array}{*{20}c } { { \mathcal e}_1^{{\rm{out } } } } \\ { { \mathcal e}_2^{{\rm{out } } } } \\ \end{array } } \right ] = { \mathbb { m}_{{\rm{real } } } } \bigotimes { \mathbb { i}}\cdot\left [ { \begin{array}{*{20}c } { { \mathcal e}_1^{{\rm{in } } } } \\ { { \mathcal e}_2^{{\rm{in } } } } \\ \end{array } } \right ] = \left [ { \begin{array}{*{20}c } { - \sqrt r } & 0 & { \sqrt t } & 0 \\ 0 & { - \sqrt r } & 0 & { \sqrt t } \\ { \sqrt t } & 0 & { \sqrt r } & 0 \\ 0 & { \sqrt t } & 0 & { \sqrt r } \\ \end{array } } \right]\cdot\left [ { \begin{array}{*{20}c } { { \mathcal e}_{1c}^{{\rm{in } } } } \\ { { \mathcal e}_{1s}^{{\rm{in } } } } \\ { { \mathcal e}_{2c}^{{\rm{in } } } } \\ { { \mathcal e}_{2s}^{{\rm{in } } } } \\ \end{array } } \right]\,.$$\end{document } the same rules apply to the sidebands of each carrier field : 31\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\left [ { \begin{array}{*{20}c } { \hat e_1^{{\rm{out}}}\left(\omega \right ) } \\ { \hat e_2^{{\rm{out}}}\left(\omega \right ) } \\ \end{array } } \right ] = { { \mathbb { m}}_{{\rm{real } } } } \bigotimes { \mathbb { i}}\cdot\left [ { \begin{array}{*{20}c } { \hat e_1^{{\rm{in}}}\left(\omega \right ) } \\ { \hat e_2^{{\rm{in}}}\left(\omega \right ) } \\ \end{array } } \right ] = \left [ { \begin{array}{*{20}c } { - \sqrt r } & 0 & { \sqrt t } & 0 \\ 0 & { - \sqrt r } & 0 & { \sqrt t } \\ { \sqrt t } & 0 & { \sqrt r } & 0 \\ 0 & { \sqrt t } & 0 & { \sqrt r } \\ \end{array } } \right]\cdot\left [ { \begin{array}{*{20}c } { \hat e_{1c}^{{\rm{in}}}(\omega ) } \\ { \hat e_{1s}^{{\rm{in}}}(\omega ) } \\ { \hat e_{2c}^{{\rm{in}}}(\omega ) } \\ { \hat e_{2s}^{{\rm{in}}}(\omega ) } \\ \end{array } } \right]\,.$$\end{document } in future , for the sake of brevity , we reduce the notation for matrices like \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathbb m}_{{\rm{real}}}}\ , \otimes { \mathbb i}$\end{document } to simply \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathbb m}_{{\rm{real}}}}$\end{document}. beam splitters : another optical element ubiquitous in the interferometers is a beamsplitter ( see figure 6 ) . in fact , it is the very same mirror considered above , but the angle of input light beams incidence is different from 0 ( if measured from the normal to the mirror surface ) . the corresponding scheme is given in figure 6 . in most cases , symmetric 50%/50% beamsplitter are used , which imply r = t = 1/2 and the coupling matrix \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathbb m}_{50/50}}$\end{document } then reads : 32\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\mathbb { m}}_{50/50 } } = \left [ { \begin{array}{*{20}c } { - 1/\sqrt 2 } & 0 & { 1/\sqrt 2 } & 0 \\ 0 & { - 1/\sqrt 2 } & 0 & { 1/\sqrt 2 } \\ { 1/\sqrt 2 } & 0 & { 1/\sqrt 2 } & 0 \\ 0 & { 1/\sqrt 2 } & 0 & { 1/\sqrt 2 } \\ \end{array } } \right]\,.$$\end{document } figure 6scheme of a beamsplitter . losses in optical elements : above , we have made one assumption that is a bit idealistic . namely , we assumed our mirrors and beamsplitters to be lossless , but it could never come true in real experiments ; therefore , we need some way to describe losses within the framework of our formalism . optical loss is a term that comprises a very wide spectrum of physical processes , including scattering on defects of the coating , absorption of light photons in the mirror bulk and coating that yields heating and so on . however , the most important features that influence the light fields , coming off the lossy optical element , can be summarized in the following two simple statements : optical loss of an optical element can be characterized by a single number ( possibly , frequency dependent ) ( usually , || 1 ) that is called the absorption coefficient . is the fraction of light power being lost in the optical element : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${e^{{\rm{out}}}}(t ) \rightarrow \sqrt { 1 - \epsilon } { e^{{\rm{out}}}}(t).$$\end{document}due to the fundamental law of nature summarized by the fluctuation dissipation theorem ( fdt ) [ 37 , 95 ] , optical loss is always accompanied by additional noise injected into the system . it means that additional noise field \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat n$\end{document } uncorrelated with the original light is mixed into the outgoing light field in the proportion of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\sqrt \epsilon$\end{document } governed by the absorption coefficient . optical loss of an optical element can be characterized by a single number ( possibly , frequency dependent ) ( usually , || 1 ) that is called the absorption coefficient . is the fraction of light power being lost in the optical element : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${e^{{\rm{out}}}}(t ) \rightarrow \sqrt { 1 - \epsilon } { e^{{\rm{out}}}}(t).$$\end{document } due to the fundamental law of nature summarized by the fluctuation dissipation theorem ( fdt ) [ 37 , 95 ] , optical loss is always accompanied by additional noise injected into the system . it means that additional noise field \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat n$\end{document } uncorrelated with the original light is mixed into the outgoing light field in the proportion of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\sqrt \epsilon$\end{document } governed by the absorption coefficient . these two rules conjure up a picture of an effective system comprising of a lossless mirror and two imaginary non - symmetric beamsplitters with reflectivity \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\sqrt { 1 - \epsilon}$\end{document } and transmissivity \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\sqrt \epsilon$\end{document } that models optical loss for both input fields , as drawn in figure 7 . model of lossy mirror . using the above model , it is possible to show that for a lossy mirror the transformation of carrier fields given by eq . ( 30 ) should be modified by simply multiplying the output fields vector by a factor 1 : 33\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\left [ { \begin{array}{*{20}c } { { \mathcal e}_1^{{\rm{out } } } } \\ { { \mathcal e}_2^{{\rm{out } } } } \\ \end{array } } \right ] = ( 1 - \epsilon){\mathbb { m}_{{\rm{real}}}}\cdot\left [ { \begin{array}{*{20}c } { { \mathcal e}_1^{{\rm{in } } } } \\ { { \mathcal e}_2^{{\rm{in } } } } \\ \end{array } } \right ] \simeq { { \mathbb { m}}_{{\rm{real}}}}\cdot\,\left [ { \begin{array}{*{20}c } { { \mathcal e}_1^{{\rm{in } } } } \\ { { \mathcal e}_2^{{\rm{in } } } } \\ \end{array } } \right],$$\end{document } where we used the fact that for low loss optics in use in gw interferometers , the absorption coefficient might be as small as 1010 . . where the noise sidebands are concerned , the transformation rule given by eq . ( 31 ) changes a bit more : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}{c } } { \left [ { \begin{array}{*{20}{c } } { \hat e_1^{out}(\omega ) } \\ { \hat e_2^{out}(\omega ) } \end{array } } \right ] = ( 1 - \epsilon){\mathbb{m}_{real } } \cdot \left [ { \begin{array}{*{20}{c } } { \hat e_1^{in}(\omega ) } \\ { \hat e_2^{in}(\omega ) } \end{array } } \right ] + \sqrt { \left ( { 1 - \epsilon } \right ) } { \mathbb{m}_{real } } \cdot \left [ { \begin{array}{*{20}{c } } { { { \hat n}_1}(\omega ) } \\ { { { \hat n}_2}(\omega ) } \end{array } } \right ] } \\ { \simeq \left ( { 1 - \epsilon } \right){\mathbb{m}_{real } } \cdot \left [ { \begin{array}{*{20}{c } } { \hat e_1^{in}(\omega ) } \\ { \hat e_2^{in}(\omega ) } \end{array } } \right ] + \sqrt \epsilon \left [ { \begin{array}{*{20}{c } } { { { \hat n'}_1}(\omega ) } \\ { { { \hat n'}_2}(\omega ) } \end{array } } \right].\;\;\ ; } \end{array}$$\end{document } here , we again used the smallness of 1 and also the fact that matrix \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathbb m}_{{\rm{real}}}}$\end{document } is unitary , i.e. , we redefined the noise that enters outgoing fields due to loss as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\{\hat n_1\prime,\hat n_2\prime\ } ^{\rm{t } } } = { \mathbb m } \cdot { ( { \hat n_1},{\hat n_2}\ } ^{\rm{t}}}$\end{document } , which keeps the new noise sources \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat n_1\prime(t)$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat n_2\prime(t)$\end{document } uncorrelated : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\left\langle { { { \hat n}_1}(t){{\hat n}_2}({t\prime } ) } \right\rangle = \left\langle { \hat n_1\prime(t)\hat n_2\prime(t ) } \right\rangle = 0$\end{document}. for full characterization of the light transformation in the gw interferometers , one significant aspect remains untouched , i.e. , the field transformation upon reflection off the movable mirror . above ( see section 2.1.1 ) , we have seen that motion of the mirror yields phase modulation of the reflected wave . consider the mirror described by the matrix \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathbb m}_{{\rm{real}}}}$\end{document } , introduced above . let us set the convention that the relations of input and output fields is written for the initial position of the movable mirror reflective surface , namely for the position where its displacement is x = 0 as drawn in figure 8 . we assume the sway of the mirror motion to be much smaller than the optical wavelength : x/0 1 . the effect of the mirror displacement x(t ) on the outgoing field \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$e_{1,2}^{{\rm{out}}}(t)$\end{document } can be straightforwardly obtained from the propagation formalism . indeed , considering the light field at a fixed spatial point , the reflected light field at any instance of time t is just the result of propagation of the incident light by twice the mirror displacement taken at time of reflection and multiplied by reflectivity \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\pm \sqrt { r}$\end{document}3 34\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { e_1^{{\rm{out}}}(t ) = - \sqrt r e_1^{{\rm{in}}}(t - 2x(t)/c ) + \sqrt t e_2^{{\rm{in}}}(t)\ , , } \\ { e_2^{{\rm{out}}}(t ) = \sqrt t e_1^{\rm in}(t ) + \sqrt r e_2^{{\rm{in}}}(t + 2x(t)/c)\ , . } \\ ( 19 ) the mirror motion modifies the quadrature amplitudes in a way that allows one to separate this effect from the reflection . it means that the result of the light reflection from the moving mirror can be represented as a sum of two independently calculable effects , i.e. , the reflection off the fixed mirror , as described above in section 2.2.4 , and the response to the mirror displacement ( see section 2.2.1 ) , i.e. , the signal presentable as a sideband vector { s1( ) , s2()}. the latter is convenient to describe in terms of the response vector { r1 , r2 } that is defined as : 35\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { s_1}(\omega ) = { r_1}x(t ) = - \sqrt r \delta { \mathbb { p}}\left [ { 2{\omega _ 0}x(t)/c } \right]\cdot{\mathcal e}_1^{{\rm{in } } } = - { { 2{\omega _ 0}\sqrt r } \over c}\left [ { \begin{array}{*{20}c } { { \mathcal e}_{1s}^{{\rm{in } } } } \\ { - { \mathcal e}_{1c}^{{\rm{in } } } } \\ \end{array } } \right]x(t ) } \\ { { s_2}(\omega ) = { r_2}x(t ) = - \sqrt r \delta { \mathbb { p}}\left [ { 2{\omega _ 0}x(t)/c } \right]\cdot{\mathcal e}_2^{{\rm{in } } } = - { { 2{\omega _ 0}\sqrt r } \over c}\left [ { \begin{array}{*{20}c } { { \mathcal e}_{2s}^{{\rm{in } } } } \\ { - { \mathcal e}_{2c}^{in } } \\ \end{array } } \right]x(t)\ , . } \\ \end{array}$$\end{document } figure 8reflection of light from the movable mirror . note that we did not include sideband fields \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat e_{1,2}^{{\rm{in}}}(\omega)$\end{document } in the definition of the response vector . in principle , sideband fields also feel the motion induced phase shift ; however , as far as it depends on the product of one very small value of 20x(t)/c = 4x(t)/0 1 by a small sideband amplitude \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\vert\hat e_{1,2}^{{\rm{in}}}(\omega)\vert \ll \varepsilon _ { 1,2}^{{\rm{in}}}\vert$\end{document } , the resulting contribution to the final response will be dwarfed by that of the classical fields . moreover , the mirror motion induced contribution ( 35 ) is itself a quantity of the same order of magnitude as the noise sidebands , and therefore we can claim that the classical amplitudes of the carrier fields are not affected by the mirror motion and that the relations ( 30 ) hold for a moving mirror too . however , the relations for sideband amplitudes must be modified . in the case of a lossless mirror , relations ( 31 ) turn : 36\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\left [ { \begin{array}{*{20}c } { \hat e_1^{{\rm{out}}}(\omega ) } \\ { \hat e_2^{{\rm{out}}}(\omega ) } \\ \end{array } } \right ] = { { \mathbb { m}}_{{\rm{real}}}}\cdot\left [ { \begin{array}{*{20}c } { \hat e_1^{{\rm{in}}}(\omega ) } \\ { \hat e_2^{{\rm{in}}}(\omega ) } \\ \end{array } } \right ] + \left [ { \begin{array}{*{20}c } { { r_1 } } \\ { { r_2 } } \\ \end{array } } \right]x(\omega)\,,$$\end{document } where x( ) is the fourier transform of the mirror displacement x(t ) \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$x(\omega ) = \,\int\nolimits_{- \infty}^\infty { dt\,x(t){e^{i\omega t}}\,.}$$\end{document } it is important to understand that signal sidebands characterized by a vector { s1( ) , s2( ) } , on the one hand , and the noise sidebands { 1( ) , 2( ) } , on the other hand , have the same order of magnitude in the real gw interferometers , and the main role of the advanced quantum measurement techniques we are talking about here is to either increase the former , or decrease the latter as much as possible in order to make the ratio of them , known as the signal - to - noise ratio ( snr ) , as high as possible in as wide as possible a frequency range . all the formulas we have derived here , though being very simple in essence , look cumbersome and not very transparent in general . in most situations , these expressions can be simplified significantly in real schemes . let us consider a simple example for demonstration purposes , i.e. , consider the reflection of a single light beam from a perfectly reflecting ( r = 1 ) moving mirror as drawn in figure 9 . the initial phase 0 of the incident wave does not matter and can be taken as zero . then \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\varepsilon _ c^{{\rm{in } } } = { \varepsilon _ 0}$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\varepsilon _ s^{{\rm{in } } } = 0$\end{document}. putting these values into eq . ( 30 ) and accounting for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\varepsilon _ 2^{{\rm{in } } } = 0$\end{document } , quite reasonably results in the amplitude of the carrier wave not changing upon reflection off the mirror , while the phase changes by : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal e}_c^{{\rm{out } } } = - { \mathcal e}_c^{{\rm{out } } } = - { { \mathcal e}_0},\quad { \mathcal e}_s^{{\rm{out } } } = 0\,.$$\end{document } since we do not have control over the laser noise , the input light has laser fluctuations in both quadratures \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat e_1^{{\rm{in } } } = \{\hat e_{1c}^{{\rm{in}}},\hat e_{1s}^{{\rm{in}}}\}$\end{document } that are transformed in full accordance with eq . ( 31 ) ) : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat e_1^{{\rm{out}}}(\omega ) = - \hat e_1^{{\rm{in}}}(\omega){.}$$\end{document } again , nothing surprising . let us see what happens with a mechanical motion induced component of the reflected wave : according to eq . ( 36 ) , the reflected light will contain a motion - induced signal in the s - quadrature : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$s(\omega ) = { { 2{\omega _ 0 } } \over c}\left [ { \begin{array}{*{20}c } 0 \\ { { { \mathcal e}_0 } } \\ \end{array } } \right]x(\omega)\,.$$\end{document } figure 9schematic view of light modulation by perfectly reflecting mirror motion . an initially monochromatic laser field e(t ) with frequency 0 = 2c/0 gets reflected from the mirror that commits slow ( compared to optical oscillations ) motion x(t ) ( blue line ) under the action of external force g. reflected the light wave phase is modulated by the mechanical motion so that the spectrum of the outgoing field e( ) contains two sidebands carrying all the information about the mirror motion . the left panel shows the spectral representation of the initial monochromatic incident light wave ( upper plot ) , the mirror mechanical motion amplitude spectrum ( middle plot ) and the spectrum of the phase - modulated by the mirror motion , reflected light wave ( lower plot ) . schematic view of light modulation by perfectly reflecting mirror motion . an initially monochromatic laser field e(t ) with frequency 0 = 2c/0 gets reflected from the mirror that commits slow ( compared to optical oscillations ) motion x(t ) ( blue line ) under the action of external force g. reflected the light wave phase is modulated by the mechanical motion so that the spectrum of the outgoing field e( ) contains two sidebands carrying all the information about the mirror motion . the left panel shows the spectral representation of the initial monochromatic incident light wave ( upper plot ) , the mirror mechanical motion amplitude spectrum ( middle plot ) and the spectrum of the phase - modulated by the mirror motion , reflected light wave ( lower plot ) . this fact , i.e. , that the mirror displacement that just causes phase modulation of the reflected field , enters only the s - quadrature , once again justifies why this quadrature is usually referred to as phase quadrature ( cf . it is instructive to see the spectrum of the outgoing light in the above considered situation . it is , expectedly , the spectrum of a phase modulated monochromatic wave that has a central peak at the carrier wave frequency and the two sideband peaks on either sides of the central one , whose shape follows the spectrum of the modulation signal , in our case , the spectrum of the mechanical displacement of the mirror x(t ) . , it enters the outgoing light in an additive manner and the typical ( though simplified ) amplitude spectrum of a noisy light reflected from a moving mirror is given in figure 10 . figure 10the typical spectrum ( amplitude spectral density ) of the light leaving the interferometer with movable mirrors . the central peak corresponds to the carrier light with frequency 0 , two smaller peaks on either side of the carrier represent the signal sidebands with the shape defined by the mechanical motion spectrum x( ) ; the noisy background represents laser noise . the typical spectrum ( amplitude spectral density ) of the light leaving the interferometer with movable mirrors . the central peak corresponds to the carrier light with frequency 0 , two smaller peaks on either side of the carrier represent the signal sidebands with the shape defined by the mechanical motion spectrum x( ) ; the noisy background represents laser noise . let us now address the question of how one can detect a gw signal imprinted onto the parameters of the light wave passing through the interferometer . the simple case of a michelson interferometer considered in section 2.1.2 where the gw signal was encoded in the phase quadrature of the light leaking out of the signal(dark ) port , does not exhaust all the possibilities . in more sophisticated interferometer setups that are covered in sections 5 and ? ? , a signal component might be present in both quadratures of the outgoing light and , actually , to different extent at different frequencies ; therefore , a detection method that allows measurement of an arbitrary combination of amplitude and phase quadrature is required . the two main methods are in use in contemporary gw detectors : these are homodyne and heterodyne detection [ 36 , 138 , 164 , 79 ] . both are common in radio - frequency technology as methods of detection of phase- and frequency - modulated signals . the basic idea is to mix a faint signal wave with a strong local oscillator wave , e.g. , by means of a beamsplitter , and then send it to a detector with a quadratic non - linearity that shifts the spectrum of the signal to lower frequencies together with amplification by an amplitude of the local oscillator . this topic is also discussed in section 4 of the living review by freise and strain with more details relevant to experimental implementation . homodyne readout . write down the signal wave as : 37\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$s(t ) = { s_c}(t)\cos { \omega _ 0}t + { s_s}(t)\sin { \omega _ 0}t$$\end{document } and the local oscillator wave as : 38\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$l(t ) = { l_c}(t)\cos { \omega _ 0}t + { l_s}(t)\sin { \omega _ 0}t\,.$$\end{document } signal light quadrature amplitudes sc , s(t ) might contain gw signal gc , s(t ) as well as quantum noise nc , s(t ) in both quadratures : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s_{c , s}}(t ) = { g_{c , s}}(t ) + { n_{c , s}}(t)\,,$$\end{document } while the local oscillator is a laser light with classical amplitudes : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$(l_c^{(0)},\,l_s^{(0 ) } ) = ( { l_0}\cos { \phi _ { lo}},\,{l_0}\sin { \phi _ { lo}})\,,$$\end{document } where we introduced a homodyne angle lo , and laser noise lc , s(t ) : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${l_{c , s}}(t ) = l_{c , s}^{(0 ) } + { l_{c , s}}(t)\,.$$\end{document } note that the local oscillator classical amplitude l0 is much larger than all other signals : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${l_0 } \gg \max \left [ { { l_{c , s}},\,{g_{c , s}},\,{n_{c , s } } } \right]\,.$$\end{document } let mix these two lights at the beamsplitter as drawn in the left panel of figure 11 and detect the two resulting outgoing waves with two photodetectors . the two photocurrents i1,2 will be proportional to the intensities i1,2 of these two lights : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { i_1 } \propto { i_1 } \propto { { \overline { { { ( l + s)}^2 } } } \over 2 } = { { l_0 ^ 2 } \over 2 } + { l_0}({g_c } + { l_c } + { n_c})\cos { \phi _ { lo } } + { l_0}({g_s } + { l_s } + { n_s})\sin { \phi _ { lo } } + { \mathcal o}\left [ { g_{c , s}^2,l_{c , s}^2,n_{c , s}^2 } \right]\ , , } \\ { { i_2 } \propto { i_2 } \propto { { \overline { { { ( l - s)}^2 } } } \over 2 } = { { l_0 ^ 2 } \over 2 } - { l_0}({g_c } - { l_c } + { n_c})\cos { \phi _ { lo } } - { l_0}({g_s } - { l_s } + { n_s})\sin { \phi _ { lo } } + { \mathcal o}\left [ { g_{c , s}^2,l_{c , s}^2,n_{c , s}^2 } \right ] , } \\ \end{array}$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\overline a$\end{document } stands for time averaging of a(t ) over many optical oscillation periods , which reflects the inability of photodetectors to respond at optical frequencies and thus providing natural low - pass filtering for our signal . the last terms in both expressions gather all the terms quadratic in gw signal and both noise sources that are of the second order of smallness compared to the local oscillator amplitude l0 and thus are omitted in further consideration . figure 11schematic view of homodyne readout ( left panel ) and dc readout ( right panel ) principle implemented by a simple michelson interferometer . schematic view of homodyne readout ( left panel ) and dc readout ( right panel ) principle implemented by a simple michelson interferometer . in a classic homodyne balanced scheme , the difference current is read out that contains only a gw signal and quantum noise of the dark port : 39\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${i_{{\rm{hom } } } } = { i_1 } - { i_2 } \propto 2{l_0}\left [ { ( { g_c } + { n_c})\cos { \phi _ { lo } } + ( { g_s } + { n_s})\sin { \phi _ { lo } } } \right]\,.$$\end{document } whatever quadrature the gw signal is in , by proper choice of the homodyne angle lo one can recover it with minimum additional noise . however , in real interferometers , the implementation of a homodyne readout appears to be fraught with serious technical difficulties . in particular , the local oscillator frequency has to be kept extremely stable , which means its optical path length and alignment need to be actively stabilized by a low - noise control system . this inflicts a significant increase in the cost of the detector , not to mention the difficulties in taming the noise of stabilising control loops , as the experience of the implementation of such stabilization in a garching prototype interferometer has shown [ 60 , 75 , 74 ] . fortunately , the search was not too long , since the suitable technique has already been used by michelson and morley in their seminal experiment . the technique is known as dc - readout and implies an introduction of a constant arms length difference , thus pulling the interferometer out of the dark fringe condition as was mentioned in section 2.1.2 . the advantage of this method is that the local oscillator is furnished by a part of the pumping carrier light that leaks into the signal port due to arms imbalance and thus shares the optical path with the signal sidebands . it automatically solves the problem of phase - locking the local oscillator and signal lights , yet is not completely free of drawbacks . the first suggestion to use dc readout in gw interferometers belongs to fritschel and then got comprehensive study by the gw community [ 138 , 164 , 79 ] . the schematic view of a michelson interferometer with dc readout is drawn in the right panel of figure 11 . as already mentioned , the local oscillator light is produced by a deliberately - introduced constant difference l of the lengths of the interferometer arms . it is also worth noting that the component of this local oscillator created by the asymmetry in the reflectivity of the arms that is always the case in a real interferometer and attributable mostly to the difference in the absorption of the northern and eastern end mirrors as well as asymmetry of the beamsplitter . all these factors can be taken into account if one writes the carrier fields at the beamsplitter after reflection off the arms in the following symmetric form : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { e_n^{{\rm{out}}}(t ) = - { { { e_0 } } \over { \sqrt 2}}(1 - { \epsilon_n})\cos { \omega _ 0}(t - 2{l_n}/c ) = - { { \overline { \mathcal e } } \over { \sqrt 2}}(1 - \delta \epsilon)\cos { \omega _ 0}(\bar t + \delta l / c)\ , , } \\ { e_e^{{\rm{out}}}(t ) = - { { { e_0 } } \over { \sqrt 2}}(1 - { \epsilon_e})\cos { \omega _ 0}(t - 2{l_e}/c ) = - { { \overline { \mathcal e } } \over { \sqrt 2}}(1 + \delta \epsilon)\cos { \omega _ 0}(\bar t - \delta l / c)\ , , } \\ \end{array}$$\end{document } where n , e and ln , e stand for optical loss and arm lengths of the corresponding interferometer arms , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\delta \epsilon = { { { \epsilon _ n } - { \epsilon _ e } } \over { 2(1 - \bar \epsilon)}}$\end{document } is the optical loss relative asymmetry with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\bar \epsilon = ( { \epsilon _ n } + { \epsilon _ e})/2,\,\bar \varepsilon = { e_0}(1 - \bar \epsilon)$\end{document } is the mean pumping carrier amplitude at the beamsplitter , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\delta l = { l_n } - { l_e}$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\bar t = t + { { { l_n } + { l_e } } \over { 2c}}$\end{document}. then the classical part of the local oscillator light in the signal ( dark ) port will be given by the following expression : 40\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$l_{{\rm{dc}}}^{(0)}(t ) = { { e_n^{{\rm{out}}}(t ) - e_e^{{\rm{out}}}(t ) } \over { \sqrt 2 } } = \underbrace { \overline { \mathcal e } \delta \epsilon\cos { { { \omega _ 0}\delta l } \over c}}_{\sqrt 2 l_c^{(0)}}\cos { \omega _ 0}\bar t + \underbrace { \overline { \mathcal e } \sin { { { \omega _ 0}\delta l } \over c}}_{\sqrt 2 l_s^{(0)}}\sin { \omega _ 0}\bar t\,,$$\end{document } where one can define the local oscillator phase and amplitude through the apparent relations : 41\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tan { \phi _ { { \rm{dc } } } } = { 1 \over { \delta \epsilon}}\tan { { { \omega _ 0}\delta l } \over c}\,\quad l_{{\rm{dc}}}^{(0 ) } \simeq \overline { \mathcal e } \sqrt { { { ( \delta \epsilon)}^2 } + { { \left({{{{\omega _ 0}\delta l } \over c } } \right)}^2 } } \simeq { { { \omega _ 0}{e_0}\delta l } \over c}\,,$$\end{document } where we have taken into account that 0l / c 1 and the rather small absolute value of the optical loss coefficient max [ n , e ] 10 1 available in contemporary interferometers . one sees that were there no asymmetry in the arms optical loss , there would be no opportunity to change the local oscillator phase . at the same time , the gw signal in the considered scheme is confined to the phase quadrature since it comprises the time - dependent part of l and thus the resulting photocurrent will be proportional to : 42\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${i_{{\rm{dc } } } } \propto \overline { { { ( l + s)}^2 } } \simeq { \left({l_{{\rm{dc}}}^{(0 ) } } \right)^2 } + 2l_{{\rm{dc}}}^{(0)}(l_c^{{\rm{out } } } + { n_c})\cos { \phi _ { { \rm{dc } } } } + 2l_{{\rm{dc}}}^{(0)}({g_s } + l_s^{{\rm{out } } } + { n_s})\sin { \phi _ { { \rm{dc}}}},$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$l_{c , s}^{{\rm{out}}}$\end{document } denote the part of the input laser noise that leaked into the output port : 43\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$l_c^{{\rm{out}}}(t ) \simeq l_c^{{\rm{in}}}\delta \epsilon\cos { { { \omega _ 0}\delta l } \over c } - l_s^{{\rm{in}}}\sin { { { \omega _ 0}\delta l } \over c } \simeq l_c^{{\rm{in}}}\delta \epsilon - l_s^{{\rm{in}}}{{{\omega _ 0}\delta l } \over c}\,,$$\end{document } 44\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$l_s^{{\rm{out}}}(t ) \simeq l_c^{{\rm{in}}}\sin { { { \omega _ 0}\delta l } \over c } + l_s^{{\rm{in}}}\delta \epsilon\cos { { { \omega _ 0}\delta l } \over c } \simeq l_c^{{\rm{in}}}{{{\omega _ 0}\delta l } \over c } + l_s^{{\rm{in}}}\delta \epsilon\,,$$\end{document } and nc , s stand for the quantum noise associated with the signal sidebands and entering the interferometer from the signal port . in the case of a small offset of the interferometer from the dark fringe condition , i.e. , for 0l / c = 2l/0 1 , the readout signal scales as local oscillator classical amplitude , which is directly proportional to the offset itself : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$l_{{\rm{dc}}}^{(0 ) } \simeq 2\pi { e_0}{{\delta l } \over { { \lambda _ 0}}}$\end{document}. the laser noise associated with the pumping carrier also leaks to the signal port in the same proportion , which might be considered as the main disadvantage of the dc readout as it sets rather tough requirements on the stability of the laser source , which is not necessary for the homodyne readout . however , this problem , is partly solved in more sophisticated detectors by implementing power recycling and/or fabry - prot cavities in the arms . these additional elements turn the michelson interferometer into a resonant narrow - band cavity for a pumping carrier with effective bandwidth determined by transmissivities of the power recycling mirror ( prm ) and/or input test masses ( itms ) of the arm cavities divided by the corresponding cavity length , which yields the values of bandwidths as low as 10 hz . since the target gw signal occupies higher frequencies , the laser noise of the local oscillator around signal frequencies turns out to be passively filtered out by the interferometer itself . dc readout has already been successfully tested at the ligo 40-meter interferometer in caltech and implemented in geo 600 [ 77 , 79 , 55 ] and in enhanced ligo [ 61 , 5 ] . it has proven a rather promising substitution for the previously ubiquitous heterodyne readout ( to be considered below ) and has become a baseline readout technique for future gw detectors . up until recently , the only readout method used in terrestrial gw detectors has been the heterodyne readout . yet with more and more stable lasers being available for the gw community , this technique gradually gives ground to a more promising dc readout method considered above . however , it is instructive to consider briefly how heterodyne readout works and learn some of the reasons , that it has finally given way to its homodyne adversary . the idea behind the heterodyne readout principle is the generalization of the homodyne readout , i.e. , again , the use of strong local oscillator light to be mixed up with the faint signal light leaking out the dark port of the gw interferometer save the fact that local oscillator light frequency is shifted from the signal light carrier frequency by rf several megahertz . in gw interferometers with heterodyne readout , local oscillator light of different than 0 frequency is produced via phase - modulation of the pumping carrier light by means of electro - optical modulator ( eom ) before it enters the interferometer as drawn in figure 12 . the interferometer is tuned so that the readout port is dark for the pumping carrier . at the same time , by introducing a macroscopic ( several centimeters ) offset l of the two arms , which is known as schnupp asymmetry , the modulation sidebands at radio frequency rf appear to be optimally transferred from the pumping port to the readout one . therefore , the local oscillator at the readout port comprises two modulation sidebands , lhet(t ) = l+(t ) + l(t ) , at frequencies 0 + rf and 0 rf , respectively . these two are detected together with the signal sidebands at the photodetector , and then the resulting photocurrent is demodulated with the rf - frequency reference signal yielding an output current proportional to gw - signal . green lines represent modulation sidebands at radio frequency rf and blue dotted lines feature signal sidebands schematic view of heterodyne readout principle implemented by a simple michelson interferometer . green lines represent modulation sidebands at radio frequency rf and blue dotted lines feature signal sidebands this method was proposed and studied in great detail in the following works [ 65 , 134 , 60 , 75 , 74 , 104 , 116 ] where the heterodyne technique for gw interferometers tuned in resonance with pumping carrier field was considered and , therefore , the focus was made on the detection of only phase quadrature of the outgoing gw signal light . this analysis was further generalized to detuned interferometer configurations in [ 36 , 138 ] where the full analysis of quantum noise in gw dualrecycled interferometers with heterodyne readout was done . let us see in a bit more detail how the heterodyne readout works as exemplified by a simple michelson interferometer drawn in figure 12 . the equation of motion at the input port of the interferometer creates two phase - modulation sideband fields ( l+(t ) and l(t ) ) at frequencies 0 rf : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { l _ + } ( t ) = \left [ { l_{c + } ^{(0 ) } + { l_{c + } } } \right]\cos ( { \omega _ 0 } + { \omega _ { { \rm{rf}}}})t + \left [ { l_{s + } ^{(0 ) } + { l_{s + } } } \right]\sin ( { \omega _ 0 } + { \omega _ { { \rm{rf}}}})t\ , , } \\ { { l _ -}(t ) = \left [ { l_{c -}^{(0 ) } + { l_{c - } } } \right]\cos ( { \omega _ 0 } - { \omega _ { { \rm{rf}}}})t + \left [ { l_{s -}^{(0 ) } + { l_{s - } } } \right]\sin ( { \omega _ 0 } - { \omega _ { { \rm{rf}}}})t\ , , } \\ \end{array}$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$l_{{\rm{(c , s ) } } \pm}^{(0)}$\end{document } stand for classical quadrature amplitudes of the modulation ( upper and lower ) sidebands4 and l(c , s)(t ) represent laser noise around the corresponding modulation frequency . unlike the homodyne readout schemes , in the heterodyne ones , not only the quantum noise components \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$n_{c , s}^{{\omega _ 0}}$\end{document } falling into the gw frequency band around the carrier frequency 0 has to be accounted for but also those rallying around twice the rf modulation frequencies 0 2rf : 45\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { s_{{\rm{het}}}}(t ) = ( { g_c } + n_c^{{\omega _ 0}})\cos { \omega _ 0}t + ( { g_s } + n_s^{{\omega _ 0}})\sin { \omega _ 0}t\quad \quad \quad \quad \quad \quad \quad \quad \quad } \\ { + n_c^{{\omega _ 0 } + 2{\omega _ { { \rm{rf}}}}}\cos ( { \omega _ 0 } + 2{\omega _ { { \rm{rf}}}})t + n_s^{{\omega _ 0 } + 2{\omega _ { { \rm{rf}}}}}\sin ( { \omega _ 0 } + 2{\omega _ { { \rm{rf}}}})t\ , } \\ \end{array}$$\end{document } 46\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$+ n_c^{{\omega _ 0 } - 2{\omega _ { { \rm{rf}}}}}\cos ( { \omega _ 0 } - 2{\omega _ { { \rm{rf}}}})t + n_s^{{\omega _ 0 } - 2{\omega _ { { \rm{rf}}}}}\sin ( { \omega _ 0 } - 2{\omega _ { { \rm{rf}}}})t\,.$$\end{document } the analysis of the expression for the heterodyne photocurrent 47\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${i_{{\rm{het } } } } \propto \overline { { { ( { l_{{\rm{het } } } } + s)}^2 } } = \,\overline { { s^2 } } + \overline { l _ + ^2 } + \overline { l _ - ^2 } + 2\overline { ( { l _ + } + { l _ -})s } + 2\overline { { l _ + } { l _ -}}$$\end{document } gives a clue to why these additional noise components emerge in the outgoing signal . it is easier to perform this kind of analysis if we represent the above trigonometric expressions in terms of scalar products of the vectors of the corresponding quadrature amplitudes and a special unit - length vector h[ ] = { cos , sin } , e.g. : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s_{{\rm{het } } } } \equiv { ( g + { n_{{\omega _ 0}}})^{\mathsf t } } \cdot h[{\omega _ 0}t ] + n_{{\omega _ 0 } + 2{\omega _ { { \rm{rf}}}}}^{\mathsf t}\cdot h[({\omega _ 0 } + { \omega _ { { \rm{rf}}}})t ] + n_{{\omega _ 0 } - 2{\omega _ { { \rm{rf}}}}}^{\mathsf t}\cdot h[({\omega _ 0 } - 2{\omega _ { { \rm{rf}}}})t]$$\end{document } where g = { gc , gs } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${n_{\omega \alpha } } = { \{n_c^\omega , n_s^\omega \ } ^{\rm{t}}}$\end{document}. another useful observation , provided that 0 max[1 , 2 ] , gives us the following relation : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline { h[({\omega _ 0 } + { \omega _ 1})t]{h^t}[({\omega _ 0 } + { \omega _ 2})t ] } = { 1 \over 2}\left [ { \begin{array}{*{20}c } { \cos ( { \omega _ 1 } - { \omega _ 2})t } & { - \sin ( { \omega _ 1 } - { \omega _ 2})t } \\ { \sin ( { \omega _ 1 } - { \omega _ 2})t } & { \cos ( { \omega _ 1 } - { \omega _ 2})t } \\ \end{array } } \right ] = { 1 \over 2}{\mathbb { p}}\left [ { ( { \omega _ 1 } - { \omega _ 2})t } \right]\,.$$\end{document } using this relation it is straightforward to see that the first three terms in eq . ( 47 ) give dc components of the photocurrent , while the fifth term oscillates at double modulation frequency 2rf . it is only the term \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$2\overline { ( { l _ + } + l -)s}$\end{document } that is linear in gw signal and thus contains useful information : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline { 2({l _ + } + { l _ -})s } \simeq { i_c}(t)\cos { \omega _ { { \rm{rf}}}}t + { i_s}(t)\sin { \omega _ { { \rm{rf}}}}t + \left\{{{\rm{oscillations at frequency}}3{\omega _ { { \rm{rf } } } } } \right\}$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { i_c}(t)\ , = { { ( g + , { n_{{\omega _ 0 } } } + { n_{{\omega _ 0 } + 2{\omega _ { { \rm{rf}}}}}})}^{{\mathsf t}}}\cdot\,l _ + ^{(0 ) } + { { ( g + { n_{{\omega _ 0 } } } + { n_{{\omega _ 0 } - 2{\omega _ { { \rm{rf}}}}}})}^{{\mathsf t}}}\cdot\,l _ - ^{(0)}\,,\quad \quad \quad \quad } \\ { { i_s}(t)\ , = - i{{(g + { n_{{\omega _ 0 } } } + { n_{{\omega _ 0 } + 2{\omega _ { { \rm{rf}}}}}})}^{{\mathsf t}}}\cdot{\sigma _ y}\cdot\,l _ + ^{(0 ) } + i{{(g + { n_{{\omega _ 0 } } } + { n_{{\omega _ 0 } - 2{\omega _ { { \rm{rf}}}}}})}^{{\mathsf t}}}\cdot\,{\sigma _ y}\cdot\,l _ - ^{(0)}\ , , } \\ \end{array}$$\end{document } and y is the 2nd pauli matrix : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\sigma _ y } = \left [ { \begin{array}{*{20}c } 0 & { - i } \\ i & 0 \\ \end{array } } \right]\,.$$\end{document } in order to extract the desired signal quadrature the photodetector readout current ihet is mixed with ( multiplied by ) a demodulation function d(t ) = d0 cos(rtf + d ) with the resulting signal filtered by a low - pass filter with upper cut - off frequency rf so that only components oscillating at gw frequencies gw rf appear in the output signal ( see figure 12 ) . it is instructive to see what the above procedure yields in the simple case of the michelson interferometer tuned in resonance with rf - sidebands produced by pure phase modulation : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$l_{c + } ^{(0 ) } = l_{c -}^{(0 ) } = 0$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$l_{s + } ^{(0 ) } = l_{s -}^{(0 ) } = { l_0}$\end{document}. the foregoing expressions simplify significantly to the following : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${i_c}(t ) = 2{l_0}\left({{g_s } + n_s^{{\omega _ 0 } } + { { n_s^{{\omega _ 0 } - 2{\omega _ { { \rm{rf } } } } } } \over 2 } + { { n_s^{{\omega _ 0 } + 2{\omega _ { { \rm{rf } } } } } } \over 2 } } \right)\quad { \rm{and}}\quad { i_s}(t ) = - { l_0}\left({n_s^{{\omega _ 0 } - 2{\omega _ { { \rm{rf } } } } } - n_s^{{\omega _ 0 } + 2{\omega _ { { \rm{rf } } } } } } \right)\,.$$\end{document } apparently , in this simple case of equal sideband amplitudes ( balanced heterodyne detection ) , only single phase quadrature of the gw signal can be extracted from the output photocurrent , which is fine , because the michelson interferometer , being equivalent to a simple movable mirror with respect to a gw tidal force as shown in section 2.1.1 and 2.1.2 , is sensitive to a gw signal only in phase quadrature . another important feature of heterodyne detection conspicuous in the above equations is the presence of additional noise from the frequency bands that are twice the rf - modulation frequency away from the carrier . as shown in this noise contributes to the total quantum shot noise of the interferometer and makes the high frequency sensitivity of the gw detectors with heterodyne readout 1.5 times worse compared to the ones with homodyne or dc readout . for more realistic and thus more sophisticated optical configurations , including fabry - prot cavities in the arms and additional recycling mirrors in the pumping and readout ports , nevertheless , it is worthwhile to note that with proper optimization of the modulation sidebands and demodulation function shapes the same sensitivity as for frequency - independent homodyne readout schemes can be obtained . however , inherent additional frequency - independent quantum shot noise brought by the heterodyning process into the detection band hampers the simultaneous use of advanced quantum non - demolition ( qnd ) techniques and heterodyne readout schemes significantly . thus far , the quantum nature of laser light being used in the gw interferometers has not been accounted for in any way . nevertheless , quantum mechanics predicts striking differences for the variances of laser light amplitude and phase fluctuations , depending on which quantum state it is in . squeezed vacuum [ 163 , 99 , 136 , 38 , 90 ] injection that has been recently implemented in the geo 600 detector and has pushed the high - frequency part of the total noise down by 3.5 db [ 151 , 1 ] serves as a perfect example of this . in this section , we provide a brief introduction into the quantization of light and the typical quantum states thereof that are common for the gw interferometers . from the point of view of quantum field theory , a freely propagating electromagnetic wave can be characterized in each spatial point with location vector r = ( x , y , z ) at time t by a heisenberg operator of an electric field strength (r , t).5 the electric field heisenberg operator of a light wave traveling along the positive direction of the z - axis can be written as a sum of a positive- and negative - frequency parts : 48\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat e(x , y , z;t ) = u(x , y , z)\left\{{{{\hat e}^{(+)}}(t ) + { { \hat e}^{(-)}}(t ) } \right\}\,,$$\end{document } where u(x , y , z ) is the spatial mode shape , slowly changing on a wavelength scale , and 49\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\hat e^{(+)}}(t ) = \int\nolimits_0^\infty { { { d\omega } \over { 2\pi } } } \sqrt { { { 2\pi \hbar \omega } \over { { \mathcal a}c } } } { \hat a_\omega}{e^{- i\omega t}}\,,\quad { \rm{and}}\quad { \hat e^{(-)}}(t ) = { \left [ { { { \hat e}^{(+)}}(t ) } \right]^\dagger}\,.$$\end{document } here , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal a}$\end{document } is the effective cross - section area of the light beam , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat a_\omega}\,(\hat a_\omega ^\dagger)$\end{document } is the single photon annihilation ( creation ) operator in the mode of the field with frequency . the meaning of eq . ( 49 ) is that the travelling light wave can be represented by an expansion over the continuum of harmonic oscillators modes of the electromagnetic field , that are , essentially , independent degrees of freedom . the latter implies the commutation relations for the operators and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat a_\omega ^\dag$\end{document } : 50\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\left [ { { { \hat a}_\omega},\,\hat a_{\omega \prime}^{\rm{\dagger } } } \right ] = 2\pi \delta ( \omega - \omega \prime)\,,\quad { \rm{and}}\quad \left [ { { { \hat a}_\omega},\,{{\hat a}_{\omega \prime } } } \right ] = \left [ { \hat a_\omega ^{\rm{\dagger}},\,\hat a_{\omega \prime}^\dagger } \right ] = 0\,.$$\end{document } in gw detectors , one deals normally with a close to monochromatic laser light with carrier frequency 0 , and a pair of modulation sidebands created by a gw signal around its frequency in the course of parametric modulation of the interferometer arm lengths . the light field coming out of the interferometer can not be considered as the continuum of independent modes anymore . the very fact that sidebands appear in pairs implies the two - photon nature of the processes taking place in the gw interferometers , which means the modes of light at frequencies 1,2 = 0 have correlated complex amplitudes and thus the new two - photon operators and related formalism is necessary to describe quantum light field transformations in gw interferometers . this was realized in the early 1980s by caves and schumaker who developed the two - photon formalism [ 39 , 40 ] , which is widely used in gw detectors as well as in quantum optics and optomechanics . one starts by defining modulation sideband amplitudes as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\hat a _ + } = { \hat a_{{\omega _ 0 } + \omega}}\,,\quad { \hat a _ - } = { \hat a_{{\omega _ 0 } - \omega}}\,,$$\end{document } and factoring out the oscillation at carrier frequency 0 in eqs . ( 48 ) , which yields : 51\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { { \hat e}^{(+)}}(t ) = { { { { \mathcal c}_0}{e^{- i{\omega _ 0}t } } } \over { \sqrt 2}}\int\nolimits_{- { \omega _ 0}}^\infty { { { d\omega } \over { 2\pi } } } { \lambda _ + } ( \omega){{\hat a } _ + } { e^{- i\omega t } } \simeq { { { { \mathcal c}_0 } } \over { \sqrt 2}}{e^{- i{\omega _ 0}t}}\int\nolimits_{- \infty}^\infty { { { d\omega } \over { 2\pi } } } { \lambda _ + } ( \omega){{\hat a } _ + } { e^{- i\omega t}}\ , , } \\ { { { \hat e}^{(-)}}(t ) = { { { { \mathcal c}_0}{e^{i{\omega _ 0}t } } } \over { \sqrt 2}}\int\nolimits_{- \infty}^{{\omega _ 0 } } { { { d\omega } \over { 2\pi } } } { \lambda _ -}(\omega)\hat a _ - ^{\rm{\dagger}}{e^{- i\omega t } } \simeq { { { { \mathcal c}_0 } } \over { \sqrt 2}}{e^{i{\omega _ 0}t}}\int\nolimits_{- \infty}^\infty { { { d\omega } \over { 2\pi}}{\lambda _ -}(\omega)\hat a _ - ^{\dagger}{e^{- i\omega t}}\ , , } } \\ \end{array}$$\end{document } where we denote \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal c}_0 } \equiv \sqrt { { { 4\pi \hbar { \omega _ 0 } } \over { { \mathcal a}c}}}$\end{document } and define functions ( ) following as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\lambda _ \pm}(\omega ) = \sqrt { { { { \omega _ 0 } \pm \omega } \over { { \omega _ 0 } } } } \,,$$\end{document } and use the fact that 0 gw enables us to expand the limits of integrals to 0 . the operator expressions in front of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${e^{\pm i{\omega _ ( 51 ) are quantum analogues to the complex amplitude and its complex conjugate * defined in eqs . ( 14 ) : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat{\mathcal e}(t ) = { { { { \mathcal c}_0 } } \over { \sqrt 2}}\hat a(t ) \equiv { { { { \mathcal c}_0 } } \over { \sqrt 2}}\int\nolimits_{- \infty}^\infty { { { d\omega } \over { 2\pi } } } { \lambda _ + } ( \omega){\hat a _ + } { e^{- i\omega t}}\,,\;{\rm{and}}\;{\hat{\mathcal e}^\dagger}(t ) = { { { { \mathcal c}_0 } } \over { \sqrt 2}}{\hat a^{\rm{\dagger}}}(t ) \equiv { { { { \mathcal c}_0 } } \over { \sqrt 2}}\int\nolimits_{- \infty}^\infty { { { d\omega } \over { 2\pi}}{\lambda _ -}(\omega)\hat a _ - ^{\dagger}{e^{- i\omega t}}\,.}$$\end{document } again using eqs . ( 14 ) , we can define two - photon quadrature amplitudes as : 52\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { { \hat{\mathcal e}}_c}(t ) = { { \hat{\mathcal e}(t ) + { { \hat{\mathcal e}}^{\rm{\dagger}}}(t ) } \over { \sqrt 2 } } = { { { { \mathcal c}_0 } } \over { \sqrt 2}}\int\nolimits_{- \infty}^\infty { { { d\omega } \over { 2\pi}}{{{\lambda _ + } { { \hat a } _ + } + { \lambda _ -}\hat a _ - ^{\rm{\dagger } } } \over { \sqrt 2 } } } { e^{- i\omega t } } \equiv { { { { \mathcal c}_0 } } \over { \sqrt 2}}\int\nolimits_{- \infty}^\infty { { { d\omega } \over { 2\pi } } } { { \hat a}_c}(\omega){e^{- i\omega t } } } \\ { { { \hat{\mathcal e}}_s}(t ) = { { \hat{\mathcal e}(t ) - { { \hat{\mathcal e}}^{\rm{\dagger}}}(t ) } \over { i\sqrt 2 } } = { { { { \mathcal c}_0 } } \over { \sqrt 2}}\int\nolimits_{- \infty}^\infty { { { d\omega } \over { 2\pi}}{{{\lambda _ + } { { \hat a } _ + } - { \lambda _ -}\hat a _ - ^{\rm{\dagger } } } \over { i\sqrt 2 } } } { e^{- i\omega t } } \equiv { { { { \mathcal c}_0 } } \over { \sqrt 2}}\int\nolimits_{- \infty}^\infty { { { d\omega } \over { 2\pi}}{{\hat a}_s}(\omega){e^{- i\omega t}}. } } \\ \end{array } \,$$\end{document } note that so - introduced operators of two - photon quadrature amplitudes c , s(t ) are hermitian and thus their frequency domain counterparts satisfy the relations for the spectra of hermitian operator : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat a_{c , s}^\dagger(t ) = { \hat a_{c , s}}(t)\ , \rightarrow \;\hat a_{c , s}^\dagger(\omega ) = { \hat a_{c , s}}(- \omega)\,.$$\end{document } now we are able to write down commutation relations for the quadrature operators , which can be derived from eq . ( 50 ) : 53\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[{\hat a_c}(\omega),\,\hat a_c^\dagger(\omega \prime)]\ , = [ { \hat a_s}(\omega),\,\,\hat a_s^\dagger(\omega \prime ) ] = 2\pi { \omega \over { { \omega _ 0}}}\delta \left({\omega - \omega \prime } \right),$$\end{document } 54\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\left [ { { { \hat a}_c}(\omega),\,\,\hat a_s^\dagger(\omega \prime ) } \right]\ , = \left [ { \hat a_c^\dagger ( \omega),\,\,{{\hat a}_s}(\omega \prime ) } \right ] = 2\pi i\delta ( \omega - \omega \prime).$$\end{document } the commutation relations represented by eqs . ( 53 ) indicate that quadrature amplitudes do not commute at different times , i.e. , [ c(t ) , c(t ) ] = [ s(t ) , s(t ) ] 0 , which imply they could not be considered for proper output observables of the detector , for a nonzero commutator , as we would see later , means an additional quantum noise inevitably contributes to the final measurement result . the detailed explanation of why it is so can be found in many works devoted to continuous linear quantum measurement theory , in particular , in chapter 6 of , appendix 2.7 of or in . where gw detection is concerned , all the authors are agreed on the point that the values of gw frequencies ( 1 hz /2 10 hz ) , being much smaller than optical frequencies 0/2 10 hz , allow one to neglect such weak commutators as those of eqs . this statement has gotten an additional ground in the calculation conducted in appendix 2.7 of where the value of the additional quantum noise arising due to the nonzero value of commutators ( 53 ) has been derived and its extreme minuteness compared to other quantum noise sources has been proven . ( 52 ) are not the real measured observables at the output of the interferometer , since the photodetectors actually measure not the energy flux 55\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat{\mathcal i}(t ) = \int { \int\nolimits_0^\infty { { { d\omega d\omega \prime } \over { { { ( 2\pi)}^2 } } } } } \,\hbar \sqrt { \omega \omega \prime } \hat a_\omega ^\dagger{\hat a_{\omega \prime}}{e^{i(\omega - \omega \prime)t}}$$\end{document } but rather the photon number flux : 56\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat { \mathcal n}(t ) = \int { \int\nolimits_0^\infty { { { d\omega d\omega \prime } \over { { { ( 2\pi)}^2 } } } } } \,\hat a_\omega ^\dagger{\hat a_{\omega \prime}}{e^{i(\omega - \omega \prime)t}}\,.$$\end{document } the former does not commute with itself : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$[\hat { \mathcal i}(t),\hat { \mathcal i}({t\prime } ) ] \ne 0$\end{document } , while the latter apparently does \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$[\hat { \mathcal n}(t),\hat { \mathcal n}({t\prime } ) ] = 0$\end{document } and therefore is the right observable for a self - consistent quantum description of the gw interferometer output signal . in the course of our review , we shall adhere to the approximate quadrature amplitude operators that can be obtained from the exact ones given by eqs . ( 52 ) by setting ( ) 1 , i.e. , 57\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { { \hat a}_c}(t)\ , = \int\nolimits_{- \infty}^\infty { { { d\omega } \over { 2\pi}}{{{{\hat a } _ + } + \hat a _ - ^\dagger } \over { \sqrt 2 } } } { e^{- i\omega t}}\ ; \leftrightarrow \;{{\hat a}_c}(\omega ) = { { { { \hat a } _ + } + \hat a _ - ^\dagger } \over { \sqrt 2}}\ , , } \\ { { { \hat a}_s}(t)\ , = \int\nolimits_{- \infty}^\infty { { { d\omega } \over { 2\pi}}{{{{\hat a } _ + } - \hat a _ - ^\dagger } \over { i\sqrt 2 } } } { e^{- i\omega t}}\ ; \leftrightarrow \;{{\hat a}_s}(\omega ) = { { { { \hat a } _ + } - \hat a _ - ^\dagger } \over { i\sqrt 2}}\ , . } \\ \end{array}$$\end{document } the new approximate two - photon quadrature operators satisfy the following commutation relations in the frequency domain : 58\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\left [ { { { \hat a}_c}(\omega),\,{{\hat a}_s}(\omega \prime ) } \right ] = 2\pi i\delta ( \omega + \omega \prime)\,,\quad { \rm{and}}\quad \left [ { { { \hat a}_c}(\omega),\,{{\hat a}_c}(\omega \prime ) } \right ] = \left [ { { { \hat a}_s}(\omega),\,{{\hat a}_s}(\omega \prime ) } \right ] = 0\,,$$\end{document } and in the time domain : 59\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\left [ { { { \hat a}_c}(t),\,{{\hat a}_s}(t\prime ) } \right ] = i\delta ( t - t\prime)\,,\quad { \rm{and}}\quad \left [ { { { \hat a}_c}(t),\,{{\hat a}_c}(t\prime ) } \right ] = \left [ { { { \hat a}_s}(t),\,{{\hat a}_s}(t\prime ) } \right ] = 0$$\end{document } then the electric field strength operator ( 48 ) can be rewritten in terms of the two - photon quadrature operators as : 60\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat e(x , y , z;t ) = u(x , y , z){{\mathcal c}_0}\left [ { { { \hat a}_c}(t)\cos { \omega _ 0}t + { { \hat a}_s}(t)\sin { \omega _ 0}t } \right].$$\end{document } hereafter , we will omit the spatial mode factor u(x , y , z ) since it does not influence the final result for quantum noise spectral densities . moreover , in order to comply with the already introduced division of the optical field into classical carrier field and to the 1st order corrections to it comprising of laser noise and signal induced sidebands , we adopt the same division for the quantum fields , i.e. , we detach the mean values of the corresponding quadrature operators via the following redefinition \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat a_{{c_s}}^{{\rm{old } } } \to { a_{c , s } } + \hat a_{c , s}^{{\rm{new}}}$\end{document } with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${a_{c , s } } \equiv \left\langle { \hat a_{c , s}^{{\rm{old } } } } \right\rangle$\end{document}. here , by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\left\langle { \hat a_{c , s}^{{\rm{old } } } } \right\rangle$\end{document } we denote an ensemble average over the quantum state | of the light wave : ( ) ||. thus , the electric field strength operator for a plain electromagnetic wave will have the following form : 61\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat e(x , y;t ) = { { \mathcal c}_0}\left [ { ( { a_c } + { { \hat a}_c}(t))\cos { \omega _ 0}t + ( { a_s } + { { \hat a}_s}(t))\sin { \omega _ 0}t } \right]\,.$$\end{document } further , we combine the two - photon quadratures into vectors in the same manner as we used to do for classical fields : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$a \equiv \left [ { \begin{array}{*{20}c } { { a_c } } \\ { { a_s } } \\ \end{array } } \right]\,,\quad { \rm{and}}\quad \hat a \equiv \left [ { \begin{array}{*{20}c } { { { \hat a}_c } } \\ { { { \hat a}_s } } \\ \end{array } } \right]\,.$$\end{document } now , when we have defined a quantum heisenberg operator of the electric field of a light wave , and introduced quantum operators of two - photon quadratures , the last obstacle on our way towards the description of quantum noise in gw interferometers is that we do not know the quantum state the light field finds itself in . since it is the quantum state that defines the magnitude and mutual correlations of the amplitude and phase fluctuations of the outgoing light , and through it the total level of quantum noise setting the limit on the future gw detectors sensitivity . in what follows , we shall consider vacuum and coherent states of the light , and also squeezed states , for they comprise the vast majority of possible states one could encounter in gw interferometers . the quantum state of the travelling wave is a subtle structure , for the system it describes comprises a continuum of modes . however , each of these modes can be viewed at as a quantum oscillator with its own generalized coordinate \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat x_\omega } = ( { \hat a_\omega } + \hat a_\omega ^\dagger)/\sqrt 2$\end{document } and momentum \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat y_\omega } = ( { \hat a_\omega } - \hat a_\omega ^\dagger)/i\sqrt 2$\end{document}. the ground state of this system , known as a vacuum state |vac , is straightforward and is simply the direct product of the ground states |0 of all modes over all frequencies : 62\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\left\vert { { \rm{vac } } } \right\rangle \equiv { \underset{\omega } \bigotimes } { \left\vert 0 \right\rangle _ \omega}\,.$$\end{document } by definition , the ground state of a mode with frequency is the state with minimum energy evac = /2 and no excitation : 63\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\hat a_\omega}{\left\vert 0 \right\rangle _ \omega } = 0\quad { \rm{and}}\quad \,{\left\langle 0 \right\vert _ the mean values of annihilation and creation operators as well as any linear combination thereof that includes quadrature amplitudes , are zero : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\left\langle { { \rm{vac}}\left\vert { { { \hat a}_\omega } } \right\vert { \rm{vac } } } \right\rangle \equiv \left\langle { { { \hat a}_\omega } } \right\rangle = \left\langle { \hat a_\omega ^\dagger } \right\rangle = 0\ ; \rightarrow \;\left\langle { { { \hat a}_c}(\omega ) } \right\rangle = \left\langle { { { \hat a}_s}(\omega ) } \right\rangle = 0\,.$$\end{document } apparently , this also holds for time domain operators : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\left\langle { { \rm{vac}}\left\vert { \hat a\left(t \right ) } \right\vert { \rm{vac } } } \right\rangle \equiv \left\langle { \hat a\left(t \right ) } \right\rangle = \left\langle { { { \hat a}^\dagger}\left(t \right ) } \right\rangle = 0 \rightarrow \left\langle { { { \hat a}_c}\left(t \right ) } \right\rangle = \left\langle { { { \hat a}_s}\left(t \right ) } \right\rangle = 0\,.$$\end{document } that the ground state of the oscillator is gaussian is evident from its q - representation , namely \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\psi _ { { \rm{vac}}}}({x_\omega } ) \equiv { \left\langle { \left . { { x_\omega } } \right\vert 0 } \right\rangle _ \omega } = { 1 \over { \sqrt [ { 4}]{\pi}}}\exp \left\{{- { { x_\omega ^2 } \over 2 } } \right\}.$$\end{document } it means that knowing the second moments of quadrature amplitudes suffices for full characterization of the state |vac. for this purpose , let us introduce a quadrature amplitudes matrix of spectral densities6 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathbb s}(\omega)$\end{document } according to the rule : 64\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\underline { \quad \quad \left [ { \begin{array}{*{20}c } { \left\langle { { { \hat a}_c}(\omega)\circ { { \hat a}_c}(\omega \prime ) } \right\rangle \left\langle { { { \hat a}_c}(\omega)\circ { { \hat a}_s}(\omega \prime ) } \right\rangle } \\ { \left\langle { { { \hat a}_s}(\omega)\circ { { \hat a}_c}(\omega \prime ) } \right\rangle \left\langle { { { \hat a}_s}(\omega)\circ { { \hat a}_s}(\omega \prime ) } \right\rangle } \\ \end{array } } \right ] } = 2\pi \delta ( \omega + \omega \prime)\left [ { \begin{array}{*{20}c } { { s_{cc}}(\omega){s_{cs}}(\omega ) } \\ { { s_{sc}}(\omega){s_{ss}}(\omega ) } \\ \end{array } } \right ] = 2\pi { \mathbb { s}}\delta ( \omega + \omega \prime)\,.$$\end{document } where sij( ) ( i , j = c , s ) denote ( cross ) power spectral densities of the corresponding quadrature amplitudes i( ) j() standing for the symmetrized product of the corresponding quadrature operators , i.e. : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\left\langle { { { \hat a}_i}(\omega ) { \circ } { { \hat a}_j}(\omega \prime ) } \right\rangle \equiv { 1 \over 2}\left\langle { { { \hat a}_i}(\omega){{\hat a}_j}(\omega \prime ) + { { \hat a}_j}(\omega \prime){{\hat a}_i}(\omega ) } \right\rangle \equiv 2\pi { s_{ij}}(\omega)\delta ( \omega - \omega \prime)\,.$$\end{document } for a vacuum state , this matrix of spectral densities can easily be obtained from the commutation relations ( 58 ) and equals to : 65\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\mathbb{s}}_{{\rm{vac}}}}(\omega ) = \,\left [ { \begin{array}{*{20}c } { 1/2 } & 0 \\ 0 & { 1/2 } \\ \end{array } } \right],$$\end{document } which implies that the ( double - sided ) power spectral densities of the quadrature amplitudes as well as their cross - spectral density are equal to : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s_{cc}}(\omega ) = { s_{ss}}(\omega ) = { 1 \over 2}\qquad { \rm{and}}\qquad { s_{cs}}(\omega ) = 0\,.$$\end{document } in time domain , the corresponding matrix of second moments , known as a covariance matrix with elements defined as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathbb v}_{ij}}\delta ( t - t\prime ) = \langle { \hat a_i}(t ) \circ { \hat a_j}(t\prime)\rangle$\end{document } , is absolutely the same as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathbb s}_{{\rm{vac}}}}(\omega)$\end{document } : 66\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\mathbb{v}}_{{\rm{vac } } } } = \left [ { \begin{array}{*{20}c } { 1/2 } & 0 \\ 0 & { 1/2 } \\ \end{array } } \right].$$\end{document } it is instructive to discuss the meaning of these matrices , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathbb s}$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathbb v}$\end{document } , and of the values they comprise . to do so , let us think of the light wave as a sequence of very short square - wave light pulses with infmitesimally small duration 0 . the delta function of time in eq . ( 66 ) tells us that the noise levels at different times , i.e. , the amplitudes of the different square waves , are statistically independent . to put it another way , ( 65 ) that quadrature amplitudes fluctuations are stationary , and it is this stationarity , as noted in that makes quadrature amplitudes such a convenient language for describing the quantum noise of light in parametric systems exemplified by gw interferometers . it is instructive to pay some attention to a pictorial representation of the quantum noise described by the covariance and spectral density matrices \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathbb v}$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathbb s}$\end{document}. with this end in view let us introduce quadrature operators for each short light pulse as follows : 67\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\hat x_\varepsilon}(t ) \equiv { 1 \over { \sqrt \varepsilon}}\int\nolimits_{t - \varepsilon /2}^{t + \varepsilon /2 } d \tau \,{\hat a_c}(\tau)\,,\qquad { \rm{and}}\qquad { \hat y_\varepsilon}(t ) \equiv { 1 \over { \sqrt \varepsilon}}\int\nolimits_{t - \varepsilon /2}^{t + \varepsilon /2 } d \tau \,{\hat a_s}(\tau)\,.$$\end{document } these operators \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat x_\varepsilon}(t)$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat y_\varepsilon}(t)$\end{document } are nothing else than dimensionless displacement and momentum of the corresponding mode ( called quadratures in quantum optics ) , normalized by zero point fluctuation amplitudes x0 and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${p_0}:{\hat x_\varepsilon}(t ) \equiv { \hat x_\varepsilon}/{x_0}$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat x_\varepsilon}(t ) \equiv { \hat p_\varepsilon}/{p_0}$\end{document}. this fact is also justified by the value of their commutator : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\left [ { { { \hat x}_\varepsilon}(t),\,{{\hat y}_\varepsilon}(t ) } \right ] = i\,.$$\end{document } there is no difficulty in showing that diagonal elements of the covariance matrix \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathbb v}_{ii}}$\end{document } are equal to the variances of the corresponding mode displacement \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat x_\varepsilon}$\end{document } and momentum \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat y_\varepsilon}$\end{document } : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\mathbb{v}}_{cc } } = \,\left\langle { \hat x_\varepsilon ^2(t ) } \right\rangle = 1/2\,,\quad { \rm{and}}\quad { { \mathbb{v}}_{ss } } = \,\left\langle { \hat y_\varepsilon ^2(t ) } \right\rangle = 1/2\,,$$\end{document } while non - diagonal terms represent correlations between these operators ( zero in our case ) : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathbb v}_{cs } } = { { \mathbb v}_{sc } } = \left\langle { { { \hat x}_\varepsilon}(t)\ , \circ \,{{\hat y}_\varepsilon}(t ) } \right\rangle = 0$\end{document}. at the same time , we see that there is no correlation between the pulses , justifying the markovianity of the quantum noise of light in vacuum state : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\left\langle { { { \hat x}_\varepsilon}(t){{\hat x}_\varepsilon}(t\prime ) } \right\rangle = \,\left\langle { { { \hat y}_\varepsilon}(t){{\hat y}_\varepsilon}(t\prime ) } \right\rangle = \,\left\langle { { { \hat x}_\varepsilon}(t ) \circ { { \hat y}_\varepsilon}(t\prime ) } \right\rangle = 0\,,\;t \neq t\prime \,.$$\end{document } an attempt to measure the light field amplitude as a function of time will give the result depicted in figure 13 . figure 13light field in a vacuum quantum state |vac. left panel ( a ) features a typical result one could get measuring the ( normalized ) electric field strength of the light wave in a vacuum state as a function of time . right panel ( b ) represents a phase space picture of the results of measurement . a red dashed circle displays the error ellipse for the state |vac that encircles the area of single standard deviation for a two - dimensional random vector of measured light quadrature amplitudes . the principal radii of the error ellipse ( equal in vacuum state case ) are equal to square roots of the covariance matrix \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathbb v}_{{\rm{vac}}}}$\end{document } eigenvalues , i.e. , to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$1/\sqrt 2$\end{document}. light field in a vacuum quantum state |vac. left panel ( a ) features a typical result one could get measuring the ( normalized ) electric field strength of the light wave in a vacuum state as a function of time . right panel ( b ) represents a phase space picture of the results of measurement . a red dashed circle displays the error ellipse for the state |vac that encircles the area of single standard deviation for a two - dimensional random vector of measured light quadrature amplitudes . the principal radii of the error ellipse ( equal in vacuum state case ) are equal to square roots of the covariance matrix \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathbb v}_{{\rm{vac}}}}$\end{document } eigenvalues , i.e. , to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$1/\sqrt 2$\end{document}. the measurement outcome at each instance of time will be a random variable with zero mean and variance defined by a covariance matrix \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathbb v}_{{\rm{vac}}}}$\end{document } of eq . ( 66 ) : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\rm{var}}[\hat e(t ) ] = \{\cos { \omega _ 0}t,\,\,\sin { \omega _ 0}t\ } { { \mathbb{v}}_{{\rm{vac}}}}{\{\cos { \omega _ 0}t,\,\sin { \omega _ 0}t\ } ^{\mathsf t } } = { 1 \over 2}\,.$$\end{document } in quantum mechanics , it is convenient to describe a quantum state in terms of a wigner function , a quantum version of joint ( quasi ) probability distribution for particle displacement and momentum ( x and y in our case ) : 68\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { w_{\vert { { \rm{vac } } } \rangle}}({x_\varepsilon},\,{y_\varepsilon } ) = \int\nolimits_{- \infty}^\infty { { { d\xi } \over { 2\pi } } } \exp \{{- i\xi { y_\varepsilon } } \}\langle { { x_\varepsilon } + \xi /2\vert { { \rm{vac } } } } \rangle \langle { \rm{vac}}\vert { x_\varepsilon } - { \xi /2 } \rangle \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad } \\ { = { 1 \over { 2\pi \sqrt { \det { { \mathbb { v}}_{{\rm{vac}}}}}}}\exp \left\{{- { 1 \over 2}{{\{{x_\varepsilon},\,{y_\varepsilon}\}}^{{\mathsf t}}}{\mathbb { v}}_{{\rm{vac}}}^{- 1}\{{x_\varepsilon},\,{y_\varepsilon}\ } } \right\ } = { 1 \over \pi}\exp \left\{{- ( x_\varepsilon ^2 + y_\varepsilon ^2 ) } \right\ } , } \\ \end{array}$$\end{document } where is simply the variable of integration . the above wigner function describes a gaussian state , which is simply the ground state of a harmonic oscillator represented by a mode with displacement x and momentum y. the corresponding plot is given in the left panel of figure 14 . gaussian states are traditionally pictured by error ellipses on a phase plane , as drawn in the right panel of figure 14 ( cf . as well as in figure 13 , a red line in both plots circumscribes all the values of x and y that fall inside the standard deviation region of the wigner function , i.e. , the region where all pertinent points are within 1 standard deviation from the center of the distribution . for a vacuum state , such a region is a circle with radius \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\sqrt { { { \mathbb v}_{cc } } } = \sqrt { { { \mathbb v}_{ss } } } = 1/\sqrt 2$\end{document}. the area of this circle , equal to 1/2 in dimensionless units and to /2 in case of dimensional displacement and momentum , is the smallest area a physical quantum state can occupy in a phase space . this fact yields from a very general physical principle , the heisenberg uncertainty relation , that limits the minimal uncertainty product for canonically conjugate observables ( displacement x and momentum y , in our case ) to be less than 1/2 in -units : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\left({{\rm{var}}[{{\hat x}_\varepsilon } ] } \right)^{1/2}}{\left({{\rm{var}}[{{\hat y}_\varepsilon } ] } \right)^{1/2}}\geqslant{1 \over 2}\,.$$\end{document } the fact that for a ground state this area is exactly equal to 1/2 is due to the fact that it is a pure quantum state , i.e. , the state of the particle that can be described by a wave function | , rather than by a density operator \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat \rho$\end{document}. for more sophisticated gaussian states with a non - diagonal covariance matrix \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathbb v}$\end{document } , the heisenberg uncertainty relation reads : 69\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\det { \mathbb{v}}\geqslant{1 \over 4}\,,$$\end{document } and noise ellipse major semi - axes are given by the square root of the matrix \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathbb v}$\end{document } eigenvalues . figure 14wigner function w|vac ( x , y ) of a ground state of harmonic oscillator ( left panel ) and its representation in terms of the noise ellipse ( right panel ) . wigner function w|vac ( x , y ) of a ground state of harmonic oscillator ( left panel ) and its representation in terms of the noise ellipse ( right panel ) . note the difference between figures 13 and 14 ; the former features the result of measurement of an ensemble of oscillators ( subsequent light pulses with infmitesimally short duration ) , while the latter gives the probability density function for a single oscillator displacement and momentum . another important state of light is a coherent state ( see , e.g. , [ 163 , 136 , 99 , 132 ] ) . it is straightforward to introduce a coherent state | of a single mode or a harmonic oscillator as a result of its ground state |0 shift on a complex plane by the distance and in the direction governed by a complex number = ||e . this can be caused , e.g. , by the action of a classical effective force on the oscillator . such a shift can be described by a unitary operator called a displacement operator , since its action on a ground state |0 inflicts its shift in a phase plane yielding a state that is called a coherent state : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\left\vert \alpha \right\rangle = \hat d\left [ \alpha \right]\left\vert 0 \right\rangle \equiv { e^{\alpha { { \hat a}^{\dagger } } - { \alpha ^{\ast}}\hat a}}\left\vert 0 \right\rangle \,,$$\end{document } or , more vividly , in q - representation of a corresponding mode of the field \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\psi _ { coh}}({x_\omega } ) \equiv \,\left\langle { \left . { { x_\omega } } \right\vert \alpha } \right\rangle = { 1 \over { \sqrt [ { 4}]{\pi}}}\exp \left\{{- { { { { ( { x_\omega } - \sqrt 2 \alpha)}^2 } } \over 2 } } \right\}\,.$$\end{document } the shift described by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat d[\alpha ] $ \end{document } is even more apparent if one writes down its action on an annihilation ( creation ) operator : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\hat d^\dagger}[\alpha ] \hat a\hat d[\alpha ] = \hat a + \alpha \,,\quad \left({{{\hat d}^\dagger}[\alpha ] { { \hat a}^\dagger}\hat d[\alpha ] = { { \hat a}^\dagger } + { \alpha ^{\ast } } } \right)\,.$$\end{document } moreover , a coherent state is an eigenstate of the annihilation operator : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat a\left\vert \alpha \right\rangle = \alpha \left\vert \alpha \right\rangle \,.$$\end{document } using the definitions of the mode quadrature operators \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat x_\omega } \equiv \hat x$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat y_\varepsilon } \equiv \hat y$\end{document } ( dimensionless oscillator displacement and momentum normalized by zero - point oscillations amplitude ) given above , one immediately obtains for their mean values in a coherent state : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\langle \alpha \vert \hat x\vert \alpha \rangle = \langle 0\vert { \hat d^\dagger}[\alpha ] \hat x\hat d[\alpha ] \vert 0\rangle = \sqrt 2 { \rm{re}}[\alpha ] \,,\qquad \langle \alpha \vert \hat y\vert \alpha \rangle = \langle 0{\hat d^\dagger}[\alpha ] \hat y\hat d[\alpha ] \vert 0\rangle = \sqrt 2 { \rm{im}}[\alpha ] \,.$$\end{document } further calculation shows that quadratures variances : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\rm{var}}[\hat x ] = \langle \alpha \vert { \hat x^2}\vert \alpha \rangle - { \left({\langle \alpha \vert \hat x\vert \alpha \rangle } \right)^2 } = { 1 \over 2}\,,\qquad { \rm{var}}[\hat y ] = \langle \alpha \vert { \hat y^2}\vert \alpha \rangle - { \left({\langle \alpha \vert \hat y\vert \alpha \rangle } \right)^2 } = { 1 \over 2}$$\end{document } have the same values as those for a ground state . these two facts unequivocally testify in favour of the statement that a coherent state is just the ground state shifted from the origin of the phase plane to the point with coordinates \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\left({{{\left\langle { \hat x } \right\rangle}_a},\,{{\left\langle { \hat y } \right\rangle}_\alpha } } \right ) = \sqrt 2 ( { \rm re}[\alpha ] , \,{\rm re}[\alpha ] ) $ \end{document}. it is instructive to calculate a wigner function for the coherent state using a definition of eq . ( 68 ) : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad { w_{\left\vert \alpha \right\rangle}}(x,\,y ) = \,\int\nolimits_{- \infty}^\infty { { { d\xi } \over { 2\pi } } } \exp \left\{{- i\xi y } \right\}\left\langle { x + \xi /\left . \alpha \right\vert x - \xi /2 } \right\rangle = } \\ { { 1 \over { 2\pi \sqrt { \det { { \mathbb { v}}_{{\rm{vac}}}}}}}\exp \left\{{- { 1 \over 2}{{\{x - \sqrt 2 { \rm{re}}[\alpha ] , \,y - \sqrt 2 { \rm{im}}[\alpha ] \}}^{\mathsf t}}{\mathbb { v}}_{{\rm{vac}}}^{- 1}\{x - \sqrt 2 { \rm{re}}[\alpha ] , \,y - \sqrt 2 { \rm{im}}[\alpha ] \ } } \right\ } = } \\ { \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad { 1 \over \pi}\exp \left\{{- \left [ { { { ( x - \sqrt 2 { \rm{re}}[\alpha ] ) } ^2 } + { { ( y - \sqrt 2 { \rm{im}}[\alpha ] ) } ^2 } } \right ] } \right\}\ , , } \\ \end{array}$$\end{document } which once again demonstrates the correctness of the former statement . generalization to the case of continuum of modes comprising a light wave is straightforward and goes along the same lines as the definition of the field vacuum state , namely ( see eq . ( 62 ) ) : 70\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\left\vert { \alpha ( \omega ) } \right\rangle \equiv { \underset{\omega } \bigotimes}{\left\vert \alpha \right\rangle _ \omega } = { \underset{\omega } \bigotimes } \hat d[\alpha ( \omega)]{\left\vert 0 \right\rangle _ \omega } = \exp \left\{{\int\nolimits_{- \infty}^\infty { { { d\omega } \over { 2\pi } } } ( \alpha ( \omega)\hat a_\omega ^{\rm{\dagger } } - { \alpha ^{\ast}}(\omega){{\hat a}_\omega } ) } \right\}\left\vert { { \rm{vac } } } \right\rangle , $ $ \end{document } where | is the coherent state that the mode of the field with frequency is in , and ( ) is the distribution of complex amplitudes over frequencies . basically , ( ) is the spectrum of normalized complex amplitudes of the field , i.e. , ( ) ( ) . for example , the state of a free light wave emitted by a perfectly monochromatic laser with emission frequency p and mean optical power \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}_0}$\end{document } will be defined by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\alpha ( \omega ) = \pi \sqrt { { { 2{{\mathcal i}_0 } } \over { \hbar { \omega _ p } } } } \delta ( \omega - \omega { - _ p})$\end{document } , which implies that only the mode at frequency p will be in a coherent state , while all other modes of the field will be in their ground states . operator \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat d[\alpha ] $ \end{document } is unitary , i.e. , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat d^\dagger}[\alpha ] \hat d[\alpha ] = \hat d[\alpha ] { \hat d^\dagger}[\alpha ] = \hat i$\end{document } with the identity operator , while the physical meaning is in the translation and rotation of the hilbert space that keeps all the physical processes unchanged . therefore , one can simply use vacuum states instead of coherent states and subtract the mean values from the corresponding operators in the same way we have done previously for the light wave classical amplitudes , just below eq . ( 60 ) . the covariance matrix and the matrix of power spectral densities for the quantum noise of light in a coherent state is thus the same as that of a vacuum state case . the typical result one can get measuring the electric field strength of light emitted by the aforementioned ideal laser is drawn in the left panel of figure 15 . figure 15light field in a coherent quantum state |(). left panel a ) features a typical result one could get measuring the ( normalized ) electric field strength of the light wave in a coherent state as a function of time . the red dashed line in the left panel marks the mean value (t). the red arrow in the right panel features the vector of the mean values of quadrature amplitudes , i.e. , a , while the red dashed circle displays the error ellipse for the state |() that encircles the area of single standard deviation for a two - dimensional random vector of quadrature amplitudes . the principle radii of the error ellipse ( equal in the coherent state case ) are equal to square roots of the covariance matrix \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathbb v}_{{\rm{coh}}}}$\end{document } , i.e. , to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$1/\sqrt 2$\end{document}. light field in a coherent quantum state |(). left panel a ) features a typical result one could get measuring the ( normalized ) electric field strength of the light wave in a coherent state as a function of time . the red dashed line in the left panel marks the mean value (t). the red arrow in the right panel features the vector of the mean values of quadrature amplitudes , i.e. , a , while the red dashed circle displays the error ellipse for the state |() that encircles the area of single standard deviation for a two - dimensional random vector of quadrature amplitudes . the principle radii of the error ellipse ( equal in the coherent state case ) are equal to square roots of the covariance matrix \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathbb v}_{{\rm{coh}}}}$\end{document } , i.e. , to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$1/\sqrt 2$\end{document}. one more quantum state of light that is worth consideration is a squeezed state . to put it in simple words , it is a state where one of the oscillator quadratures variance appears decreased by some factor compared to that in a vacuum or coherent state , while the conjugate quadrature variance finds itself swollen by the same factor , so that their product still remains heisenberg - limited . squeezed states of light are usually obtained as a result of a parametric down conversion ( pdc ) process [ 92 , 172 ] in optically nonlinear crystals . this is the most robust and experimentally elaborated way of generating squeezed states of light for various applications , e.g. , for gw detectors [ 149 , 152 , 141 ] , or for quantum communications and computation purposes . however , there is another way to generate squeezed light by means of a ponderomotive nonlinearity inherent in such optomechanical devices as gw detectors . this method , first proposed by corbitt et al . , utilizes the parametric coupling between the resonance frequencies of the optical modes in the fabry - prot cavity and the mechanical motion of its mirrors arising from the quantum radiation pressure fluctuations inflicting random mechanical motion on the cavity mirrors . further , we will see that the light leaving the signal port of a gw interferometer finds itself in a ponderomotively squeezed state ( see , e.g. , for details ) . a dedicated reader might find it illuminating to read the following review articles on this topic [ 133 , 101 ] . worth noting is the fact that generation of squeezed states of light is the process that inherently invokes two modes of the field and thus naturally calls for usage of the two - photon formalism contrived by caves and schumaker [ 39 , 40 ] . to demonstrate this let us consider the physics of a squeezed state generation in a nonlinear crystal . here photons of a pump light with frequency p = 20 give birth to pairs of correlated photons with frequencies 1 and 2 ( traditionally called signal and idler ) by means of the nonlinear dependence of polarization in a birefringent crystal on electric field . such a process can be described by the following hamiltonian , provided that the pump field is in a coherent state |ap with strong classical amplitude |p| 1 ( see , e.g. , section 5.2 of for details ) : 71\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{{{\hat h}_{{\rm{pdc } } } } } \over \hbar } = { \omega _ 1}\hat a_1^\dagger{\hat a_1 } + { \omega _ 2}\hat a_2^\dagger{\hat a_2 } + i\left({\chi \hat a_1^\dagger\hat a_2^\dagger{e^{- 2i{\omega _ 0}t } } - { \chi ^{\ast}}{{\hat a}_1}{{\hat a}_2}{e^{2i{\omega _ 0}t } } } \right)\,,$$\end{document } where 1,2 describe annihilation operators for the photons of the signal and idler modes and = e is the complex coupling constant that is proportional to the second - order susceptibility of the crystal and to the pump complex amplitude . worth noting is the meaning of t in this hamiltonian : it is a parameter that describes the duration of a pump light interaction with the nonlinear crystal , which , in the simplest situation , is either the length of the crystal divided by the speed of light c , or , if the crystal is placed between the mirrors of the optical cavity , the same as the above but multiplied by an average number of bounces of the photon inside this cavity , which is , in turn , proportional to the cavity finesse \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f}$\end{document}. it is straightforward to obtain the evolution of the two modes in the interaction picture ( leaving apart the obvious free evolution time dependence \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${e^{- i{\omega _ { s , i}}t}}$\end{document } ) solving the heisenberg equations : 72\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\hat a_1}(t ) = { \hat a_1}\cosh \rho t + \hat a_2^\dagger{e^{2i\phi}}\sinh \rho t\,,\qquad { \hat a_2}(t ) = { \hat a_2}\cosh \rho t + \hat a_1^\dagger{e^{2i\phi}}\sinh \rho t\,.$$\end{document } let us then assume the signal and idler mode frequencies symmetric with respect to the half of pump frequency 0 = p/2 : 1 + = 0 + and 2 = 0 ( 1 + and 2 ) . then the electric field of a two - mode state going out of the nonlinear crystal will be written as ( we did not include the pump field here assuming it can be ruled out by an appropriate filter ) : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat e(t ) = { { \mathcal c}_0}\left [ { \hat x(t)\cos { \omega _ 0}t + \hat y(t)\sin { \omega _ 0}t } \right],$$\end{document } where two - mode quadrature amplitudes \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat x(t)$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat y(t)$\end{document } are defined along the lines of eqs . ( 57 ) , keeping in mind that only idler and signal components at the frequencies 0 = should be kept in the integral , which yields : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { \hat x(t ) = { 1 \over { \sqrt 2}}\left [ { { { \hat a } _ + } ( t){e^{i\omega t } } + \hat a -}(t){e^{- i\omega t } } + \hat a _ - ^{\rm{\dagger}}(t){e^{i\omega t } } } \right ] = \hat a_c^{{\rm{sqz}}}{e^{- i\omega t } } + \hat a_c^{{\rm{sqz\dagger}}}{e^{i\omega t}}\ , , } \\ { \hat y(t ) = { 1 \over { i\sqrt 2}}\left [ { { { \hat a } _ + } ( t){e^{i\omega t } } - \hat a _ -}(t){e^{- i\omega t } } - \hat a _ - ^{\rm{\dagger}}(t){e^{i\omega t } } } \right ] = \hat a_s^{{\rm{sqz}}}{e^{- i\omega t } } + \hat a_s^{{\rm{sqz\dagger}}}{e^{i\omega t}}\ , , } \\ \end{array}$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat a_c^{{\rm{sqz } } } = ( { { \hat a } _ + } ( t ) + \hat a _ - ^\dagger ( t))/\sqrt 2$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat a_s^{{\rm{sqz } } } = ( { \hat a _ + } ( t ) - \hat a _ - ^\dagger ( t))/(i\sqrt 2)$\end{document } are the spectral quadrature amplitudes of the two - mode field at sideband frequency ( cf . ( 72 ) into the above expressions yields transformation rules for quadrature amplitudes : 73\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { \hat a_c^{{\rm{sqz } } } = { { \hat a}_c}\left({\cosh \rho t + \cos 2\phi \sinh \rho t } \right ) + { { \hat a}_s}\sin 2\phi \sinh \rho t\ , , } \\ { \hat a_s^{{\rm{sqz } } } = { { \hat a}_c}\sin 2\phi \sinh \rho t + { { \hat a}_s}\left({\cosh \rho t - \cos 2\phi \sinh \rho t } \right)\ , , } \\ \end{array}$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat a_c } = ( { \hat a _ + } + \hat a _ - ^\dagger)/\sqrt 2$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat a_s } = ( { \hat a _ + } - \hat a _ - ^\dagger)/(i\sqrt { 2)}$\end{document } stand for initial values of spectral quadrature amplitudes of the two - mode light wave created in the pdc process . a close look at these transformations written in the matrix form reveals that it can be represented as the following sequence : 74\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\hat a^{{\rm{sqz } } } } = \left({\begin{array}{*{20}c } { \hat a_c^{{\rm{sqz } } } } \\ { \hat a_s^{{\rm{sqz } } } } \\ \end{array } } \right ) = { { \mathbb { s}}_{{\rm{sqz } } } [ \rho t,\phi ] \hat a = { \mathbb { p}}[\phi ] { \mathbb { s}}_{{\rm{sqz}}}}[\rho t,0]{\mathbb { p } } [ - \phi ] \hat a$$\end{document } where 75\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathbb { s}_{{\rm{sqz}}}}[\rho t,\phi ] \equiv \left [ { \begin{array}{*{20}c } { \cosh \rho t + \cos 2\phi \sinh \rho t } & { \sin 2\phi \sinh \rho t } \\ { \sin 2\phi \sinh \rho t } & { \cosh \rho t - \cos 2\phi \sinh \rho t } \\ \end{array } } \right]\ ; \rightarrow \;{\mathbb { s}_{{\rm{sqz}}}}[\rho t,0 ] = \left [ { \begin{array}{*{20}c } { { e^{\rho t } } } & 0 \\ 0 & { { e^{- \rho t } } } \\ \end{array } } \right]$$\end{document } are squeezing matrices in general and in special = 0 ) case , while [ ] stands for a counterclockwise 2d - rotation matrix by angle defined by ( 17 ) . therefore , the evolution of a two - mode light quadrature amplitude vector in a pdc process described by the hamiltonian ( 71 ) consists of a clockwise rotation by an angle followed by a deformation along the main axes ( stretching along the ac - axis and proportional squeezing along the as - axis ) and rotation back by the same angle . it is straightforward to show that vector \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat x^{{\rm{sqz } } } } = { \left\{{\hat x(t),\,\hat y(t ) } \right\}^{\rm{t } } } = { \hat a^{{\rm{sqz}}}}{e^{- i\omega t } } + { \hat a^{{\rm{sqz*}}}}{e^{i\omega t}}$\end{document } transforms similarly \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$({\rm{here}}\,{\hat a^{{\rm{sqz * } } } } = { \left\{{\hat a_c^{{\rm{sqz}}\dagger},\,\hat a_s^{{\rm{sqz}}\dagger } } \right\}^{\rm{t}}})$\end{document}. this geometric representation is rather useful , particularly for the characterization of a squeezed state . if the initial state of the two - mode field is a vacuum state then the outgoing field will be in a squeezed vacuum state . one can define it as a result of action of a special squeezing operator [ , t , ] on the vacuum state 76\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\left\vert { { \rm{sqz } } } _ { 0}(\rho t , \phi ) \rangle = \hat s[\rho t , \phi ] \right\vert{\rm{vac } } \rangle { .}$$\end{document } this operator is no more and no less than the evolution operator for the pdc process in the interaction picture , i.e. , 77\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat s[\rho t,\phi ] \equiv \exp \left\{{\rho t({{\hat a } _ + } { { \hat a } _ _ + ^\dagger \hat a _ - ^\dagger { e^{2i\phi } } ) } \right\}.$$\end{document } action of this operator on the two - photon quadrature amplitudes is fully described by eqs . ( 76 ) : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\hat a^{{\rm{sqz } } } } = { \hat s^\dagger}[\rho t,\phi ] \hat a\hat s[\rho t,\phi ] = \mathbb{p}[\phi ] { \mathbb{s}_{{\rm{sqz}}}}[\rho t,0]\mathbb{p } [ - \phi ] \hat a,$$\end{document } while annihilation operators of the corresponding modes are transformed in accordance with eqs . the linearity of the squeezing transformations implies that the squeezed vacuum state is gaussian since it is obtained from the gaussian vacuum state and therefore can be fully characterized by the expectation values of operators \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat x$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat y$\end{document } and their covariance matrix \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathbb v}_{{\rm{sqz}}}}$\end{document}. let us calculate these values : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\left\langle { \hat x } \right\rangle _ { { \rm{sqz } } } } = \left\langle { { \rm{sq}}{{\rm{z}}_0}(r,\phi)\vert\hat x\vert{\rm{sq}}{{\rm{z}}_0}(r,\phi ) } \right\rangle = \left\langle { { \rm{vac}}\vert{{\hat s}^\dagger}[r,\phi ] \hat x\hat s[r,\phi ] \vert{\rm{vac } } } \right\rangle = \left\langle { { \rm{vac}}\vert\hat x(t)\vert{\rm{vac } } } \right\rangle = 0,\quad { \left\langle { \hat y } \right\rangle _ { { \rm{sqz } } } } = 0,$$\end{document } and for a covariance matrix one can get the following expression : 78\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { \mathbb{v}_{{\rm{sqz } } } } = \left\langle { \rm{sq}}{\rm{z}}_{0}(r,\phi)\vert\hat x \circ { \hat x}^ { { \mathsf { t}}}\vert{\rm{sq}}{\rm{z}}_{0}(r,\phi ) \right\rangle = \mathbb{p } [ - \phi ] { \mathbb{s}_{{\rm{sqz}}}}[r,0]\mathbb{v}_{\rm{vac}}\mathbb{s}_{\rm{sqz}}[r,0]\mathbb{p}[\phi ] = \,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\ , } \\ { 1 \over 2}\mathbb{p } [ - \phi ] \mathbb{s}_{\rm{sqz}}[2r,0]\mathbb{p}[\phi ] = { 1 \over 2}\left [ \begin{array}{*{20}c } { \cos \phi } & { \sin \phi } \\ { - \sin \phi } & { \cos \phi}\\ \end{array } \right]\,\,\,\left [ \begin{array}{*{20}c } { { e^{2r } } } & 0 \\ 0 & { { e^{- 2r}}}\\ \end{array } \right]\,\,\,\left [ \begin{array}{*{20}c } { \cos \phi } & { - \sin \phi } \\ { \sin \phi } & { \cos \phi}\\ \end{array } \right ] , \end{array}$$\end{document } where we introduced squeezing parameter r t and used a short notation for the symmetrized outer product of vector \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat x$\end{document } with itself : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat x \circ { \hat x^\mathsf { t } } \equiv \left [ { \begin{array}{*{20}c } { \hat x \circ \hat x\,\,\hat x \circ \hat y } \\ { \hat y \circ \right].$$\end{document } the squeezing parameter r is the quantity reflecting the strength of the squeezing . this way of characterizing the squeezing strength , though convenient enough for calculations , is not very ostensive . conventionally , squeezing strength is measured in decibels ( db ) that are related to the squeezing parameter r through the following simple formula : 79\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${r_{{\rm{db } } } } = 10\;{\log _ { 10}}{e^{2r } } = 20r\;{\log _ { 10}}e\ ; \leftrightarrow r = { r_{{\rm{db}}}}/(20\;{\log _ { 10}}e){.}$$\end{document } for example , 10 db squeezing corresponds to r 1.15 . the covariance matrix ( 78 ) refers to a unique error ellipse on a phase plane with semi - major axis \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${e^r}/\sqrt 2$\end{document } and semi - minor axis \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${e^{- r}}/\sqrt 2$\end{document } rotated by angle clockwise as featured in figure 16 . figure 16schematic plot of a vacuum state transformation under the action of the squeezing operator [r , ] . ( 74 ) demonstrate the equivalence of the general squeezing operator [r , ] to a sequence of phase plane counterclockwise rotation by an angle ( transition from a ) to b ) ) , phase plane squeezing and stretching by a factor e ( transition from b ) to c ) ) and rotation back by the same angle ( transition from c ) to d ) ) . point p tracks how transformations change the initial state marked with point p. schematic plot of a vacuum state transformation under the action of the squeezing operator [r , ] . ( 74 ) demonstrate the equivalence of the general squeezing operator [r , ] to a sequence of phase plane counterclockwise rotation by an angle ( transition from a ) to b ) ) , phase plane squeezing and stretching by a factor e ( transition from b ) to c ) ) and rotation back by the same angle ( transition from c ) to d ) ) . point p tracks how transformations change the initial state marked with point p. it would be a wise guess to make , that a squeezed vacuum wigner function can be obtained from that of a vacuum state , using these simple geometric considerations . indeed , for a squeezed vacuum state it reads : 80\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${w_{\left\vert { { \rm{sqz } } } \right\rangle}}(x,\,y ) = { 1 \over { 2\pi \sqrt { \det { \mathbb{v}_{{\rm{sqz}}}}}}}\exp \left\{{- { 1 \over 2}{{\{x,\,y\}}^ { \mathsf { t}}}\mathbb{v}_{{\rm{sqz}}}^{- 1}\{x,\,y\ } } \right\},$$\end{document } where the error ellipse refers to the level where the wigner function value falls to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$1/\sqrt e$\end{document } of the maximum . the corresponding plot and phase plane picture of the squeezed vacuum wigner function are featured in figure 17 . figure 17left panel : wigner function of a squeezed vacuum state with squeeze parameter r = 0.5 ( 5 db ) and rotation angle = /4 . right panel : error ellipse corresponding to that wigner function . left panel : wigner function of a squeezed vacuum state with squeeze parameter r = 0.5 ( 5 db ) and rotation angle = /4 . another important state that arises in gw detectors is the displaced squeezed state |sqz(r , ) that is obtained from the squeezed vacuum state in the same manner as the coherent state yields from the vacuum state , i.e. , by the application of the displacement operator ( equivalent to the action of a classical force ) : 81\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\left\vert { { \rm{sq}}{{\rm{z}}_\alpha}(r,\phi ) } \right\rangle = \hat d[\alpha ] \left\vert { { \rm{sq}}{{\rm{z}}_0}(r,\phi ) } \right\rangle = \hat d[\alpha ] \hat s[r,\phi ] \left\vert { { \rm{vac } } } \right\rangle .$$\end{document } the light leaving a gw interferometer from the signal port finds itself in such a state , if a classical gw - like force changes the difference of the arm lengths , thus displacing a ponderomotively squeezed vacuum state in phase quadrature y by an amount proportional to the magnitude of the signal force . such a displacement has no other consequence than simply to shift the mean values of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat x$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat y$\end{document } by some constant values dependent on shift complex amplitude : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\left\langle { \hat x } \right\rangle _ { { \rm{sqz } } } } = \sqrt 2 { \rm{re}}[\alpha ] \,,\qquad { \left\langle { \hat y } \right\rangle _ { { \rm{sqz } } } } = \sqrt 2 { \rm{im}}[\alpha ] .$$\end{document } let us now generalize the results of a two - mode consideration to a continuous spectrum case . apparently , quadrature operators \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat x(t)$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat y(t)$\end{document } are similar to c(t ) and s(t ) for the traveling wave case . utilizing this similarity , let us define a squeezing operator for the continuum of modes as : 82\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat s[r(\omega),\phi ( \omega ) ] \equiv \exp \left\{{\int\nolimits_{- \infty}^\infty { { { d\omega } \over { 2\pi}}r(\omega)\left [ { { { \hat a } _ + } { { \hat a } _ _ + ^\dagger \hat a _ - ^\dagger { e^{2i\phi ( \omega ) } } } \right ] } } \right\},$$\end{document } where r( ) and ( ) are frequency - dependent squeezing factor and angle , respectively . acting with this operator on a vacuum state of the travelling wave yields a squeezed vacuum state of a continuum of modes in the very same manner as in eq . the result one could get in the measurement of the electric field amplitude of light in a squeezed state as a function of time is presented in figure 18 . thus , we are free to use these formulas for calculation of the power spectral density matrix for a traveling wave squeezed vacuum state . indeed , substituting \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat a_{c , s}}(\omega ) \to \hat a_{c , s}^{{\rm{sqz}}}(\omega)$\end{document } in eq . ( 74 ) one immediately gets : 83\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathbb{s}_{{\rm{sqz}}}}(\omega ) = \mathbb{p } [ - \phi ( \omega)]{\mathbb{s}_{{\rm{sqz}}}}[r(\omega),0]{\mathbb{s}_{{\rm{vac}}}}(\omega){\mathbb{s}_{{\rm{sqz}}}}[r(\omega),0]\mathbb{p}[\phi ( \omega ) ] = { \mathbb{s}_{{\rm{sqz}}}}(r(\omega),\phi ( \omega)).$$\end{document } note that entries of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathbb s}_{{\rm{sqz}}}}(\omega)$\end{document } might be frequency dependent if squeezing parameter r( ) and squeezing angle ( ) are frequency dependent as is the case in all physical situations . this indicates that quantum noise in a squeezed state of light is not markovian and this can easily be shown by calculating the the covariance matrix , which is simply a fourier transform of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathbb s}(\omega)$\end{document } according to the wiener - khinchin theorem : 84\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathbb{v}_{\rm{s}}}_{{\rm{qz}}}(t - t{\prime } ) = \int\nolimits_{- \infty}^\infty { { { d\omega } \over { 2\pi}}{\mathbb{s}_{\rm{s}}}_{{\rm{qz}}}(\omega){e^{- i\omega ( t - t{\prime})}}.}$$\end{document } of course , the exact shape of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathbb v}_{{\rm{sqz}}}}(t - { t\prime})$\end{document } could be obtained only if we specify r( ) and ( ) . note that the noise described by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathbb v}_{{\rm{sqz}}}}(t - { t\prime})$\end{document } is stationary since all the entries of the covariance matrix ( correlation functions ) depend on the difference of times t t. figure 18light field in a squeezed state |sqz(r , ). upper row features time dependence of the electric field strength e(t ) in three different squeezed states ( 10 db squeezing assumed for all ) : a ) squeezed vacuum state with squeezing angle = /4 ; b ) displaced squeezed state with classical amplitude ac = 5 ( mean field strength oscillations (t)sqz are given by red dashed line ) and amplitude squeezing ( = /2 ) ; c ) displaced squeezed state with classical amplitude ac = 5 and phase squeezing ( = 0 ) . lower row features error ellipses ( red dashed lines ) for the corresponding plots in the upper row . light field in a squeezed state |sqz(r , ). upper row features time dependence of the electric field strength e(t ) in three different squeezed states ( 10 db squeezing assumed for all ) : a ) squeezed vacuum state with squeezing angle = /4 ; b ) displaced squeezed state with classical amplitude ac = 5 ( mean field strength oscillations (t)sqz are given by red dashed line ) and amplitude squeezing ( = /2 ) ; c ) displaced squeezed state with classical amplitude ac = 5 and phase squeezing ( = 0 ) . lower row features error ellipses ( red dashed lines ) for the corresponding plots in the upper row . the spectral density matrix allows for pictorial representation of a multimode squeezed state where an error ellipse is assigned to each sideband frequency . this effectively adds one more dimension to a phase plane picture already used by us for the characterization of a two - mode squeezed states . figure 19 exemplifies the state of a ponderomotively squeezed light that would leave the speed - meter type of the interferometer ( see section 6.2 ) . figure 19example of a squeezed state of the continuum of modes : output state of a speed - meter interferometer . left panel shows the plots of squeezing parameter rdb( ) and squeezing angle ( ) versus normalized sideband frequency / ( here is the interferometer half - bandwidth ) . right panel features a family of error ellipses for different sideband frequencies that illustrates the squeezed state defined by rdb( ) and ( ) drawn in the left panel . example of a squeezed state of the continuum of modes : output state of a speed - meter interferometer . left panel shows the plots of squeezing parameter rdb( ) and squeezing angle ( ) versus normalized sideband frequency / ( here is the interferometer half - bandwidth ) . right panel features a family of error ellipses for different sideband frequencies that illustrates the squeezed state defined by rdb( ) and ( ) drawn in the left panel . in this section , we give a brief introduction to calculation of the power spectral densities of quantum noise one usually encounters in linear optical measurement . in optomechanical sensors , as we have discussed earlier , the outgoing light carries the information about the measured quantity ( e.g. , the displacement due to gw tidal forces ) in its phase and ( sometimes ) amplitude quadratures . the general transformation from the input light characterized by a vector of quadrature amplitudes ( ) = { c( ) , s( ) } tto the readout quantity of a meter is linear and can be written in spectral form as : 85\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat y(\omega ) = { { \mathcal y}^\dagger}(\omega)\hat a(\omega ) + g(\omega ) = { \mathcal y}_c^{\ast}(\omega){\hat a_c}(\omega ) + { \mathcal y}_s^{\ast}(\omega){\hat a_s}(\omega ) + g(\omega),$$\end{document } where g( ) is the spectrum of the measured quantity , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal y}_{c , s}}(\omega)$\end{document } are some complex - valued functions of that characterize how the light is transformed by the device . quantum noise is represented by the terms of the above expression not dependent on the measured quantity g , i.e. , 86\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\hat n_y}(\omega ) = { { \mathcal y}^\dagger}(\omega)\hat a(\omega ) = \left({{\mathcal y}_c^{\ast}(\omega)\,\,{\mathcal y}_s^{\ast}(\omega ) } \right)\,\,.\,\,\left({\begin{array}{*{20}c } { { { \hat a}_c}(\omega ) } \\ { { { \hat a}_s}(\omega ) } \\ \end{array } } \right).$$\end{document } the measure of quantum noise is the power spectral density sy ( ) that is defined by the following expression : 87\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$2\pi { s_y}(\omega)\delta ( \omega - \omega \prime ) = \left\langle { \psi \vert{{\hat n}_y}(\omega ) \circ \hat n_y^\dagger ( \omega \prime){\rm{\vert}}\psi } \right\rangle = \left\langle { { { \hat n}_y}(\omega ) \circ \hat n_y^\dagger ( \omega \prime ) } \right\rangle .$$\end{document } here | is the quantum state of the light wave . in our review , we will encounter two types of quantum states that we have described above , i.e. , vacuum |vac and squeezed vacuum |sqz0(r , ) states . let us show how to calculate the power ( double - sided ) spectral density of a generic quantity \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat y(\omega)$\end{document } in a vacuum state . to do so , one should substitute eq . ( 87 ) and obtain that : 88\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { s_y^{{\rm{vac}}}(\omega ) = { { \mathcal y}^\dagger}(\omega){{\left\langle { \,\hat a(\omega ) \circ { { \hat a}^\dagger}(\omega ) } \right\rangle}_{{\rm{vac}}}}{\mathcal y}(\omega ) = { { \mathcal y}^\dagger}(\omega){\mathbb{s}_{{\rm{vac}}}}(\omega){\mathcal y}(\omega ) } \\ { = { { { { \mathcal y}^\dagger}(\omega){\mathcal y}(\omega ) } \over 2 } = { { \vert{{\mathcal y}_c}(\omega){\vert^2 } + \vert{{\mathcal y}_s}(\omega){\vert^2 } } \over 2},\quad \quad \quad \ , } \\ \end{array}$$\end{document } where we used the definition of the power spectral density matrix of light in a vacuum state ( 65)7 . similarly , one can calculate the spectral density of quantum noise if the light is in a squeezed state |sqz0(r , ) } , utilizing the definition of the squeezed state density matrix given in eq . ( 83 ) : 89\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { s_y^{{\rm{sqz}}}(\omega ) = { { \mathcal y}^\dagger}(\omega){{\left\langle { \hat a(\omega)^\circ { { \hat a}^\dagger}(\omega ) } \right\rangle}_{{\rm{sqz}}}}{\mathcal y}(\omega ) = { { \mathcal y}^\dagger}(\omega){\mathbb{s}_{{\rm{sqz}}}}(\omega){\mathcal y}(\omega ) = { 1 \over 2}{{\mathcal y}^\dagger}(\omega)\mathbb{p } [ - \phi ] { \mathbb{s}_{{\rm{sqz}}}}[2r,0]\mathbb{p}[\phi ] { \mathcal y}(\omega ) } \\ { = { { \vert{{\mathcal y}_c}(\omega){\vert^2 } } \over 2}(\cosh 2r + \sinh 2r\cos 2\phi ) + { { \vert{{\mathcal y}_s}(\omega){\vert^2 } } \over 2}(\cosh 2r - \sinh 2r\cos 2\phi)\quad \quad \quad \quad \,\ , } \\ { - { \rm{re}}\left [ { { { \mathcal y}_c}(\omega){\mathcal y}_s^{\ast}(\omega ) } \right]\sinh 2r\sin 2\phi .\quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad } \\ \end{array}$$\end{document } it might also be necessary to calculate also cross - correlation spectral density syz( ) of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat y(\omega)$\end{document } with some other quantity \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat z(\omega)$\end{document } with quantum noise defined as : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\hat n_z}(\omega ) = { \mathcal z}{(\omega)^\dagger}\hat a(\omega ) = { \mathcal z}_c^{\ast}(\omega){\hat a_c}(\omega ) + { \mathcal z}_s^{\ast}(\omega){\hat a_s}(\omega){.}$$\end{document } using the definition of cross - spectral density syz( ) similar to ( 87 ) : 90\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$2\pi { s_{yz}}(\omega)\delta ( \omega - \omega \prime ) = \left\langle { \psi \vert{{\hat n}_y}(\omega)^\circ \hat n_z^\dagger ( \omega \prime){\rm{\vert}}\psi } \right\rangle = \left\langle { { { \hat n}_y}(\omega)^\circ \hat n_z^\dagger ( \omega \prime ) } \right\rangle , $ $ \end{document } one can get the following expressions for spectral densities in both cases of the vacuum state : 91\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { s_{y\,z}^{{\rm{vac}}}(\omega ) = { { \mathcal y}^\dagger}(\omega){{\left\langle { \hat a(\omega ) \circ { { \hat a}^\dagger}(\omega ) } \right\rangle}_{{\rm{vac}}}}{\mathcal z}(\omega ) = { { \mathcal y}^\dagger}(\omega){\mathbb{s}_{{\rm{vac}}}}(\omega){\mathcal z}(\omega)\quad \quad \quad \quad \quad \,\,\,\ , } \\ { = { { { { \mathcal y}^\dagger}(\omega){\mathcal z}(\omega ) } \over 2 } = { { { \mathcal y}_c^{\ast}(\omega){{\mathcal z}_c}(\omega ) + { \mathcal y}_s^{\ast}(\omega){{\mathcal z}_s}(\omega ) } \over 2},\quad \quad \quad \quad \quad \,\,\ , } \\ \end{array}$$\end{document } and the squeezed state : 92\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { s_{y\,z}^{{\rm{sqz}}}(\omega ) = { { \mathcal y}^\dagger}(\omega){{\left\langle { \hat a(\omega ) \circ { { \hat a}^\dagger}(\omega ) } \right\rangle}_{{\rm{sqz}}}}{\mathcal z}(\omega ) = { { \mathcal y}^\dagger}(\omega){\mathbb{s}_{{\rm{sqz}}}}(\omega){\mathcal z}(\omega)\quad \,\,\,\quad \quad \,\,\,\quad \quad \,\,\,\quad \quad \,\,\,\quad \quad \,\,\,\quad \,\,\,\,\ , } \\ { = { 1 \over 2}{{\mathcal y}^\dagger}(\omega)\mathbb{p } [ - \phi ] { \mathbb{s}_{{\rm{sqz}}}}[2r,0]\mathbb{p}[\phi ] { \mathcal z}(\omega)\quad \quad \quad \,\,\,\,\quad \quad \quad \quad \,\,\,\,\quad \quad \quad \quad \,\,\,\,\quad \quad \quad \quad \ , } \\ { = { { { \mathcal y}_c^{\ast}(\omega){{\mathcal z}_c}(\omega ) } \over 2}(\cosh 2r + \sinh 2r\cos 2\phi ) + { { { \mathcal y}_s^{\ast}(\omega){{\mathcal z}_s}(\omega ) } \over 2}(\cosh 2r - \sinh 2r\cos 2\phi ) } \\ { - { { { \mathcal y}_s^{\ast}(\omega){{\mathcal z}_c}(\omega ) + { \mathcal y}_c^{\ast}(\omega){{\mathcal z}_s}(\omega ) } \over 2}\sinh 2r\sin 2\phi .\quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \,\,\ , } \\ \end{array}$$\end{document } note that since the observables \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat y(t)$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat z(t)$\end{document } that one calculates spectral densities for are hermitian , it is compulsory , as is well known , for any operator to represent a physical quantity , then the following relation holds for their spectral coefficients \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal y}_{c , s}}(\omega)$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal z}_{c , s}}(\omega)$\end{document } : 93\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal y}_{c , s}^{\ast}(\omega ) = { { \mathcal y}_{c , s}}(- \omega)\,,\qquad { \rm{and}}\qquad { \mathcal z}_{c , s}^{\ast}(\omega ) = { { \mathcal z}_{c , s}}(- \omega){.}$$\end{document } this leads to an interesting observation that the coefficients \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal y}_{c , s}}(\omega)$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal z}_{c , s}}(\omega)$\end{document } should be real - valued functions of variable s = i. now we can make further generalizations and consider multiple light and vacuum fields comprising the quantity of interest : 94\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat y(\omega)\ ; \rightarrow \;{\hat n_y}(\omega ) = \sum\limits_{i = 1}^n { { \mathcal y}_i^\dagger ( \omega){{\hat a}_i}(\omega),}$$\end{document } where i( ) stand for quadrature amplitude vectors of n independent electromagnetic fields , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal y}_i^\dagger ( \omega)$\end{document } are the corresponding complex - valued coefficient functions indicating how these fields are transmitted to the output . in reality , the readout observable of a gw detector is always a combination of the input light field and vacuum fields that mix into the output optical train as a result of optical loss of various origin . this statement can be exemplified by a single lossy mirror i / o - relations given by eq . thus , to calculate the spectral density for such an observable , one needs to know the initial state of all light fields under consideration . since we assume i( ) independent from each other , the initial state will simply be a direct product of the initial states for each of the fields : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\vert \psi \rangle = \underset{i=1}{\overset{n}\otimes}\vert { { \psi _ i } } \rangle , $ $ \end{document } and the formula for the corresponding power ( double - sided ) spectral density reads : 95\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s_y}(\omega ) = \sum\limits_{i = 1}^n { { \mathcal y}_i^\dagger ( \omega ) } \left\langle { { \psi _ i}\vert{{\hat a}_i}(\omega ) \circ \hat a_i^\dagger ( \omega)\vert{\psi _ i } } \right\rangle { { \mathcal y}_i}(\omega ) = \sum\limits_{i = 1}^n { { \mathcal y}_i^\dagger ( \omega){\mathbb{s}_i}(\omega){{\mathcal y}_i}(\omega ) = \sum\limits_{i = 1}^n { { s_{{y_i } } } } ( \omega),}$$\end{document } with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathbb s}_i}(\omega)$\end{document } standing for the i - th input field spectral density matrix . hence , the total spectral density is just a sum of spectral densities of each of the fields . the cross - spectral density for two observables \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat y(\omega)$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat z(\omega)$\end{document } can be built by analogy and we leave this task to the reader . in section 3 , we discussed the quantum nature of light and fluctuations of the light field observables like phase and amplitude that stem thereof and yield what is usually called the quantum noise of optical measurement . in gw detection applications , where a sensitivity of the phase measurement is essential , as discussed in section 2.1.3 , the natural question arises : is there a limit to the measurement precision imposed by quantum mechanics ? a seemingly simple answer would be that such a limit is set by the quantum fluctuations of the outgoing light phase quadrature , which are , in turn , governed by the quantum state the outgoing light finds itself in . the difficult part is that on its way through the interferometer , the light wave inflicts an additional back - action noise that adds up to the phase fluctuations of the incident wave and contaminates the output of the interferometer . the origin of this back action is in amplitude fluctuations of the incident light , giving rise to a random radiation pressure force that acts on the interferometer mirrors along with the signal gw force , thus effectively mimicking it . and it is the fundamental principle of quantum mechanics , the heisenberg uncertainty principle , that sets a limit on the product of the phase and amplitude uncertainties ( since these are complementary observables ) , thus leading up to the lower bound of the achievable precision of phase measurement . this limit appears to be a general feature for a very broad class of measurement known as linear measurement and is referred to as the sql [ 16 , 22 ] . in this section , we try to give a brief introduction to quantum measurement theory , starting from rather basic examples with discrete measurement and then passing to a general theory of continuous linear measurement . we introduce the concept of the sql and derive it for special cases of probe bodies . we also discuss briefly possible ways to overcome this limit by contriving smarter ways of weak force measurement then direct coordinate monitoring . let us consider a very simple measurement scheme , which , nevertheless , embodies all key features of a general position measurement . in the scheme shown in figure 20 , a sequence of very short light pulses are used to monitor the displacement of a probe body m. the position x of m is probed periodically with time interval . in order to make our model more realistic , we suppose that each pulse reflects from the test mass f > 1 times , thus increasing the optomechanical coupling and thereby the information of the measured quantity contained in each reflected pulse . we also assume mass m large enough to neglect the displacement inflicted by the pulses radiation pressure in the course of the measurement process . then each j - th pulse , when reflected , carries a phase shift proportional to the value of the test - mass position x(tj ) at the moment of reflection : 96\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat \phi _ j^{{\rm{refl } } } = { \hat \phi _ j } - 2\digamma{k_p}\hat x({t_j}),$$\end{document } where kp = p / c , p is the light frequency , j = , 1 , 0 , 1 , is the pulse number and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat \phi _ j}$\end{document } is the initial ( random ) phase of the j - th pulse . we assume that the mean value of all these phases is equal to zero , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\left\langle { { { \hat \phi}_j } } \right\rangle = 0$\end{document } , and their root mean square ( rms ) uncertainty \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\left\langle { ( { { \hat \phi}^2 } } \right\rangle - { \left\langle { \hat \phi } \right\rangle ^2}{/^{1/2}}$\end{document } is equal to . the reflected pulses are detected by a phase - sensitive device ( the phase detector ) . the implementation of an optical phase detector is considered in detail in section 2.3.1 . here we suppose only that the phase \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat \phi _ j^{{\rm{refl}}}$\end{document } measurement error introduced by the detector is much smaller than the initial uncertainty of the phases . in this case , the initial uncertainty will be the only source of the position measurement error : 97\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta { x_{{\rm{meas } } } } = { { \delta \phi } \over { 2\digamma{k_p}}}.$$\end{document } for convenience , we renormalize eq . ( 96 ) as the equivalent test - mass displacement : 98\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\tilde x_j } \equiv - { { \hat \phi _ j^{{\rm{refl } } } } \over { 2\digamma{k_p } } } = \hat x({t_j } ) + { \hat x_{{\rm{fl}}}}({t_j}),$$\end{document } where 99\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\hat x_{{\rm{fl}}}}({t_j } ) = - { { { { \hat \phi}_j } } \over { 2\digamma{k_p}}}$$\end{document } are the independent random values with the rms uncertainties given by eq . , each light pulse kicks the test mass , transferring to it a back - action momentum equal to 100\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat p_j^{{\rm{after } } } - \hat p_j^{{\rm{before } } } = \hat p_j^{{\rm{b{.}a{. } } } } = { { 2\digamma } \over c}{\hat{\mathcal w}_j},$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat p_j^{{\rm{before}}}$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat p_j^{{\rm{after}}}$\end{document } are the test - mass momentum values just before and just after the light pulse reflection , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal w}_j}$\end{document } is the energy of the j - th pulse . the major part of this perturbation is contributed by classical radiation pressure : 101\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\langle \hat p_j^{{\rm{b}}{.}{\rm{a}}{.}}\rangle = { { 2}\digamma \over c}{\mathcal w}\,,$$\end{document } with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal w}$\end{document } the mean energy of the pulses . therefore , one could neglect its effect , for it could be either subtracted from the measurement result or compensated by an actuator . the random part , which can not be compensated , is proportional to the deviation of the pulse energy : 102\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\hat p^{{\rm{b}}{\rm{.a}}{.}}}({t_j } ) = \hat p_j^{{\rm{b}}{\rm{.a}}{. } } - \langle \hat p_j^{{\rm{b}}{\rm{.a}}{.}}\rangle = { { 2}\digamma \over c}\left({{{\hat{\mathcal w}}_j } - { \mathcal w } } \right)\,,$$\end{document } and its rms uncertainly is equal to 103\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta { p_{{\rm{b}}{\rm{.a}}{. } } } = { { 2\digamma\delta { \mathcal w } } \over c}\,,$$\end{document } with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\delta { \mathcal w}$\end{document } the rms uncertainty of the pulse energy . the energy and the phase of each pulse are canonically conjugate observables and thus obey the following uncertainty relation : 104\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta { \mathcal w}\delta \phi \geq { { \hbar { \omega _ p } } \over 2}\,.$$\end{document } therefore , it follows from eqs . ( 97 and 103 ) that the position measurement error xmeas and the momentum perturbation pb.a . due to back action also satisfy the uncertainty relation : 105\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta { x_{{\rm{meas}}}}\delta { p_{{\rm{b}}{\rm{.a}}{. } } } \geq { \hbar \over 2}\,.$$\end{document } this example represents a simple particular case of a linear measurement . this class of measurement schemes can be fully described by two linear equations of the form ( 98 ) and ( 100 ) , provided that both the measurement uncertainty and the object back - action perturbation \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$({\hat x_{{\rm{fl}}}}({t_j})$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat p^{{\rm{b}}{\rm{.a}}}}({t_j})$\end{document } in this case ) are statistically independent of the test object initial quantum state and satisfy the same uncertainty relation as the measured observable and its canonically conjugate counterpart ( the object position and momentum in this case ) . suppose the test mass to be heavy enough for a single pulse to either perturb its momentum noticeably , or measure its position with the required precision ( which is a perfectly realistic assumption for the kilogram - scale test masses of gw interferometers ) . in this case , many pulses should be used to accumulate the measurement precisions ; at the same time , the test - mass momentum perturbation will be accumulated as well choose now such a time interval t , which , on the one hand , is long enough to comprise a large number of individual pulses : 106\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$n = { t \over \vartheta } \gg 1\,,$$\end{document } and , on the other hand , is sufficiently short for the test - mass position x not to change considerably during this time due to the test - mass self - evolution . then one can use all the n measurement results to refine the precision of the test - mass position x estimate , thus getting \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\sqrt n$\end{document } times smaller uncertainty 107\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta { x_t } = { { \delta { x_{{\rm{meas } } } } } \over { \sqrt n } } = \delta { x_{{\rm{meas}}}}\sqrt { { \vartheta \over t } } \,.$$\end{document } at the same time , the accumulated random kicks the object received from each of the pulses random kicks , see eq . ( 102 ) , result in random change of the object s momentum similar to that of brownian motion , and thus increasing in the same diffusive manner : 108\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta { x_{{\rm{meas } } } } \rightarrow \infty \leftrightarrow \delta { p_{{\rm{b}}{\rm{.a}}{. } } } \rightarrow 0\,,$$\end{document } if we now assume the interval between the measurements to be infinitesimally small ( 0 ) , keeping at the same time each single measurement strength infinitesimally weak : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta { x_{{\rm{meas } } } } \rightarrow \infty \leftrightarrow \delta { p_{{\rm{ba } } } } \to 0\,,$$\end{document } then we get a continuous measurement of the test - mass position \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat x(t)$\end{document } as a result . strength than xmeas and pb.a .. for continuous measurement we introduce the following parameters instead : 109\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s_x } = { \lim\limits_{\vartheta\rightarrow 0 } } { ( \delta { x_{{\rm{meas}}}})^2}\vartheta = { s_{\phi } \over 4\digamma^{2}k_p^2}\,,\qquad { s_f } = { \lim\limits_{\vartheta\rightarrow 0 } } { { { { ( \delta { p_{{\rm{b}}{\rm{.a}}{.}}})}^2 } } \over \vartheta } = { { 4\digamma^{2}{s_{\mathcal i } } } \over { { c^2}}}\,,$$\end{document } with 110\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s_\phi } = { \lim\limits_{\vartheta\rightarrow 0 } } { ( \delta \phi)^2}\vartheta \,,\qquad { s_{\mathcal i } } = { \lim\limits_{\vartheta\rightarrow 0 } } { { { { ( \delta { \mathcal w})}^2 } } \over \vartheta}\,.$$\end{document } this allows us to rewrite eqs . ( 107 ) and ( 108 ) in a form that does not contain the time interval : 111\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta { x_t } = \sqrt { { { { s_x } } \over t } } \,,\qquad \delta { p_t } = \sqrt { { s_f}t } \,.$$\end{document } to clarify the physical meaning of the quantities sx and s let us rewrite eq . ( 98 ) in the continuous limit : 112\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tilde x(t ) = \hat x(t ) + { \hat x_{{\rm{fl}}}}(t)\,,$$\end{document } where 113\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\hat x_{{\rm{fl}}}}(t ) = - { { \hat \phi ( t ) } \over { 2\digamma{k_p}}}$$\end{document } stands for measurement noise , proportional to the phase \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat \phi ( t)$\end{document } of the light beam ( in the continuous limit the sequence of individual pulses transforms into a continuous beam ) . then there is no difficulty in seeing that sx is a power ( double - sided ) spectral density of this noise , and s is a power double - sided spectral density of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat \phi ( t)$\end{document}. if we turn to eq . ( 100 ) , which describes the meter back action , and rewrite it in a continuous limit we will get the following differential equation for the object momentum : 114\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{d\hat p(t ) } \over { dt } } = { \hat f_{{\rm{fl}}}}(t ) + \ldots$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat f_{{\rm{fl}}}}(t)$\end{document } is a continuous markovian random force , defined as a limiting case of the following discrete markov process : 115\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\hat f_{{\rm{fl}}}}({t_j } ) = { \lim\limits_{\vartheta\rightarrow 0 } } { { { { \hat p}^{{\rm{b}}{\rm{.a}}{.}}}({t_j } ) } \over \vartheta } = { { 2}\digamma \over c } { \lim\limits_{\vartheta\rightarrow 0 } } { { { { \hat{\mathcal w}}_j } - { \mathcal w } } \over \vartheta } = { { 2}\digamma \over c}[\hat{\mathcal i}({t_j } ) - { { \mathcal i}_0}]\,,$$\end{document } with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat { \mathcal i}(t)$\end{document } the optical power , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}_0}$\end{document } its mean value , and here meaning all forces ( if any ) , acting on the object but having nothing to do with the meter ( light , in our case ) . double - sided power spectral density of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat f_{{\rm{b}}{\rm{.a}}{\rm{.}}}}$\end{document } is equal to sf , and double - sided power spectral density of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat { \mathcal i}$\end{document } is \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${s_{\mathcal i}}$\end{document}. we have just built a simple model of a continuous linear measurement , which nevertheless comprises the main features of a more general theory , i.e. , it contains equations for the calculation of measurement noise ( 112 ) and also for back action ( 114 ) . the precision of this measurement and the object back action in this case are described by the spectral densities sx and sf of the two meter noise sources , which are assumed to not be correlated in our simple model , and thus satisfy the following relation ( cf . ( 109 ) ) : 116\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s_x}{s_f } = { { { s_\phi}{s_{\mathcal i } } } \over { \omega _ p^2 } } \geq { { { \hbar ^2 } } \over 4}\,.$$\end{document } this relation ( as well as its more general version to be discussed later ) for continuous linear measurements plays the same role as the uncertainty relation ( 105 ) for discrete measurements , establishing a universal connection between the accuracy of the monitoring and the perturbation of the monitored object . simple case : light in a coherent state . recall now that scheme of representing the quantized light wave as a sequence of short statistically - independent pulses with duration we referred to in section 3.2 . it is the very concept we used here , and thus we can use it to calculate the spectral densities of the measurement and back - action noise sources for our simple device featured in figure 20 assuming the light to be in a coherent state with classical amplitude \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${a_c } = \sqrt { 2{{\mathcal i}_0}/(\hbar { \omega _ p})}$\end{document } ( we chose as = 0 thus making the mean phase of light \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\left\langle { \hat \phi } \right\rangle = 0$\end{document } ) . to do so we need to express phase \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat \phi$\end{document } and energy \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat { \mathcal w}$\end{document } in the pulse in terms of the quadrature amplitudes c , s(t ) ( 61 ) and make use of the following definition of the mean electromagnetic energy of the light wave contained in the volume \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\upsilon _ \vartheta } \equiv { \mathcal a}c\vartheta$\end{document } ( here , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal a}$\end{document } is the effective cross - sectional area of the light beam ) : 117\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat{\mathcal w } = { { { v_\vartheta } } \over { 4\pi}}\overline { { { \hat e}^2}(t ) } = { { { v_\vartheta } } \over { 4\pi \vartheta}}\int\nolimits_{- \vartheta /2}^{\vartheta /2 } d \tau \,{\hat e^2}(\tau ) = { \mathcal w } + \delta \hat{\mathcal w}\,,$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal w } = { \upsilon _ \vartheta}{\mathcal c}_0 ^ 2a_c^2/(8\pi ) = { { \mathcal i}_0}\vartheta$\end{document } is the mean pulse energy , and 118\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta \hat{\mathcal w } \simeq { { { \mathcal a}c{\mathcal c}_0 ^ 2 } \over { 4\pi}}2{a_c}\int\nolimits_{- \vartheta /2}^{\vartheta /2 } d \tau \,{\hat a_c}(\tau ) = \sqrt { 2\hbar { \omega _ p}{{\mathcal i}_0}\vartheta } { \hat x_\vartheta}(t ) = \sqrt { 2\hbar { \omega _ p}{\mathcal w } } { \hat x_\vartheta}(t)$$\end{document } is a fluctuating part of the pulse energy8 . we used here the definition of the mean pulse quadrature amplitude operators introduced in eqs . ( 14 ) and with the assumption of small phase fluctuations ( 1 ) one can get : 119\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat \phi \simeq { 1 \over { { a_c}\vartheta}}\int\nolimits_{- \vartheta /2}^{\vartheta /2 } d \tau { \hat a_s}(\tau ) = \sqrt { { { \hbar { \omega _ p } } \over { 2{{\mathcal i}_0}\vartheta } } } { \hat y_\vartheta } = \sqrt { { { \hbar { \omega _ p } } \over { 2{\mathcal w } } } } { \hat y_\vartheta}\,.$$\end{document } thus , since in a coherent state \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\delta \hat x_\vartheta ^2 = \delta \hat y_\vartheta ^2 = 1/2$\end{document } the phase and energy uncertainties are equal to 120\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta \phi = { 1 \over 2}\sqrt { { { \hbar { \omega _ p } } \over { \mathcal w } } } \,,\qquad \delta { \mathcal w } = \sqrt { \hbar { \omega _ p}{\mathcal w } } \,,$$\end{document } and hence 121\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta { x_{{\rm{meas } } } } = { c \over { 4}\digamma}\sqrt { { \hbar \over { { \omega _ p}{\mathcal w } } } } \,,\qquad \delta { p_{{\rm{b}}{\rm{.a}}{. } } } = { { 2}\digamma \over c}\sqrt { \hbar { \omega _ p}{\mathcal w } } \,.$$\end{document } substituting these expressions into eqs . ( 110 , 109 ) , we get the following expressions for the power ( double - sided ) spectral densities of the measurement and back - action noise sources : 122\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s_\phi } = { { \hbar { \omega _ p } } \over { 4{{\mathcal i}_0}}}\,,\qquad { s_{\mathcal i } } = \hbar { \omega _ p}{{\mathcal i}_0}\,,$$\end{document } and 123\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s_x } = { { \hbar { c^2 } } \over { 16{\omega _ p}{{\mathcal i}_0}{\digamma^2}}}\,,\qquad { s_f } = { { 4\hbar { \omega _ p}{{\mathcal i}_0}{\digamma^2 } } \over { { c^2}}}\,.$$\end{document } we should emphasize that this simple measurement model and the corresponding uncertainty relation ( 116 ) are by no means general . we have made several rather strong assumptions in the course of derivation , i.e. , we assumed : energy and phase fluctuations in each of the light pulses uncorrelated : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\left\langle { \hat { \mathcal w}({t_j})\hat \phi ( { t_j } ) } \right\rangle = 0$\end{document};all pulses to have the same energy and phase uncertainties \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\delta { \mathcal w}$\end{document } and , respectively;the pulses statistically independent from each other , particularly taking \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\left\langle { \hat { \mathcal w}({t_j})\hat { \mathcal w}({t_j } ) } \right\rangle = \left\langle { \hat \phi ( { t_i})\hat \phi ( { t_j } ) } \right\rangle = \left\langle { \hat { \mathcal w}({t_i})\hat \phi ( { t_i } ) } \right\rangle = 0$\end{document } with ti tj . energy and phase fluctuations in each of the light pulses uncorrelated : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\left\langle { \hat { \mathcal w}({t_j})\hat \phi ( { t_j } ) } \right\rangle = 0$\end{document } ; all pulses to have the same energy and phase uncertainties \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\delta { \mathcal w}$\end{document } and , respectively ; the pulses statistically independent from each other , particularly taking \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\left\langle { \hat { \mathcal w}({t_j})\hat { \mathcal w}({t_j } ) } \right\rangle = \left\langle { \hat \phi ( { t_i})\hat \phi ( { t_j } ) } \right\rangle = \left\langle { \hat { \mathcal w}({t_i})\hat \phi ( { t_i } ) } \right\rangle = 0$\end{document } with ti tj . these assumptions can be mapped to the following features of the fluctuations \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat x_{{\rm{fl}}}}(t)$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat f_{{\rm{b}}{\rm{.a}}{\rm{.}}}}(t)$\end{document } in the continuous case : these noise sources are mutually not correlated;they are stationary ( invariant to the time shift ) and , therefore , can be described by spectral densities sx and sf;they are markovian ( white ) with constant ( frequency - independent ) spectral densities . these noise sources are mutually not correlated ; they are stationary ( invariant to the time shift ) and , therefore , can be described by spectral densities sx and sf ; they are markovian ( white ) with constant ( frequency - independent ) spectral densities . the features 1 and 2 , in turn , lead to characteristic fundamentally - looking sensitivity limitations , the sql . we will call linear quantum meters , which obey these limitations ( that is , with mutually non - correlated and stationary noises \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat x_{{\rm{fl}}}}$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat f_{{\rm{b}}{\rm{.a}}{\rm{.}}}}$\end{document } ) , simple quantum meters ( sqm ) . in this section , we generalize the concept of linear quantum measurement discussed above and give a comprehensive overview of the formalism introduced in and further elaborated in [ 33 , 43 ] . this formalism can be applied to any system that performs a transformation from some unknown classical observable ( e.g. , gw tidal force in gw interferometers ) into another classical observable of a measurement device that can be measured with ( ideally ) arbitrarily high precision ( e.g. , in gw detectors , the readout photocurrent serves such an observable ) and its value depends on the value of unknown observable linearly . for definiteness , let us keep closer to gw detectors and assume the continuous measurement of a classical force . it consists of a probe \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal p}$\end{document } that is exposed to the action of a classical force g(t ) , and the meter . the action of this force on the probe causes its displacement \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat x$\end{document } that is monitored by the meter ( e.g. , light , circulating in the interferometer ) . the output observable of the meter is monitored by some arbitrary classical device that makes a measurement record o(t ) . the quantum nature of the probe - meter interaction is reflected by the back - action force \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat f$\end{document } that randomly kicks the probe on the part of the meter ( e.g. , radiation pressure fluctuations ) . at the same time , the meter itself is the source of additional quantum noise fl(t ) in the readout signal . quantum mechanically , this system can be described by the following hamiltonian : 124\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat{\mathcal h } = \hat{\mathcal h}_{{\rm{probe}}}^{(0 ) } + \hat{\mathcal h}_{{\rm{meter}}}^{(0 ) } + \hat v(t)\,,$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat { \mathcal h}_{{\rm{probe}}}^{(0)}$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat { \mathcal h}_{{\rm{meter}}}^{(0)}$\end{document } are the hamiltonians describing the free evolution of the probe and the meter , respectively , i.e. , when there is no coupling between these systems , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat v(t ) = - \hat x(g(t ) + \hat f)$\end{document } is the interaction hamiltonian . the evolution of this system can be found , in general , by solving heisenberg equations for all of the system observables . however , it is convenient to rewrite it first in the interaction picture factoring out the free evolution of the probe and the meter ( see appendix 3.7 of for detailed derivation ) : 125\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\hat{\mathcal h}_i}(t ) = \exp \left\{{{i \over \hbar}{{\hat{\mathcal h}}^{(0)}}(t - { t_0 } ) } \right\}\hat v(t)\exp \left\{{- { i \over \hbar}{{\hat{\mathcal h}}^{(0)}}(t - { t_0 } ) } \right\ } = - { \hat x^{(0)}}(t)(g(t ) + { \hat f^{(0)}}(t))\,,$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal h}^{(0 ) } } = { \mathcal h}_{{\rm{probe}}}^{(0 ) } + { \mathcal h}_{{\rm{meter}}}^{(0)}$\end{document } , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat x^{(0)}}(t)$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat f^{(0)}}(t)$\end{document } are the heisenberg operators of the probe s displacement and the meter back - action force , respectively , in the case of no coupling between these systems , i.e. , the solution to the following system of independent heisenberg equations : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{d{{\hat x}^{(0)}}(t ) } \over { dt } } = { i \over \hbar}\left [ { \hat{\mathcal h}_{{\rm{probe}}}^{(0)},{{\hat x}^{(0)}}(t ) } \right]\,,\qquad { { d{{\hat f}^{(0)}}(t ) } \over { dt } } = { i \over \hbar}\left [ { \hat{\mathcal h}_{{\rm{meter}}}^{(0)},{{\hat f}^{(0)}}(t ) } \right]\,,$$\end{document } and t0 is the arbitrary initial moment of time that can be set to without loss of generality . figure 21general scheme of the continuous linear measurement with g standing for measured classical force , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat { \mathcal x}$\end{document } the measurement noise , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat { \mathcal f}$\end{document } the back - action noise , the meter readout observable , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat o$\end{document } the actual probe s displacement . general scheme of the continuous linear measurement with g standing for measured classical force , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat { \mathcal x}$\end{document } the measurement noise , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat { \mathcal f}$\end{document } the back - action noise , the meter readout observable , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat o$\end{document } the actual probe s displacement . the following statement can be proven ( see , section vi of , and theorems 3 and 4 in appendix 3.7 of for proof ) : for a linear system with hamiltonian ( 124 ) , for any linear observable of the probe and for any linear observable \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat b$\end{document } of the meter , their full heisenberg evolutions are given by : 126\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { \hat a(t ) = { { \hat a}^{(0)}}(t ) + \int\nolimits_{{t_0}}^t d t{\prime } \,{\chi _ { ax}}(t , t{\prime})[\hat f(t{\prime } ) + g(t{\prime})]\ , , } \\ { \hat b(t ) = { { \hat b}^{(0)}}(t ) + \int\nolimits_{{t_0}}^t d t{\prime } \,{\chi _ { bf}}(t , t{\prime})\hat x(t{\prime})\,,\quad \quad \quad \quad } \\ \end{array}$$\end{document } where (t ) and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat b^{(0)}}(t)$\end{document } stand for the free heisenberg evolutions in the case of no coupling , and the functions ax(t , t ) and bf(t , t ) are called ( time - domain ) susceptibilities and defined as : 127\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { \chi _ { ax}}(t , t{\prime } ) \equiv \left\{{\begin{array}{*{20}c } { { i \over \hbar}\left [ { { { \hat a}^{(0)}}(t),{{\hat x}^{(0)}}(t{\prime } ) } \right]\,,\;\;\;t \geqslant t{\prime } \quad \;\ ; } \\ { 0,\;\quad t \\ { { \chi _ { bf}}(t , t{\prime } ) \equiv \left\{{\begin{array}{*{20}c } { { i \over \hbar}\left [ { { { \hat b}^{(0)}}(t),{{\hat f}^{(0)}}(t{\prime } ) } \right],\;\;\;t \geqslant t{\prime } \quad \;\ ; } \\ { 0,\;\quad for time independent hamiltonian \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat { \mathcal h}^{(0)}}$\end{document } and operator \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat f$\end{document } ( in the schrdinger picture ) , the susceptibilities are invariant to time shifts , i.e. , (t , t ) = (t + ,t + ) , therefore they depend only on the difference of times : (t , t ) (t t ) . in this case , one can rewrite eqs . ( 126 ) in frequency domain as : 128\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat a(\omega ) = { \hat a^{(0)}}(\omega ) + { \chi _ { ax}}(\omega)[\hat f(\omega ) + g(\omega)]\,,\qquad \hat b(\omega ) = { \hat b^{(0)}}(\omega ) + { \chi _ { bf}}(\omega)\hat x(\omega)\,,$$\end{document } where the fourier transforms of all of the observables are defined in accordance with eq . ( 6 ) . let us now use these theorems to find the full set of equations of motion for the system of linear observables \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat x$\end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat f$\end{document } and that fully characterize our linear measurement process in the scheme featured in figure 21 : 129\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { \hat o(t ) = { { \hat o}^{(0)}}(t ) + \int\nolimits_{{t_0}}^t d t{\prime } \,{\chi _ { of}}(t - t{\prime})\hat x(t{\prime})\,,\quad \quad \quad \;\;\ ; } \\ { \hat f(t ) = { { \hat f}^{(0)}}(t ) + \int\nolimits_{{t_0}}^t d t{\prime } \,{\chi _ { ff}}(t - t{\prime})\hat x(t{\prime})\,,\quad \quad \quad \quad } \\ { \hat x(t ) = { { \hat x}^{(0)}}(t ) + \int\nolimits_{{t_0}}^t d t{\prime } \,{\chi _ { xx}}(t - t{\prime})\left [ { g(t{\prime } ) + \hat f(t{\prime } ) } \right]\ , , } \\ \end{array}$$\end{document } where time - domain susceptibilities are defined as 130\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { \chi _ { of}}(t - t{\prime } ) = { i \over \hbar}\left [ { { { \hat o}^{(0)}}(t),{{\hat f}^{(0)}}(t{\prime } ) } \right]\ , , } \\ { { \chi _ { ff}}(t - t{\prime } ) = { i \over \hbar}\left [ { { { \hat f}^{(0)}}(t),{{\hat f}^{(0)}}(t{\prime } ) } \right]\ , , } \\ { { \chi _ { xx}}(t - t{\prime } ) = { i \over \hbar}\left [ { { { \hat x}^{(0)}}(t),{{\hat x}^{(0)}}(t{\prime } ) } \right]\ , . } \\ ( 129 ) describes how the readout observable (t ) of the meter , say the particular quadrature of the outgoing light field measured by the homodyne detector ( cf . ( 39 ) ) , depends on the actual displacement \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat x(t)$\end{document } of the probe , and the corresponding susceptibility of(t t ) is the transfer function for the meter from \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat x$\end{document } to . the term (t ) stands for the free evolution of the readout observable , provided that there was no coupling between the probe and the meter . in the case of the gw detector , this is just a pure quantum noise of the outgoing light that would have come out were all of the interferometer test masses fixed . it was shown explicitly in and we will demonstrate below that this noise is fully equivalent to that of the input light except for the insignificant phase shift acquired by the light in the course of propagation through the interferometer . the following important remark should be made concerning the meter s output observable (t ) . as we have mentioned already , the output observable in the linear measurement process should be precisely measurable at any instance of time . this implies a simultaneous measurability condition [ 30 , 41 , 147 , 22 , 43 , 33 ] on the observable (t ) requiring that it should commute with itself at any moment of time : 131\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\left [ { \hat o(t),\,\hat o(t{\prime } ) } \right ] = 0\,,\quad \forall t,\,t{\prime } \,.$$\end{document } initially , this condition was introduced as the definition of the quantum non - demolition ( qnd ) observables by braginsky et al . it means that the measurement of (t1 ) at some moment of time t1 shall not disturb the measurement result at any other moments of time and therefore the sample data { (t1 ) , (t1 ) , , (tn ) } can be stored directly as bits of classical data in a classical storage medium , and any noise from subsequent processing of the signal can be made arbitrarily small . it means that all noise sources of quantum origin are already included in the quantum fluctuations of (t ) [ 43 , 33 ] . and the fact that due to ( 131 ) this susceptibility turns out to be zero reflects the fact that (t ) should be a classical observable . the second equation in ( 129 ) describes how the back - action force exerted by the meter on the probe system evolves in time and how it depends on the probe s displacement . the first term , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat f^{(0)}}(t)$\end{document } , meaning is rather obvious . in gw interferometer , it is the radiation pressure force that the light exerts on the mirrors while reflecting off them . it depends only on the mean value and quantum fluctuations of the amplitude of the incident light and does not depend on the mirror motion . the second term here stands for a dynamical back - action of the meter and since , by construction , it is the part of the back - action force that depends , in a linear way , from the probe s displacement , the meaning of the susceptibility ff(t t ) becomes apparent : it is the generalized rigidity that the meter introduces , effectively modifying the dynamics of the probe . we will see later how this effective rigidity can be used to improve the sensitivity of the gw interferometers without introducing additional noise and thus enhancing the snr of the gw detection process . the third equation of ( 129 ) concerns the evolution of the probe s displacement in time . let us start with the second and the third ones : 132\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${x_s}(t ) = \int\nolimits_{{t_0}}^t d t{\prime } \,{\chi _ { xx}}(t - t{\prime})g(t{\prime})\,,\quad { \rm{and}}\quad { \hat x_{{\rm{b}}{.}{\rm{a}}{.}}}(t ) = \int\nolimits_{{t_0}}^t d t{\prime } \,{\chi _ { xx}}(t - t{\prime})\hat f(t{\prime})\,.$$\end{document } here xs(t ) is the probe s response on the signal force g(t ) and is , actually , the part we are mostly interested in . this expression also unravels the role of susceptibility xx(t t ) : it is just the green s function of the equation of motion governing the probe s bare dynamics ( also known as impulse response function ) that can be shown to be a solution of the following initial value problem : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { \bf{d}}{\chi _ { xx}}(t - t{\prime } ) = \delta ( t - t{\prime})\,,\quad { \chi _ { xx}}(t - t{\prime})\vert _ { { t \rightarrow t{\prime } } } = 0\,,\quad \ldots } \\ { \ldots \;{{{\partial ^{n - 2}}{\chi _ { xx}}(t - t{\prime } ) } \over { \partial { t^{n - 2}}}}\vert _ { { t \rightarrow t{\prime } + 0 } } = 0\,,\quad { { { \partial ^{n - 1}}{\chi _ { xx}}(t - t{\prime } ) } \over { \partial { t^{n - 1}}}}\vert _ { { t \rightarrow t{\prime } + 0 } } = { 1 \over { { a_n}}}\,,\quad \quad \quad \quad } \\ \end{array}$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\bf{d } } = \sum\nolimits_{k = 0}^n { { a_k}{{{d^k } } \over { d{t^k}}}}$\end{document } is the linear differential operator that is governed by the dynamics of the probe , e.g. , it is equal to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\bf{d}}_{{\rm{f}}{\rm{.m}}{\rm{. } } } } = m{{{d^2 } } \over { d{t^2}}}$\end{document } for a free mass m and to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\omega _ m}$\end{document } for a harmonic oscillator with eigenfrequency m . apparently , operator d is an inverse of the integral operator xx whose kernel is xx(t t ) : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${x_s}(t ) = { { \bf{d}}^{- 1}}g(t ) = { \chi _ { xx}}g(t ) = \int\nolimits_{{t_0}}^t d t{\prime } \,{\chi _ { xx}}(t - t{\prime})g(t{\prime})\,.$$\end{document } the second value , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat x_{{\rm{b}}{\rm{.a}}{\rm{.}}}}(t)$\end{document } , is the displacement of the probe due to the back - action force exerted by the meter on the probe . since it enters the probe s response in the very same way the signal does , it is the most problematic part of the quantum noise that , as we demonstrate later , imposes the sql [ 16 , 22 ] . and finally , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat x^{(0)}}(t)$\end{document } simply features a free evolution of the probe in accordance with its equations of motion and thus depends on the initial values of the probe s displacement \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat x^{(0)}}({t_0})$\end{document } , momentum \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat p^{(0)}}({t_0})$\end{document } , and , possibly , on higher order time derivatives of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat x^{(0)}}(t)$\end{document } taken at t0 , as per the structure of the operator d governing the probe s dynamics . it is this part of the actual displacement that bears quantum uncertainties imposed by the initial quantum state of the probe . one could argue that these uncertainties might become a source of additional quantum noise obstructing the detection of gws , augmenting the noise of the meter . this is not the case as was shown explicitly in , since our primary interest is in the detection of a classical force rather than the probe s displacement . therefore , performing over the measured data record o(t ) the linear transformation corresponding to first applying the operator \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal x}_{of}^{- 1}$\end{document } on the readout quantity that results in expressing o(t ) in terms of the probe s displacement : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tilde x(t ) = \chi _ { of}^{- 1}o(t ) = { x_{{\rm{fl}}}}(t ) + { x^{(0)}}(t ) + { x_s}(t ) + { x_{{\rm{b}}{.}{\rm{a}}{.}}}(t)\,,$$\end{document } with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${x_{{\rm{fl}}}}(t ) = { \mathcal x}_{of}^{- 1}{o^{(0)}}(t)$\end{document } standing for the meter s own quantum noise ( measurement uncertainty ) , and then applying a probe dynamics operator d that yields a force signal equivalent to the readout quantity o(t ) : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tilde f(t ) = { \bf{d}}\tilde x(t ) = { \bf{d}}{x_{{\rm{fl}}}}(t ) + { f_{{\rm{b}}{.}{\rm{a}}{.}}}(t ) + g(t)\,.$$\end{document } the term dx(t ) vanishes since x(t ) is the solution of a free - evolution equation of motion . thus , we see that the result of measurement contains two noise sources , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat x_{{\rm{fl}}}}(t)$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat f_{{\rm{b}}{\rm{.a}}{\rm{.}}}}(t)$\end{document } , which comprise the sum noise masking the signal force g(t ) . since we can remove initial quantum uncertainties associated with the state of the probe , it would be beneficial to turn to the fourier domain and rewrite eqs . ( 129 ) in the spectral form : 133\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { \hat o(\omega ) = { { \hat o}^{(0)}}(\omega ) + { \chi _ { of}}(\omega)\hat x(\omega)\ , , } \\ { \hat f(\omega ) = { { \hat f}^{(0)}}(\omega ) + { \chi _ { ff}}(\omega)\hat x(\omega)\ , , } \\ { \hat x(\omega ) = { \chi _ { xx}}(\omega)[\hat f(\omega ) + g(\omega)]\ , , } \\ \end{array}$$\end{document } where spectral susceptibilities are defined as : 134\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\chi _ { ab}}(\omega ) = \int\nolimits_0^\infty d \tau \,{\chi _ { ab}}(\tau){e^{i\omega \tau}},$$\end{document } with ( a , b ) ( o , f , x ) , and we omit the term \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat x^{(0)}}(\omega)$\end{document } in the last equation for the reasons discussed above . the solution of these equations is straightforward to get and reads : 135\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat o(\omega ) = { \hat o^{(0)}}(\omega ) + { { { \chi _ { xx}}(\omega){\chi _ { of}}(\omega ) } \over { 1 - { \chi _ { xx}}(\omega){\chi _ { ff}}(\omega)}}\left [ { g(\omega ) + { { \hat f}^{(0)}}(\omega ) } \right]\,,$$\end{document } 136\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat f(\omega ) = { 1 \over { 1 - { \chi _ { xx}}(\omega){\chi _ { ff}}(\omega)}}\left [ { { { \hat f}^{(0)}}(\omega ) + { \chi _ { ff}}(\omega){\chi _ { xx}}(\omega)g(\omega ) } \right]\,,$$\end{document } 137\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat x(\omega ) = { { { \chi _ { xx}}(\omega ) } \over { 1 - { \chi _ { xx}}(\omega){\chi _ { ff}}(\omega)}}\left [ { g(\omega ) + { { \hat f}^{(0)}}(\omega ) } \right]{.}$$\end{document } it is common to normalize the output quantity of the meter ( ) to unit signal . in gw interferometers , the first one tends to consider the tidal force g as a signal and thus set to 1 the coefficient in front of g( ) in eq . the other one takes gw spectral amplitude h( ) as a signal and sets the corresponding coefficient in ( ) to unity . ( 12 ) as : 138\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$- m{\omega ^2}{x_h}(\omega ) \equiv - m{\omega ^2}{{lh(\omega ) } \over 2 } = g(\omega)\quad \rightarrow \quad h(\omega ) = - 2g(\omega)/(ml{\omega ^2})\,.$$\end{document } in both cases , the renormalized output quantities can be considered as a sum of the noise and signal constituents : 139\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\hat o^f } = { \hat{\mathcal n}^f } + g\qquad { \rm{or}}\qquad { \hat o^h } = { \hat{\mathcal n}^h } + h(\omega)\,.$$\end{document } and it is the noise term in both cases that we are seeking to calculate to determine the sensitivity of the gw detector . let us rewrite ( ) in force normalization : 140\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { { \hat o}^f}(\omega ) = { { 1 - { \chi _ { ff}}(\omega){\chi _ { xx}}(\omega ) } \over { { \chi _ { of}}(\omega){\chi _ { xx}}(\omega)}}\hat o(\omega ) = { { { { \hat o}^{(0)}}(\omega ) } \over { { \chi _ { of}}(\omega){\chi _ { xx}}(\omega ) } } + \left({{{\hat f}^{(0)}}(\omega ) - { { { \chi _ { ff}}(\omega ) } \over { { \chi _ { of}}(\omega)}}{{\hat o}^{(0)}}(\omega ) } \right ) + g(\omega ) } \\ { \equiv { { \hat{\mathcal x}(\omega ) } \over { { \chi _ { xx}}(\omega ) } } + \hat{\mathcal f}(\omega ) + g(\omega)\,,\quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \;\;\ ; } \\ \end{array}$$\end{document } where we introduce two new linear observables \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat { \mathcal x}$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat { \mathcal f}$\end{document } of the meter defined as : 141\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat{\mathcal x}(\omega ) \equiv { { { { \hat o}^{(0)}}(\omega ) } \over { { \chi _ { of}}(\omega)}}\,,\qquad \hat{\mathcal f}(\omega ) \equiv { \hat f^{(0)}}(\omega ) - { { { \chi _ { ff}}(\omega ) } \over { { \chi _ { of}}(\omega)}}{\hat o^{(0)}}(\omega)\,,$$\end{document } that have the following meaning : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat { \mathcal x}$\end{document } is the effective output fluctuation of the meter not dependent on the probe . henceforth , we will refer to it as the effective measurement noise ( shot noise , in the gw interferometer common terminology);\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat { \mathcal f}$\end{document } is the effective response of the output at time t to the meter s back - action force at earlier times t < t. in the following we will refer to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat { \mathcal f}$\end{document } as the effective back - action noise ( radiation - pressure noise , in the gw interferometer common terminology ) . these two new observables that embody the two types of noise inherent in any linear measurement satisfy the following commutation relations : 142\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\left [ { \hat{\mathcal x}(\omega),{{\hat{\mathcal x}}^\dagger}(\omega { \prime } ) } \right ] = \left [ { \hat{\mathcal f}(\omega),{{\hat{\mathcal f}}^\dagger}(\omega { \prime } ) } \right ] = 0\,,\quad \leftrightarrow \quad \left [ { \hat{\mathcal x}(t),\hat{\mathcal x}(t{\prime } ) } \right ] = \left [ { \hat{\mathcal f}(t),\hat{\mathcal f}(t{\prime } ) } \right ] = 0\,,$$\end{document } 143\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\left [ { \hat{\mathcal x}(\omega),{{\hat{\mathcal f}}^{{\dagger}}}(\omega { \prime } ) } \right ] = - 2\pi i\hbar \delta ( \omega - \omega { \prime})\,,\quad \leftrightarrow \quad \left [ { \hat{\mathcal x}(t),{{\hat{\mathcal f}}^{\dagger}}(t{\prime } ) } \right ] = - i\hbar \delta ( t - t{\prime})\,,$$\end{document } that can be interpreted in the way that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat { \mathcal x}(t)$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat { \mathcal f}(t)$\end{document } can be seen at each instance of time as the canonical momentum and the coordinate of different effective linear measuring devices ( meter+probe ) , thus defining an infinite set of subsequent measurements similar to the successive independent monitors of von neumann s model . in this case , however , the monitors described by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat { \mathcal x}(t)$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat { \mathcal f}(t)$\end{document } are not , generally speaking , independent . in gw detectors , these monitors appear correlated due to the internal dynamics of the detector , i.e. , the noise processes they describe are non - markovian . \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat { \mathcal x}$\end{document } is the effective output fluctuation of the meter not dependent on the probe . henceforth , we will refer to it as the effective measurement noise ( shot noise , in the gw interferometer common terminology ) ; \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat { \mathcal f}$\end{document } is the effective response of the output at time t to the meter s back - action force at earlier times t < t. in the following we will refer to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat { \mathcal f}$\end{document } as the effective back - action noise ( radiation - pressure noise , in the gw interferometer common terminology ) . in particular , this can be seen when one calculates the power ( double - sided ) spectral density of the sum noise \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat { \mathcal n}^f}(t)$\end{document } : 144\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s^f}(\omega ) = \int\nolimits_{- \infty}^\infty { dt } \,\langle { \hat{\mathcal n}^f}(t)\circ { \hat{\mathcal n}^f}(t{\prime})\rangle { e^{i\omega ( t - t{\prime } ) } } = { { { s_{{\mathcal x}{\mathcal x}}}(\omega ) } \over { \vert { \chi _ { xx}}(\omega)\vert ^{2 } } } + { s_{{\mathcal f}{\mathcal f}}}(\omega ) + 2{\rm{re}}\left [ { { { { s_{{\mathcal x}{\mathcal f}}}(\omega ) } \over { { \chi _ { xx}}(\omega ) } } } \right]\,,$$\end{document } where spectral densities : 145\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s_{{\mathcal x}{\mathcal x}}}(\omega ) = \int\nolimits_{- \infty}^\infty { dt } \,\langle \hat{\mathcal x}(t)\circ \hat{\mathcal x}(t{\prime})\rangle { e^{i\omega ( t - t{\prime})}}\,,$$\end{document } 146\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s_{{\mathcal f}{\mathcal f}}}(\omega ) = \int\nolimits_{- \infty}^\infty { dt } \,\langle \hat{\mathcal f}(t)\circ \hat{\mathcal f}(t{\prime})\rangle { e^{i\omega ( t - t{\prime})}}\,,$$\end{document } 147\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s_{{\mathcal x}{\mathcal f}}}(\omega ) = \int\nolimits_{- \infty}^\infty { dt } \,\langle \hat{\mathcal x}(t)\circ \hat{\mathcal f}(t{\prime})\rangle { e^{i\omega ( t - t{\prime})}}\,,$$\end{document } are not necessarily constant and , thus , describe non - markovian random processes . it can also be shown that since \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat { \mathcal x}(t)$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat { \mathcal f}(t)$\end{document } satisfy commutation relations ( 142 ) , their spectral densities shall satisfy the schrdinger - robertson uncertainty relation : 148\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s_{{\mathcal x}{\mathcal x}}}(\omega){s_{{\mathcal f}{\mathcal f}}}(\omega ) - \vert { s_{{\mathcal x}{\mathcal f}}}(\omega)\vert ^{2}\geqslant{{{\hbar ^2 } } \over 4}$$\end{document } that is the generalization of a heisenberg uncertainty relation in the case of correlated observables . the general structure of quantum noise in the linear measurement process , comprising two types of noise sources whose spectral densities are bound by the uncertainty relation ( 148 ) , gives a clue to several rather important corollaries . one of the most important is the emergence of the sql , which we consider in detail below . the sqm has non - correlated effective measurement and back - action noises that results in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${s_{{\mathcal x}{\mathcal f}}}(\omega ) = 0$\end{document}. apparently , under these conditions \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat { \mathcal x}$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat { \mathcal f}$\end{document } turn into \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat x_{{\rm{fl}}}}$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat f_{{\rm{fl}}}}$\end{document } of eqs . hence , we will use sx( ) instead of s ( ) and sf( ) instead of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${s_{{\mathcal f}{\mathcal f}}}(\omega)$\end{document } then the uncertainty relation ( 148 ) transforms into : 149\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s_x}(\omega){s_f}(\omega ) \geqslant{{{\hbar ^2 } } \over 4}\,.$$\end{document } the sql is the name for an ultimate lower bound of a sum noise spectral density the sqm can , in principle , have at any given frequency . to derive this limit we assume noise sources xfl and fb.a . to have minimal values allowed by quantum mechanics , i.e. 150\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s_x}(\omega){s_f}(\omega ) = { { { \hbar ^2 } } \over 4}\,.$$\end{document } then , using this condition , one can minimize sqm s sum noise : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s^f}(\omega ) = { { { s_x}(\omega ) } \over { \vert { \chi _ { xx}}(\omega)\vert ^{2 } } } + { s_f}(\omega ) = { { { s_x}(\omega ) } \over { \vert { \chi _ { xx}}(\omega)\vert ^{2 } } } + { { { \hbar ^2 } } \over { 4{s_x}(\omega)}}$$\end{document } to yield : 151\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$s_{{\rm{sql}}}^f(\omega ) = { \hbar \over { \vert { \chi _ { xx}}(\omega)\vert}}$$\end{document } that is achieved when contributions of measurement noise and back - action noise to the sum noise are equal to each other , i.e. , when 152\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s_x}(\omega ) = { \hbar \over 2}\vert { \chi _ { xx}}(\omega)\vert \,,\quad \quad \leftrightarrow \quad \quad { s_f}(\omega ) = { \hbar \over { 2\vert { \chi _ { xx}}(\omega)\vert}}\,.$$\end{document } it is instructive to cite the forms of the sql in other normalizations , i.e. , for h - normalization and for x - normalization . the former is obtained from ( 151 ) via multiplication by 4/(ml ) : 153\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$s_{{\rm{sql}}}^h(\omega ) = { { 4s_{{\rm{sql}}}^f(\omega ) } \over { { m^2}{l^2}{\omega ^4 } } } = { { 4\hbar } \over { { m^2}{l^2}{\omega ^4}\vert { \chi _ { xx}}(\omega)\vert}}\,.$$\end{document } the latter is obtained from eq . ( 151 ) using the obvious connection between force and displacement x( ) = xx()f( ) : 154\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$s_{{\rm{sql}}}^x(\omega ) = \vert { \chi _ { xx}}(\omega)\vert ^{2}s_{{\rm{sql}}}^f(\omega ) = \hbar \vert { \chi _ { xx}}(\omega)\vert \,.$$\end{document } these limits look fundamental . there are no parameters of the meter ( only as a reminder of the uncertainty relation ( 116 ) ) , and only the probe s dynamics is in there . nevertheless , this is not the case and , actually , this limit can be beaten by more sophisticated , but still linear , position meters . at the same time , the sql represents an important landmark beyond which the ordinary brute - force methods of sensitivity improving cease working , and methods that allow one to blot out the back - action noise \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat { \mathcal f}(t)$\end{document } from the meter output signal have to be used instead . due to this reason , the sql , and especially the sql for the simplest test object free mass is usually considered as a borderline between the classical and the quantum domains . in the rest of this section , we consider in more detail the sqls for a free mass and for a harmonic oscillator . the free mass is not only the simplest model for the probe s dynamics , but also the most important class of test objects for gw detection . test masses of gw detectors must be isolated as much as possible from the noisy environment . to this end , the design of gw interferometers implies suspension of the test masses on thin fibers . the real suspensions are rather sophisticated and comprise several stages slung one over another , with mechanical eigenfrequencies fm in 1hz range . the sufficient degree of isolation is provided at frequencies much higher than fm , where the dynamics of test masses can be approximated with good precision by that of a free mass . let us introduce the following convenient measure of measurement strength ( precision ) in the first place : 155\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\omega _ q } = { \left({{{{s_f } } \over { { m^2}{s_x } } } } \right)^{1/4}}.$$\end{document } using the uncertainty relation ( 150 ) , the noise spectral densities sx and sf can be expressed through q as follows : 156\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s_x } = { \hbar \over { 2m\omega _ q^2}}\,,\qquad { s_f } = { { \hbar m\omega _ q^2 } \over 2}\,.$$\end{document } therefore , the larger q is , the smaller sx is ( the higher is the measurement precision ) , and the larger sf is ( the stronger the meter back action is ) . in the case of interferometers , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\omega _ q^2$\end{document } is proportional to the circulating optical power . for example , for the toy optical meter considered above , 157\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\omega _ q } = \sqrt { { { 8{\omega _ p}{{\mathcal i}_0}{\digamma^2 } } \over { m{c^2 } } } } \,,$$\end{document } see eqs . ( 123 ) . using this notation , and taking into account that for a free mass m , 158\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\chi _ { xx}^{{\rm{f}}{.}{\rm{m}}{.}}(\omega ) = - { 1 \over { m{\omega ^2}}}\,,$$\end{document } the sum quantum noise power ( double - sided ) spectral density can be written as follows : 159\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$s_{{\rm{f}}{.}{\rm{m}}{.}}^f(\omega ) = { m^2}{\omega ^4}{s_x } + { s_f } = { { \hbar m\omega _ q^2 } \over 2}\left({{{{\omega ^4 } } \over { \omega _ q^4 } } + 1 } \right)\,.$$\end{document } the sql optimization ( 152 ) takes the following simple form in this case : 160\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\omega _ q } = \omega \,,$$\end{document } giving : 161\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$s_{{\rm{sql}}\,{\rm{f}}{.}{\rm{m}}{.}}^f(\omega ) = \hbar m{\omega ^2}\,.$$\end{document } this consideration is illustrated by figure 22 ( left ) , where power ( double - sided ) spectral density ( 159 ) is plotted for three different values of q . it is easy to see that these plots never dive under the sql line ( 161 ) , which embodies a common envelope for them . due to this reason , the sensitivities area above this line classical domain , and below it as the quantum domain. figure 22sum quantum noise power ( double - sided ) spectral densities of the simple quantum meter for different values of measurement strength q(red ) < q(green ) < q(blue ) . right : harmonic oscillator sum quantum noise power ( double - sided ) spectral densities of the simple quantum meter for different values of measurement strength q(red ) < q(green ) < right : harmonic oscillator the simplest way to overcome the limit ( 161 ) , which does not require any quantum tricks with the meter , is to use a harmonic oscillator as a test object , instead of the free mass . it is easy to see from eq.(151 ) that the more responsive the test object is at some given frequency ( that is , the bigger xx( ) ) is , the smaller its force sql at this frequency is . in the harmonic oscillator case , 162\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\chi _ { xx}^{{\rm{osc}}}(\omega ) = { 1 \over { m(\omega _ 0 ^ 2 - { \omega ^2})}}\,,$$\end{document } with 0 standing for the oscillator mechanical eigenfrequency , and the sum quantum noise power ( double - sided ) spectral density equal to 163\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$s_{{\rm{osc}}}^f(\omega ) = { m^2}{(\omega _ 0 ^ 2 - { \omega ^2})^2}{s_x } + { s_f } = { { \hbar m\omega _ q^2 } \over 2}\left [ { { { { { ( \omega _ 0 ^ 2 - { \omega ^2})}^2 } } \over { \omega _ q^4 } } + 1 } \right].$$\end{document } due to a strong response of the harmonic oscillator near resonance , the first ( measurement noise ) term in eq . ( 159 ) goes to zero in the vicinity of 0 . therefore , reducing the value of q , that is , using weaker measurement , it is possible to increase the sensitivity in a narrow band around 0 . at the same time , the smaller q is , the more narrow the bandwidth is where this sensitivity is achieved , as can be seen from the plots drawn in figure 22 ( right ) . consider , in particular , the following minimax optimization of the narrow - band sensitivity . suppose also 164\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\vert \nu \vert \ll { \omega _ 0}\,.$$\end{document } in this case , the sum noise power ( double - sided ) spectral density ( 163 ) can be approximated as follows : 165\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$s_{{\rm{osc}}}^f({\omega _ 0 } + \nu ) = { { \hbar m\omega _ q^2 } \over 2}\left({{{4\omega _ 0 ^ 2{\nu ^2 } } \over { \omega _ q^4 } } + 1 } \right).$$\end{document } then require the maximum of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$s_{{\rm{osc}}}^f$\end{document } in a given frequency range be as small as possible . it is evident that this frequency range has to be centered around the resonance frequency 0 , with the maximums at its edges , = /2 . the sum noise power ( double - sided ) spectral density at these points is equal to 166\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$s_{{\rm{osc}}}^f({\omega _ 0 } \pm \delta \omega /2 ) = { { \hbar m\omega _ q^2 } \over 2}\left({{{\omega _ 0 ^ 2\delta { \omega ^2 } } \over { \omega _ q^4 } } + 1 } \right).$$\end{document } the minimum of this expression is provided by 167\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\omega _ q } = \sqrt { { \omega _ 0}\delta \omega } \,.$$\end{document } substitution of this value back into eq . ( 165 ) gives the following optimized power ( double - sided ) spectral density : 168\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$s_{{\rm{osc}}}^f({\omega _ 0 } + \nu ) = { { \hbar m{\omega _ 0 } } \over 2}\left({{{4{\nu ^2 } } \over { \delta \omega } } + \delta \omega } \right),$$\end{document } with 169\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$s_{{\rm{osc}}}^f({\omega _ 0 } \pm \delta \omega /2 ) = \hbar m{\omega _ 0}\delta \omega \,.$$\end{document } therefore , the harmonic oscillator can provide a narrow - band sensitivity gain , compared to the free mass sql ( 161 ) , which reads 170\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\xi _ { { \rm{osc}}}^2 = { { s_{{\rm{osc}}}^f({\omega _ 0 } + \nu ) } \over { s_{{\rm{sql}}\,{\rm{f}}{.}{\rm{m}}{.}}^f({\omega _ 0 } ) } } \simeq { 1 \over 2}\left({{{4{\nu ^2 } } \over { \omega _ q^2 } } + { { \omega _ q^2 } \over { \omega _ 0 ^ 2 } } } \right)\,,$$\end{document } and can be further written accounting for the above optimization as : 171\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\left . { { { s_{{\rm{osc}}}^f({\omega _ 0 } + \nu ) } \over { s_{{\rm{sql}}\,{\rm{f}}{.}{\rm{m}}{.}}^f({\omega _ 0 } ) } } } \right\vert _ { \vert \nu \vert \leq \delta \omega /2 } } \leq { { s_{{\rm{osc}}}^f({\omega _ 0 } \pm \delta \omega /2 ) } \over { s_{{\rm{sql}}\,{\rm{f}}{.}{\rm{m}}{.}}^f({\omega _ 0 } ) } } = { { \delta \omega } \over { { \omega _ 0}}}\,.$$\end{document } of course , the oscillator sql , equal to 172\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$s_{{\rm{sql}}\,{\rm{osc}}}^f = \hbar m\vert \omega _ 0 ^ 2 - { \omega ^2}\vert \approx 2\hbar m{\omega _ 0}\vert \nu \vert$$\end{document } can not be beaten is this way , and the question of whether the sensitivity ( 171 ) is the true beating of the sql or not , is the question to answer ( and the subject of many discussions ) . above , we have discussed , in brief , different normalizations of the sum noise spectral density and derived the general expressions for the sql in these normalizations ( cf . eqs . let us consider how these expressions look for the free mass and harmonic oscillator and how the sensitivity curves transform when changing to different normalizations . h - normalization : the noise spectral density in h - normalization can be obtained using eq . ( 12 ) . where the sqm is concerned , the sum noise in h - normalization reads \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${h_{{\rm{sum}}}}(\omega ) \equiv { \hat{\mathcal n}^h}(\omega ) = - { 2 \over { ml{\omega ^2}}}\left [ { { { { { \hat x}_{{\rm{fl}}}}(\omega ) } \over { { \chi _ { xx}}(\omega ) } } + { { \hat f}_{{\rm{fl}}}}(\omega ) } \right]\,.$$\end{document } in the case of a free mass with xx( ) = 1/(m ) the above expression transforms as : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h_{{\rm{sum}}}^{{\rm{f}}{.}{\rm{m}}{.}}(\omega ) = { { 2{{\hat x}_{{\rm{fl}}}}(\omega ) } \over l } - { { 2{{\hat f}_{{\rm{fl } } } } } \over { ml{\omega ^2}}}$$\end{document } and that results in the following power ( double - sided ) spectral density formula : 173\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$s_{{\rm{f}}{.}{\rm{m}}{.}}^h(\omega ) = { 4 \over { { l^2}}}\left [ { { s_x } + { { { s_f } } \over { { m^2}{\omega ^4 } } } } \right ] = { { 2\hbar } \over { m{l^2}\omega _ q^2}}\left({1 + { { \omega _ q^4 } \over { { \omega ^4 } } } } \right)$$\end{document } and results in the following formula for free mass sql in h - normalization : 174\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$s_{{\rm{sql}}\,{\rm{f}}{.}{\rm{m}}{.}}^h(\omega ) = { { 4\hbar } \over { m{l^2}{\omega ^2}}}\,.$$\end{document } the plots of these spectral densities at different values of q are given in the left panel of figure 23 . figure 23sum quantum noise power ( double - sided ) spectral densities of the simple quantum meter in the h - normalization for different values of measurement strength : q ( red ) < q ( green ) lt ; q(blue ) . sum quantum noise power ( double - sided ) spectral densities of the simple quantum meter in the h - normalization for different values of measurement strength : q ( red ) < q ( green ) lt ; q(blue ) . thin black line : sql . left : free mass . , similar formulas can be obtained taking into account that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal x}_{xx}^{{\rm{osc}}}(\omega ) = 1/(m(\omega _ 0 ^ 2 - { \omega ^2}))$\end{document}. thus , one has : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h_{{\rm{sum}}}^{{\rm{osc}}}(\omega ) = - { { 2(\omega _ 0 ^ 2 - { \omega ^2}){{\hat x}_{{\rm{fl}}}}(\omega ) } \over { l{\omega ^2 } } } - { { 2{{\hat f}_{{\rm{fl } } } } } \over { ml{\omega ^2}}}$$\end{document } that results in the following power ( double - sided ) spectral density formula : 175\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$s_{{\rm{osc}}}^h(\omega ) = { 4 \over { { l^2}}}\left [ { { { \left({1 - { { \omega _ 0 ^ 2 } \over { { \omega ^2 } } } } \right)}^2}{s_x } + { { { s_f } } \over { { m^2}{\omega ^4 } } } } \right ] = { { 2\hbar } \over { m{l^2}\omega _ q^2}}\left [ { { { \omega _ q^4 } \over { { \omega ^4}}}\left({1 + { { { { ( \omega _ 0 ^ 2 - { \omega ^2})}^2 } } \over { \omega _ q^4 } } } \right ) } \right]$$\end{document } and results in the following formula for free mass sql in h - normalization : 176\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$s_{{\rm{sql}}\,{\rm{osc}}}^h(\omega ) = { { 4\hbar \vert \omega _ 0 ^ 2 - { \omega ^2}\vert } \over { m{l^2}{\omega ^4}}}\,.$$\end{document } the corresponding plots are drawn in the right panel of figure 23 . despite a quite different look , in essence , these spectral densities are the same force spectral densities as those drawn in figure 22 , yet tilted rightwards by virtue of factor 1/. in particular , they are characterized by the same minimum at the resonance frequency , created by the strong response of the harmonic oscillator on a near - resonance force , as the corresponding force - normalized spectral densities ( 163 , 172 ) . x - normalization : another normalization that is worth considering is the actual probe displacement , or x - normalization . in this normalization , the sum noise spectrum is obtained by multiplying noise term \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat { \mathcal n}^f}(\omega)$\end{document } in eq . ( 139 ) by the probe s susceptibility 177\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\hat x_{{\rm{sum}}}}(\omega ) = { \hat x_{{\rm{fl}}}}(\omega ) + { \chi _ { xx}}(\omega){\hat f_{{\rm{fl}}}}(\omega)\,.$$\end{document } it looks rather natural at a first glance ; however , as we have shown below , it is less heuristic than the force normalization and could even be misleading . spectral density of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat x_{{\rm{sum}}}}(\omega)$\end{document } and the corresponding sql are equal to 178\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s^x}(\omega ) = { s_x}(\omega ) + \vert { \chi _ { xx}}(\omega)\vert ^{2}{s_f}(\omega)\,,$$\end{document } 179\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$s_{{\rm{sql}}}^x(\omega ) = \hbar \vert { \chi _ { xx}}(\omega)\vert \,.$$\end{document } in the free mass case , the formulas are the same as in h - normalization except for the multiplication by 4/l : 180\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$s_{{\rm{f}}{.}{\rm{m}}{.}}^x(\omega ) = \left [ { { s_x } + { { { s_f } } \over { { m^2}{\omega ^4 } } } } \right ] = { \hbar \over { 2m\omega _ q^2}}\left({1 + { { \omega _ q^4 } \over { { \omega ^2 } } } } \right)$$\end{document } with sql equal to : 181\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$s_{{\rm{sql}}\,{\rm{f}}{.}{\rm{m}}{.}}^x(\omega ) = { \hbar \over { m{\omega ^2}}}\,.$$\end{document } in the harmonic oscillator case , these equations have the following form : 182\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$s_{{\rm{osc}}}^x(\omega ) = { s_x } + { { { s_f } } \over { { m^2}{{(\omega _ 0 ^ 2 - { \omega ^2})}^2 } } } = { \hbar \over { 2m\omega _ q^2}}\left [ { { { \omega _ q^4 } \over { { \omega ^4}}}\left({1 + { { { { ( \omega _ 0 ^ 2 - { \omega ^2})}^2 } } \over { \omega _ q^4 } } } \right ) } \right]\,,$$\end{document } 183\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$s_{{\rm{sql}}\,{\rm{osc}}}^x(\omega ) = { \hbar \over { m\vert \omega _ 0 ^ 2 - { \omega ^2}\vert}}\,.$$\end{document } the corresponding plot of the harmonic oscillator power ( double - sided ) spectral density in x - normalization is given in figure 24 . figure 24sum quantum noise power ( double - sided ) spectral densities of simple quantum meter and harmonic oscillator in displacement normalization for different values of measurement strength : q(red ) < q(green ) sum quantum noise power ( double - sided ) spectral densities of simple quantum meter and harmonic oscillator in displacement normalization for different values of measurement strength : q(red ) < q(green ) < however , the resonance growth of the displacement due to signal force have a long start over this seeming noise outburst , as we have shown already , leads to the substantial sensitivity gain for a near - resonance force . this sensitivity increase is clearly visible in the force and equivalent displacement normalization , see figures 22 and 23 , but completely masked in figure 24 . the sql is not a fundamental limitation as we have mentioned already , and the clue to how to overcome it can be devised from the expression for the general linear measurement sum noise spectral density ( 144 ) . one can see that a properly constructed cross - correlation between the measurement noise \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat { \mathcal x } = { \hat o^{(0)}}/{{\mathcal x}_{of}}(\omega)$\end{document } and back - action noise \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat f^{(0)}}$\end{document } , i.e. , the right choice of ff( ) that should be at any frequency equal to : 184\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\chi _ { ff}}(\omega ) = { s_{{\mathcal x}f}}(\omega)/{s_{{\mathcal x}{\mathcal x}}}(\omega)$$\end{document } can compensate the back - action term and leave only the measurement noise - related contribution to the final sum quantum noise : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s^f}(\omega ) = { s_{{\mathcal x}{\mathcal x}}}(\omega)/\vert { \chi _ { xx}}(\omega)\vert ^{2}{.}$$\end{document } this spectral density could be arbitrarily small , providing the unbound measurement strength . however , there is a significant obstacle on the way towards back - action free measurement : the optimal correlation should be frequency dependent in the right and rather peculiar way . another drawback of such back - action evasion via noise cancellation resides in the dissipation that is always present in real measurement setups and , according to fluctuation - dissipation theorem [ 37 , 95 ] is a source of additional noise that undermines any quantum correlations that might be built in the ideal system . the simplest way is to make the relation ( 4.4 ) hold at some fixed frequency , which can always be done either ( i ) by preparing the meter in some special initial quantum state that has measurement and back - action fluctuations correlated ( unruh [ 148 , 147 ] proposed to prepare input light in a squeezed state to achieve such correlations ) , or ( ii ) by monitoring a linear combination of the probe s displacement and momentum [ 162 , 159 , 158 , 160 , 161 , 51 , 53 ] that can be accomplished , e.g. , via homodyne detection , as we demonstrate below . we consider the basic principles of the schemes , utilizing the noise cancellation via building cross - correlations between the measurement and back - action noise . we start from the very toy example discussed in section 4.1.1 . the advanced version of that example is shown in figure 25 . the only difference between this scheme and the initial one ( see figure 20 ) is that here the detector measures not the phase of light pulses , but linear combination of the phase and energy , parametrized by the homodyne angle lo ( cf . ( 39 ) ) : 185\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat o({t_j } ) = - \hat \phi _ j^{{\rm{refl}}}\sin { \phi _ { { \rm{lo } } } } + { { { { \hat{\mathcal w}}_j } - { \mathcal w } } \over { 2{\mathcal w}}}\cos { \phi _ { { \rm{lo}}}}\,,$$\end{document } ( we subtracted the regular term proportional to the mean energy \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal w}$\end{document } from the output signal (tj ) ) . figure 25toy example of a linear optical position measurement . ( 98 ) , renormalize this output signal as the equivalent test object displacement : 186\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tilde x({t_j } ) \equiv { { { { \hat o}_j } } \over { 2\digamma{k_p}\sin { \phi _ { { \rm{lo } } } } } } = \hat x({t_j } ) + { \hat x_{{\rm{fl}}}}({t_j})\,,$$\end{document } where the noise term has in this case the following form : 187\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\hat x_{{\rm{fl}}}}({t_j } ) = { 1 \over { 2\digamma{k_p}}}\left({- \hat \phi ( { t_j } ) + { { { { \hat{\mathcal w}}_j } - { \mathcal w } } \over { 2{\mathcal w}}}\cot { \phi _ { { \rm{lo } } } } } \right).$$\end{document } rms uncertainty of this value ( the measurement error ) is equal to 188\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta { x_{{\rm{meas } } } } = { 1 \over { 2\digamma{k_p}}}\sqrt { { { ( \delta \phi)}^2 } + { { { { ( \delta { \mathcal w})}^2 } } \over { 4{{\mathcal w}^2}}}{{\cot}^2}{\phi _ { { \rm{lo } } } } } \,.$$\end{document } at first glance , it seems like we just obtained the increased measurement error , for the same value of the test object perturbation , which is still described by eq . ( 185 ) can be viewed not only as the additional noise , but as the source of information about the test object perturbation . it can be used to subtract , at least in part , the terms induced by this perturbation from the subsequent measurement results . quantitatively , this information is characterized by the cross - correlation of the measurement error and the back action : 189\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta ( xp ) = \langle { \hat x_{{\rm{fl}}}}({t_j})\circ { \hat p^{{\rm{b}}{.}{\rm{a}}{.}}}({t_j})\rangle = { { { { ( \delta { \mathcal w})}^2 } } \over { 2{\omega _ p}{\mathcal w}}}\cot { \phi _ { { \rm{lo}}}}\,.$$\end{document } it is easy to see that the uncertainties ( 103 , 188 , 189 ) satisfy the following schrdinger - robertson uncertainty relation : 190\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${(\delta { x_{{\rm{meas}}}})^2}{(\delta { p_{{\rm{b}}{.}{\rm{a}}{.}}})^2 } - { [ \delta ( xp)]^2 } = { { { { ( \delta \phi)}^2}{{(\delta { \mathcal w})}^2 } } \over { \omega _ p^2 } } \geq { { { \hbar ^2 } } \over 4}\,.$$\end{document } now we can perform the transition to the continuous measurement limit as we did in section 4.1.2 : 191\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { s_x } = { \lim\limits_{\vartheta \rightarrow 0 } } { { ( \delta { x_{{\rm{meas}}}})}^2}\vartheta = { 1 \over { 4\digamma^{2}k_p^2}}\left({{s_\phi } + { { { s_{\mathcal i } } } \over { 4{{\mathcal i}^2}}}{{\cot}^2}{\phi _ { { \rm{lo } } } } } \right)\,,\,\ , } \\ { { s_f } = { \lim\limits _ { \vartheta \rightarrow 0}}{{{{(\delta { p_{{\rm{b}}{.}{\rm{a}}{.}}})}^2 } } \over \vartheta } = { { 4\digamma^{2}{s_{\mathcal i } } } \over { { c^2}}},\quad \quad \quad \quad \quad \quad \quad \quad \quad \ , } \\ { { s_{xf } } = { \lim\limits_{\vartheta \rightarrow 0}}\delta ( xp ) = { { { s_{\mathcal i } } } \over { 2{\omega _ p}{\mathcal i}}}\cot { \phi _ { { \rm{lo}}}}\,.,\quad \quad \quad \quad \quad \quad \quad \,\,\ , } \\ \end{array}$$\end{document } which transform inequality ( 190 ) to the schodinger - robertson uncertainty relation for continuous measurements : 192\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s_x}{s_f } - s_{xf}^2 = { { { s_\phi}{s_{\mathcal i } } } \over { \omega _ p^2 } } \geq { { { \hbar ^2 } } \over 4}\,.$$\end{document } in the particular case of the light pulses in a coherent quantum state ( 120 ) , the measurement error ( 188 ) , the momentum perturbation ( 103 ) , and the cross - correlation term ( 189 ) are equal to : 193\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta { x_{{\rm{meas } } } } = { c \over { 4}\digamma}\sqrt { { \hbar \over { { \omega _ p}{\mathcal w}{{\sin}^2}{\phi _ { { \rm{lo } } } } } } } \,,\qquad \delta { p_{{\rm{b}}{.}{\rm{a}}{. } } } = { { 2 } \over c}\sqrt { \hbar { \omega _ p}{\mathcal w } } \,,\qquad \delta ( xp ) = { \hbar \over 2}\cot { \phi _ { { \rm{lo}}}}$$\end{document } ( the momentum perturbation pb.a . evidently remains the same as in the uncorrelated case , and we provided its value here only for convenience ) , which gives the exact equality in the schrdinger - robertson uncertainty relation : 194\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${(\delta { x_{{\rm{meas}}}})^2}{(\delta { p_{{\rm{b}}{.}{\rm{a}}{.}}})^2 } - { [ \delta ( xp)]^2 } = { { { \hbar ^2 } } \over 4}\,.$$\end{document } correspondingly , substituting the coherent quantum state power ( double - sided ) spectral densities ( 122 ) into eqs . ( 191 ) , we obtain : 195\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s_x } = { { \hbar { c^2 } } \over { 16{\omega _ p}{{\mathcal i}_0}{\digamma^2}{{\sin}^2}{\phi _ { { \rm{lo}}}}}}\,,\qquad { s_f } = { { 4\hbar { \omega _ p}{{\mathcal i}_0}{\digamma^2 } } \over { { c^2}}}\qquad , { s_{xf } } = { \hbar \over 2}\cot { \phi _ { { \rm{lo}}}}\,,$$\end{document } with 196\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s_x}{s_f } - s_{xf}^2 = { { { \hbar ^2 } } \over 4}$$\end{document } [ compare with eqs . ( 123 ) ] . the cross - correlation between the measurement and back - action fluctuations is equivalent to the virtual rigidity \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal x}_{ff}^{{\rm{virt}}}(\omega ) \equiv - { k_{{\rm{virt } } } } = { \rm{const}}{\rm{.}}$\end{document } as one can conclude looking at eqs . \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\hat x_{{\rm{fl}}}}(t ) = \hat{\mathcal x}(t)\,,\qquad { \rm{and}}\qquad { \hat f^{(0 ) } } = \hat{\mathcal f}(t ) + { k_{{\rm{virt}}}}{\hat x_{{\rm{fl}}}}(t)\,.$$\end{document } the sum noise does not change under the above transformation and can be written as : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\hat f_{{\rm{sum}}}}(t ) = { \bf{d}}{\hat x_{{\rm{fl}}}}(t ) + \hat f_{{\rm{fl}}}^{(0)}(t ) = { { \bf{d}}_{{\rm{eff}}}}{\hat x_{{\rm{fl}}}}(t ) + \hat{\mathcal f}(t)\,,$$\end{document } where the new effective dynamics that correspond to the new noise are governed by the following differential operator 197\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\bf{d}}_{{\rm{eff } } } } = { \bf{d } } + { k_{{\rm{virt}}}}\,.$$\end{document } the above explains why we refer to kvirt as virtual rigidity. to see how virtual rigidity created by cross - correlation of noise sources can help beat the sql consider a free mass as a probe body in the above considered toy example . the modified dynamics : 198\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\bf{d}}_{{\rm{eff } } } } = m{{{d^2 } } \over { d{t^2 } } } + { k_{{\rm{virt}}}}\,,\qquad { s_{xf } } = { k_{{\rm{virt}}}}{s_x } \neq 0\,,$$\end{document } correspond to a harmonic oscillator with eigenfrequency \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\omega _ 0 ^ 2 = { k_{{\rm{virt}}}}/m$\end{document } , and as we have demonstrated in eq . ( 171 ) provide a narrow - band sensitivity gain versus a free mass sql near the resonance frequency 0 . however , there is a drawback of virtual rigidity compared to the real one : it requires higher measurement strength , and therefore higher power , to reach the same gain in sensitivity as provided by a harmonic oscillator . this becomes evident if one weighs the back - action spectral density sf , which is a good measure of measurement strength according to eqs . ( 156 ) , for the virtual rigidity against the real one . for the latter , to overcome the free mass sql by a factor 199\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\xi ^2 } = { { \delta \omega } \over { { \omega _ 0}}}$$\end{document } ( see eq . ( 171 ) ) at a given frequency 0 , the back - action noise spectral density has to be reduced by this factor : 200\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s_f } = { { \hbar m\omega _ q^2 } \over 2 } = { \xi ^2}s_f^{{\rm{opt}}\,{\rm{f}}{.}{\rm{m}}{.}}\,,$$\end{document } see eqs . ( 156 , 160 , 167 ) . here , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$s_f^{{\rm{opt}}\,{\rm{f}}{\rm{.m}}{\rm{. } } } = \hbar m\omega/2$\end{document } is the back - action noise spectral density , which allows one to reach the free mass sql ( 161 ) at frequency 0 . such a sensitivity gain is achieved at the expense of proportionally reduced bandwidth : 201\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta \omega = { \xi ^2}{\omega _ 0}\,.$$\end{document } for the virtual rigidity , the optimal value of sf results from eq . ( 167 ) : 202\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s_f } = { { { \hbar ^2}/4 + s_{xf}^2 } \over { { s_x } } } = { { \hbar m } \over 2}\left({\omega _ q^2 + { { \omega _ 0 ^ 4 } \over { \omega _ q^2 } } } \right ) = s_f^{{\rm{opt}}\,{\rm{f}}{.}{\rm{m}}{.}}\left({{\xi ^2 } + { 1 \over { { \xi ^2 } } } } \right).$$\end{document } hence , the better the sensitivity ( the smaller ) , the larger sf must be and , therefore , measurement strength . another evident flaw of the virtual rigidity , which it shares with the real one , is the narrow - band character of the sensitivity gain it provides around 0 and that this band shrinks as the sensitivity gain rises ( cf . ( 199 ) ) . in order to provide a broadband enhancement in sensitivity , either the real rigidity \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$k = m\omega _ 0 ^ 2$\end{document } , or the virtual one kvirt = sxf / sf should depend on frequency in such a way as to be proportional ( if only approximately ) to in the frequency band of interest . of all the proposed solutions providing frequency dependent virtual rigidity , the most well known are the quantum speed meter and the filter cavities schemes . the toy scheme that demonstrates a bare idea of the quantum speed meter is shown in figure 26 . the main difference of this scheme from the position meters considered above ( see figures 20 , 25 ) is that each light pulse reflects from the test mass twice : first from the front and then from the rear face after passing the delay line with delay time . an outgoing pulse acquires a phase shift proportional to the difference of the test - object positions at time moments separated by , which is proportional to the test - mass average velocity \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat \bar \upsilon ( { t_j})$\end{document } in this time interval ( tj indicates the time moment after the second reflection ) : 203\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\hat \phi ^{{\rm{refl}}}}({t_j } ) = \hat \phi ( { t_j } ) + 2\digamma { k_p}\tau \hat \bar v({t_j})\,,$$\end{document } where 204\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat \bar v({t_j } ) = { { \hat x({t_j } ) - \hat x({t_j } - \tau ) } \over \tau}\,.$$\end{document } figure 26toy example of the quantum speed - meter scheme . we omit here mathematical details of the transition to the continuous measurement limit as they are essentially the same as in the position measurement case , see section 4.1.2 , and start directly with the continuous time equations . the output signal of the homodyne detector in the speed - meter case is described by the following equations : 205\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat o(t ) = - { \hat \phi ^{{\rm{refl}}}}(t)\sin { \phi _ { { \rm{lo } } } } + { { \hat{\mathcal i}(t ) - { { \mathcal i}_0 } } \over { 2{{\mathcal i}_0}}}\,\cos { \phi _ { { \rm{lo}}}},$$\end{document } 206\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\hat \hat x(t - \tau)]\,.$$\end{document } in spectral representation these equations yield : 207\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tilde x(\omega ) \equiv - { { \hat o(\omega ) } \over { 2\digamma{k_p}\sin { \phi _ { { \rm{lo } } } } } } = \hat x(\omega ) + { \hat x_{{\rm{fl}}}}(\omega)\,,$$\end{document } where 208\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\hat x_{{\rm{fl}}}}(\omega ) = { 1 \over { 2\digamma{k_p}(1 - { e^{i\omega \tau}})}}\left [ { \hat \phi ( \omega ) - { { \hat{\mathcal i}(\omega ) - { { \mathcal i}_0 } } \over { 2{{\mathcal i}_0}}}\,\cot { \phi _ { { \rm{lo } } } } } \right]$$\end{document } is the equivalent displacement measurement noise . the back - action force with account for the two subsequent light reflections off the faces of the probe , can be written as : 209\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\hat f_{{\rm{b}}{.}{\rm{a}}{.}}}(t ) = { { 2}\digamma \over c}[\hat{\mathcal i}(t + \tau ) - \hat{\mathcal i}(t)]$$\end{document } and in spectral form as : 210\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\hat f_{{\rm{b}}{.}{\rm{a}}{.}}}(\omega ) = { { 2}\digamma \over c}\hat{\mathcal i}(\omega)({e^{- i\omega \tau } } - 1){.}$$\end{document } then one can make a reasonable assumption that the time between the two reflections is much smaller than the signal force variation characteristic time ( ) that spills over into the following condition : 211\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\omega \tau \ll 1\,,$$\end{document } and allows one to expand the exponents in eqs . ( 208 , 209 ) into a taylor series : 212\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\hat x_{{\rm{fl}}}}(\omega ) = { { { { \hat v}_{{\rm{fl}}}}(\omega ) } \over { - i\omega}}\,,\qquad { \hat f_{{\rm{b}}{.}{\rm{a}}{.}}}(t ) = - i\omega { \hat p_{{\rm{b}}{.}{\rm{a}}{.}}}(\omega)\,,$$\end{document } where 213\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\hat v_{{\rm{fl}}}}(\omega ) = { 1 \over { 2\digamma{k_p}\tau}}\left [ { \hat \phi ( \omega ) - { { \hat{\mathcal i}(\omega ) - { \mathcal i } } \over { 2{\mathcal i}}}\,\cot { \phi _ { { \rm{lo } } } } } \right],$$\end{document } 214\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\hat p_{{\rm{b}}{.}{\rm{a}}{.}}}(\omega ) = { { { { \hat f}_{{\rm{b}}{.}{\rm{a}}{.}}}(\omega ) } \over { - i\omega } } = { { 2\digamma\tau \hat{\mathcal i}(\omega ) } \over c}\,.$$\end{document } spectral densities of theses noises are equal to 215\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s_x}(\omega ) = { { { s_v } } \over { { \omega ^2}}}\,,\qquad { s_f}(\omega ) = { \omega ^2}{s_p}(\omega)\,,\qquad { s_{xf}}(\omega ) = - { s_{vp}}(\omega)\,,$$\end{document } where 216\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { s_v } = { 1 \over { 4\digamma^{2}k_p^2{\tau ^2}}}\left({{s_\phi } + { { { s_{\mathcal i } } } \over { 4{{\mathcal i}^2}}}{{\cot}^2}{\phi _ { { \rm{lo } } } } } \right)\,,}\\ { { s_p } = { { 4\digamma^{2}{\tau ^2}{s_{\mathcal i } } } \over { { c^2}}}\,,\quad \quad \quad \quad \quad \quad \quad \quad } \\ { { s_{vp } } = - { { { s_{\mathcal i } } } \over { 2{\omega _ p}{\mathcal i}}}\cot { \phi _ { { \rm{lo}}}}\quad \quad \quad \quad \quad \quad } \\ \end{array}$$\end{document } note also that 217\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s_x}(\omega){s_f}(\omega ) - s_{xf}^2(\omega ) = { s_v}{s_p } - s_{vp}^2 = { { { s_\phi}{s_{\mathcal i } } } \over { \omega _ p^2 } } \geq { { { \hbar ^2 } } \over 4}\,.$$\end{document } the apparent difference of the spectral densities presented in eq . ( 215 ) from the ones describing the ordinary position meter ( see eqs . ( 191 ) ) is that they now have rather special frequency dependence . it is this frequency dependence that together with the cross - correlation of the measurement and back - action fluctuations , sxf 0 , allows the reduction of the sum noise spectral density to arbitrarily small values . ( 215 ) into eq . ( 144 ) with a free mass xx( ) = 1/(m ) in mind : 218\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s^f } = { m^2}{\omega ^4}{s_x}(\omega ) + { s_f}(\omega ) - 2m{\omega ^2}{s_{xf}}(\omega ) = { \omega ^2}({m^2}{s_v } + 2m{s_{vp } } + { s_p})\,.$$\end{document } if there was no correlation between the back - action and measurement fluctuations , i.e. , svp = 0 , then by virtue of the uncertainty relation , the sum sensitivity appeared limited by the sql ( 161 ) : 219\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s^f } = { \omega ^2}\left({{{{\hbar ^2}{m^2 } } \over { 4{s_p } } } + { s_p } } \right)\geqslant\hbar m{\omega ^2}\,.$$\end{document } one might wonder , what is the reason to implement such a complicated measurement strategy to find ourselves at the same point as in the case of a simple coordinate measurement ? however , recall that in the position measurement case , a constant cross - correlation sxf cot lo allows one to get only a narrow - band sub - sql sensitivity akin to that of a harmonic oscillator . this effect we called virtual rigidity , and showed that for position measurement this rigidity kvirt = sxf / sx is constant . in the speed - meter case , the situation is totally different ; it is clearly seen if one calculates virtual rigidity for a speed meter : 220\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$k_{{\rm{virt}}}^{{\rm{sm } } } = { { { s_{xf } } } \over { { s_x}(\omega ) } } = - { \omega ^2}{{{s_{vp } } } \over { { s_v}}}\,.$$\end{document } it turns out to be frequency dependent exactly in the way that is necessary to compensate the free mass dynamical response to the back - action fluctuations . indeed , in order to minimize the sum noise spectral density ( 218 ) conditioned on uncertainty relation ( 217 ) , one needs to set 221\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s_{xf } } = - { s_{vp } } = { { { s_f } } \over { m{\omega ^2 } } } = { { { s_p } } \over m } = { \rm{const}}.,$$\end{document } which allows one to overcome the sql simply by choosing the right fixed homodyne angle : 222\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\cot { \phi _ { { \rm{lo } } } } = { { 8{\digamma^2}{\tau ^2}{\omega _ p}{{\mathcal i}_0 } } \over { m{c^2}}}\,.$$\end{document } then the sum noise is equal to 223\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s^f}(\omega ) = { { { s_\phi}{s_{\mathcal i } } } \over { \omega _ p^2}}\,{{{m^2}{\omega ^2 } } \over { { s_p } } } = { { { m^2}{\omega ^2 } } \over { 4{\digamma^2}k_p^2{\tau ^2}}}{s_\phi}\,.$$\end{document } and , in principle , can be made arbitrarily small , if a sufficient value of sp is provided ; that is , if the optomechanical coupling is sufficiently strong . let us consider how the spectral density of a speed meter will appear if the light field is in a coherent state . ( 122 ) , hence the sum noise power ( double - sided ) spectral density for the speed meter takes the following form : 224\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s^f}(\omega ) = { { \hbar { m^2}{c^2}{\omega ^2 } } \over { 16{\omega _ p}{{\mathcal i}_0}{\digamma^2}{\tau ^2 } } } = { { \hbar m } \over { 2{\tau ^2}}}{\left({{\omega \over { { \omega _ q } } } } \right)^2 } = { { s_{{\rm{sql}}\,{\rm{f}}{.}{\rm{m}}{.}}^f } \over { 2\omega _ q^2{\tau ^2}}}\,,$$\end{document } where q for our scheme is defined in eq . this formula indicates the ability of a speed meter to have a sub - sql sensitivity in all frequencies provided high enough optical power and no optical loss . the initial motivation to consider speed measurement rested on the assumption that a velocity \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat \upsilon$\end{document } of a free mass is directly proportional to its momentum \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat p = m\hat \upsilon$\end{document}. and the momentum in turn is , as an integral of motion , a qnd - observable , i.e. , it satisfies the simultaneous measurability condition ( 131 ) : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[\hat p(t),\,\hat p(t{\prime } ) ] = 0\quad \forall t,\,t{\prime } .$$\end{document } but this connection between and holds only if one considers an isolated free mass not coupled to a meter . as the measurement starts , the velocity value gets perturbed by the meter and it is not proportional to the momentum anymore . the distinctive feature of this example is that the meter probes the test position \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat x$\end{document } twice , with opposite signs of the coupling factor . therefore , the lagrangian of this scheme can be written as : 225\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat{\mathcal l } = { { m{{\hat v}^2 } } \over 2 } + \beta ( t)\hat x\hat{\mathcal n } + { \hat{\mathcal l}_{{\rm{meter}}}}\,,$$\end{document } where 226\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat v = { { d\hat x } \over { dt}}$$\end{document } is the test - mass velocity , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal n}$\end{document } stands for the meter s observable , which provides coupling to the test mass , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal l}_{{\rm{meter}}}}$\end{document } is the self - lagrangian of the meter , and (t ) is the coupling factor , which has the form of two short pulses with the opposite signs , separated by the time , see figure 27 . we suppose for simplicity that the evolution of the meter observable \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal n}$\end{document } can be neglected during the measurement ( this is a reasonable assumption , for in real schemes of the speed meter and in the gedanken experiment considered above , this observable is proportional to the number of optical quanta , which does not change during the measurement ) . this assumption allows one to omit the term \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal l}_{{\rm{meter}}}}$\end{document } from consideration . figure 27real ( (t ) ) and effective ( (t ) ) coupling constants in the speed - meter scheme . real ( (t ) ) and effective ( (t ) ) coupling constants in the speed - meter scheme . this lagrangian does not satisfy the most well - known sufficient ( but not necessary ! ) condition of the qnd measurement , namely the commutator of the interaction hamiltonian \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat { \mathcal h}_{{\rm{int } } } } = - \beta ( t)\hat x\hat { \mathcal n}$\end{document } with the operator of measured observable \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat x$\end{document } does not vanish . however , it can be shown that a more general condition is satisfied : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[\hat u(0,\tau),\hat x ] = 0$$\end{document } where (0 , ) is the evolution operator of probe - meter dynamics from the initial moment tstart = 0 when the measurement starts till the final moment tend = when it ends . basically , the latter condition guarantees that the value of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat x$\end{document } before the measurement will be equal to that after the measurement , but does not say what it should be in between ( see section 4.4 of for details ) . moreover , using the following nice trick9 , the lagrangian ( 225 ) can be converted to the form , satisfying the simple condition of : 227\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat{\mathcal l}\prime = \hat{\mathcal l } - { { d\alpha ( t)\hat x } \over { dt}}\hat{\mathcal n}\,,$$\end{document } where 228\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\alpha ( t ) = \int\nolimits_{- \infty}^t \beta ( t{\prime})\,dt{\prime } \,,$$\end{document } see figure 27 . it is known that two lagrangians are equivalent if they differ only by a full time derivative of and arbitrary function of the generalized coordinates . lagrangian equations of motion for the coordinates of the system are invariant to such a transformation . the new lagrangian has the required form with the interaction term proportional to the test - mass velocity : 229\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat{\mathcal l } = { { m{{\hat v}^2 } } \over 2 } - \alpha ( t)\hat v\hat{\mathcal n}\,.$$\end{document } note that the antisymmetric shape of the function (t ) guarantees that the coupling factor (t ) becomes equal to zero when the measurement ends . the canonical momentum of the mass m is equal to 230\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p = { { \partial { \mathcal l } } \over { \partial v } } = mv - \alpha ( t){\mathcal n}\,,$$\end{document } and the hamiltonian of the system reads 231\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat{\mathcal h } = pv - { \mathcal l } = { { { { [ p + \alpha ( t){\mathcal n}]}^2 } } \over { 2m}}\,.$$\end{document } the complete set of observables describing our system includes in addition apart to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat x$\end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat p$\end{document } , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat { \mathcal n}$\end{document } , the observable \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat \phi$\end{document } canonically conjugated to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat { \mathcal n}$\end{document } : 232\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[\hat \phi , \hat{\mathcal n } ] = i\hbar$$\end{document } ( if \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat { \mathcal n}$\end{document } is proportional to the number of quanta in the light pulse then \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat \phi$\end{document } is proportional to its phase ) , which represents the output signal of the meter . the heisenberg equations of motion for these observables are the following : 233\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { { d\hat x(t ) } \over { dt } } = \hat v(t ) = { { \hat p + \alpha ( t)\hat{\mathcal n } } \over m}\,,\quad \quad \quad \quad \ ; } & { { { d\hat p(t ) } \over { dt } } = 0\ , , } \\ { { { d\hat \phi ( t ) } \over { dt } } = { { \alpha ( t)[\hat p + \alpha ( t)\hat{\mathcal n } ] } \over m } = \alpha ( t)\hat v(t)\ , , } & { { { d\hat{\mathcal n}(t ) } \over { dt } } = 0\ , . } \\ \end{array}$$\end{document } these equations show clearly that ( i ) a canonical momentum \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat p$\end{document } is preserved by this measurement scheme while ( ii ) the test - mass velocity \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat \upsilon$\end{document } as well as its kinematic momentum \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${m_{\hat \upsilon}}$\end{document } , are perturbed during the measurement ( that is , while 0 ) , yet restore their initial values after the measurement , and ( iii ) the output signal of the meter \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat \phi$\end{document } carries the information about this perturbed value of the velocity . specify (t ) to be of a simple rectangular shape : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\alpha ( t ) = \left\{{\begin{array}{*{20}c } { 1 , } & { { \rm{if}}\;0 \leq t < \tau \quad \quad } \\ { 0 , } & { { \rm{if}}\;t < 0\;{\rm{or}}\;t \geq \tau \ , . } \\ \end{array } } \right.$$\end{document } this assumption does not affect our main results , but simplifies the calculation . in this case , the solution of eqs . ( 233 ) reads : 234a\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat x(\tau ) = \hat x(0 ) + \hat \bar v\tau \,,$$\end{document } 234b\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat \phi ( \tau ) = \hat \phi ( 0 ) + \hat \bar v\tau \,,$$\end{document } with 235\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat \bar v = { { \hat p + \hat{\mathcal n } } \over m}$$\end{document } the test - mass velocity during the measurement [ compare with eqs . therefore , by detecting the variable ( ) , the perturbed value of velocity \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\bar \upsilon$\end{document } is measured with an imprecision 236\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta { v_{{\rm{meas } } } } = { { \delta \phi } \over \tau}\,,$$\end{document } where is the initial uncertainty of . the test - mass position perturbation after this measurement is proportional to the initial uncertainty of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal n}$\end{document } : 237\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta { x_{{\rm{b}}{.}{\rm{a}}{. } } } = \delta { v_{{\rm{b}}{.}{\rm{a}}{.}}}\tau = { { \delta { \mathcal n}\tau } \over m}\,.$$\end{document } it follows from the last two equations that 238\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta { v_{{\rm{meas}}}}\delta { x_{{\rm{b}}{.}{\rm{a}}{. } } } = { { \delta \phi \delta { \mathcal n } } \over m } \geq { \hbar \over { 2m}}\,.$$\end{document } the sum error of the initial velocity estimate vinit = p / m yielding from this measurement is thus equal to : 239\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta { v_{{\rm{sum } } } } = \sqrt { { { \left({{{\delta \phi } \over \tau } } \right)}^2 } + { { \left({{{\delta { \mathcal n } } \over m } } \right)}^2 } } \geq \sqrt { { \hbar \over { m\tau } } } \,.$$\end{document } as we see , it is limited by a value of the velocity measurement sql : 240\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta { v_{{\rm{sql } } } } = \sqrt { { \hbar \over { m\tau } } } \,.$$\end{document } to overcome this sql one has to use cross - correlation between the measurement error and back - action . such a cross - correlation can be achieved by measuring the following combination of the meter observables 241\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat \phi ( \tau ) - \hat{\mathcal n}\tau /m = \hat \phi ( 0 ) + { { \hat p\tau } \over m}$$\end{document } instead of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat \phi ( \tau)$\end{document } , which gives a sum measurement uncertainty for the initial velocity \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat p / m$\end{document } , proportional to the initial uncertainty of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat \phi$\end{document } only : 242\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta { v_{{\rm{sum } } } } = { { \delta \phi } \over \tau}\,,$$\end{document } and hence not limited by the sql . in section 4 , we have talked about the quantum measurement , the general structure of quantum noise implied by the quantum mechanics and the restrictions on the achievable sensitivity it imposes . in this section , we turn to the application of these general and lofty principles to real life , i.e. , we are going to calculate quantum noise for several types of the schemes of gw interferometers and consider the advantages and drawbacks they possess . to grasp the main features of quantum noise in an advanced gw interferometer it would be elucidating to consider first two elementary examples : ( i ) a single movable mirror coupled to a free optical field , reflecting from it , and ( ii ) a fabry - prot cavity comprising two movable mirrors and pumped from both sides . these two systems embody all the main features and phenomena that also mold the advanced and more complicated interferometers quantum noise . should one encounter these phenomena in real - life gw detectors , knowledge of how they manifest themselves in these simple situations would be of much help in successfully discerning them . it is illuminated from both sides by the two independent laser sources with frequency p , and mean power values \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}_1}$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}_2}$\end{document}. in terms of the general linear measurement theory of section 4.2 we have two meters represented by these two incident light waves . the two arbitrary quadratures of the reflected waves are deemed as measured quantities 1 and 2 . let us analyze quantum noise in such a model keeping to the scheme given by eqs . figure 28scheme of light reflection off the single movable mirror of mass m pulled by an external force g. scheme of light reflection off the single movable mirror of mass m pulled by an external force g. the first one of eqs . ( 133 ) in our simple scheme is represented by the input - output coupling equations ( 30 , 36 ) of light on a movable mirror derived in section 2.2.4 . we choose the transfer matrix of the mirror to be real : 243\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathbb{m}_{{\rm{real } } } } = \left [ { \begin{array}{*{20}c } { - \sqrt r } & { \sqrt t } \\ { \sqrt t } & { \sqrt r } \\ . then we can write down the coupling equations , substituting electric field strength amplitudes \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\varepsilon _ { 1,2}}$\end{document } and 1,2( ) by their dimensionless counterparts as introduced by eq . ( 61 ) of section 3.1 : 244\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\left [ { \begin{array}{*{20}c } { { { \bf{\hat{b}}}_1}(\omega ) } \\ { { { \bf{\hat{b}}}_2}(\omega ) } \\ \end{array } } \right ] = { \mathbb{m}_{{\rm{real}}}}.\left [ { \begin{array}{*{20}c } { { { \bf{\hat{a}}}_1}(\omega ) } \\ { { { \bf{\hat{a}}}_2}(\omega ) } \\ \end{array } } \right],\quad { \rm{and}}\quad \left [ { \begin{array}{*{20}c } { { { \hat b}_1}(\omega ) } \\ { { { \hat b}_2}(\omega ) } \\ \end{array } } \right ] = { \mathbb{m}_{{\rm{real}}}}.\left [ { \begin{array}{*{20}c } { { { \hat a}_1}(\omega ) } \\ { { { \hat a}_2}(\omega ) } \\ \end{array } } \right ] + \left [ { \begin{array}{*{20}c } { { r_1 } } \\ { { r_2 } } \\ \end{array } } \right]\hat x(\omega),$$\end{document } where 245\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${r_1 } = 2\sqrt r { { { \omega _ p } } \over c}\left [ { \begin{array}{*{20}c } { { { \rm{a}}_{1s } } } \\ { - { { \rm{a}}_{1c } } } \\ \end{array } } \right],\quad { \rm{and}}\quad { r_2 } = 2\sqrt r { { { \omega _ p } } \over c}\left [ { \begin{array}{*{20}c } { { { \rm{a}}_{2s } } } \\ { - { { \rm{a}}_{2c } } } \\ \end{array } } \right]\;.$$\end{document } without any loss of generality one can choose the phase of the light incident from the left to be such that a1s = 0 and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\rm{a}}_{1c } } = \sqrt { 2{{\mathcal i}_1}/(\hbar { \omega _ p})}$\end{document}. then factoring in the constant phase difference between the left and the right beams equal to 0 , one would obtain for the left light \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\{{a_{2c}},{a_{2s}}\ } = \sqrt { 2{{\mathcal i}_2}/(\hbar { \omega _ p } ) } \{{\rm{cos}}\,{\phi _ 0},\,\sin \,{\phi _ 0}\}$\end{document}. the two output measured quantities will then be given by the two homodyne photocurrents : 246\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\hat o_1}(\omega ) = { h^ { \mathsf { t}}}[{\phi _ 1}]{\hat b_1 } = { \hat b_{1c}}(\omega)\cos { \phi _ 1 } + { \hat b_{1s}}(\omega)\sin { \phi _ 1}\,,$$\end{document } 247\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\hat o_2}(\omega ) = { h^{\mathsf { t}}}[{\phi _ 2}]{\hat b_2 } = { \hat b_{2c}}(\omega)\cos { \phi _ 2 } + { \hat b_{2s}}(\omega)\sin { \phi _ 2}\,,$$\end{document } where vector h[ ] was first introduced in section 2.3.2 after eq . ( 47 ) as : 248\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h[\phi ] \equiv \left [ { \begin{array}{*{20}c } { \cos \phi } \\ { \sin \phi } \\ \end{array } } \right]\;.$$\end{document } then , after substitution of ( 243 ) into ( 244 ) , and then into ( 246 ) , one gets 249\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat o_1^{(0)}(\omega ) = { h^{\mathsf { t}}}[{\phi _ 1}]\left(- \sqrt r { { \hat{a}_1 } } + \sqrt t { { \hat{a}_2}}\right)\,,$$\end{document } 250\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat o_2^{(0)}(\omega ) = { h^ { \mathsf { t}}}[{\phi _ 2}]\left(\sqrt t { { \hat{a}_1 } } + \sqrt r { { \hat{a}_2}}\right)\,,$$\end{document } and 251\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\chi _ { { o_1}f}}(\omega ) = { h^t}[{\phi _ 1}]{r_1 } = - { { 2\sqrt { 2r\hbar { \omega _ p}{{\mathcal i}_1 } } } \over { \hbar c}}\sin { \phi _ 1},$$\end{document } 252\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\chi _ { { o_2}f}}(\omega ) = { h^t}[{\phi _ 2}]{r_2 } = - { { 2\sqrt { 2r\hbar { \omega _ p}{{\mathcal i}_2 } } } \over { \hbar c}}\sin ( { \phi _ 2 } - { \phi _ 0}).$$\end{document } now we can write down the equation of motion for the mirror assuming it is pulled by a gw tidal force g : 253\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$m\ddot \hat x(t ) = { \hat f_{{\rm{r.}}{\rm{p.}}}}(t ) + g(t)\qquad \rightarrow \qquad - m{\omega ^2}\hat x(\omega ) = { \hat f_{{\rm{r.}}{\rm{p.}}}}(\omega ) + g(\omega),$$\end{document } that gives us the probe s dynamics equation ( the third one ) in ( 133 ) : 254\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat x(\omega ) = { \chi _ { xx}}(\omega)[{\hat f_{{\rm{b.}}{\rm{a.}}}}(\omega ) + g(\omega ) ] = - { 1 \over { m{\omega ^2}}}[{\hat f_{{\rm{r.}}{\rm{p.}}}}(\omega ) + g(\omega)],$$\end{document } where the free mirror mechanical susceptibility xx( ) = 1/(m ) . the term \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat f_{{\rm{b}}{\rm{.a}}{\rm{.}}}}(t)$\end{document } stands for the radiation pressure force imposed by the light that can be calculated as 255\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { { \hat f}_{{\rm{r}}.{\rm{p}}.}}(\omega ) = { f_0 } + { { \hat f}_{{\rm{b}}.{\rm{a}}.}}(\omega ) = { { { { \hat{\mathcal i}}_{{a_1}}}(\omega ) + { { \hat{\mathcal i}}_{{b_1}}}(\omega ) - { { \hat{\mathcal i}}_{{a_2}}}(\omega ) - { { \hat{\mathcal i}}_{{b_2}}}(\omega ) } \over c } \simeq \quad \quad \quad \quad \quad \quad \quad \quad \quad } \\ { { { \hbar { k_p } } \over 2}\left [ { ( { \bf{a}}_1^t{{\bf{a}}_1 } + { \bf{b}}_1^t{{\bf{b}}_1 } - { \bf{a}}_2^t{{\bf{a}}_2 } - { \bf{b}}_2^t{{\bf{b}}_2 } ) + { \bf{b}}_1^t{{\hat b}_1}(t ) - { \bf{a}}_2^t{{\hat a}_2}(t ) - { \bf{b}}_2^t{{\hat b}_2}(t ) ) } \right ] } \\ \end{array}$$\end{document } where kp p / c 256\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${f_0 } = { { \hbar { k_p } } \over 2}({\bf{a}}_1^t{{\bf{a}}_1 } + { \bf{b}}_1^t{{\bf{b}}_1 } - { \bf{a}}_2^t{{\bf{a}}_2 } - { \bf{b}}_2^t{{\bf{b}}_2 } ) = { { 2r } \over c}({{\mathcal i}_1 } - { { \mathcal i}_2 } ) - { { 4\sqrt { rt } } \over c}\sqrt { { { \mathcal i}_1}{{\mathcal i}_2 } } \cos { \phi _ 0},$$\end{document } is the regular part of the radiation pressure force10 . it is constant and thus can be compensated by applying a fixed restoring force of the same magnitude but with opposite direction , which can be done either by employing an actuator , or by turning the mirror into a low - frequency pendulum with m gw by suspending it on thin fibers , as is the case for the gw interferometers , that provides a necessary gravity restoring force in a natural way . however , it does not change the quantum noise and thus can be omitted from further consideration . the latter term represents a quantum correction to the former one 257\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\hat f_{{\rm{b.}}{\rm{a.}}}}(\omega ) \simeq \hbar { k_p}({\bf{a}}_1^t{\hat a_1}(\omega ) + { \bf{b}}_1^t{\hat b_1}(\omega ) - { \bf{a}}_2^t{\hat a_2}(\omega ) - { \bf{b}}_2^t{\hat b_2}(\omega ) ) = { \bf{f}}_1^t{\hat a_1}(\omega ) + { \bf{f}}_2^t{\hat a_2}(\omega ) - k\hat x(\omega)$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat f_{{\rm{b.a.}}}^{(0 ) } \equiv { \bf{f}}_1^t{\hat{\bf{a}}_1}(\omega ) + { \bf{f}}_2^t{\hat{\bf{a}}_2}(\omega)$\end{document } is the random part of the radiation pressure that depends on the input light quantum fluctuations described by quantum quadrature amplitudes vectors 1( ) and 2( ) with coefficients given by vectors : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${f_1 } = { { 2\sqrt { 2\hbar { \omega _ p}r } } \over c}\left [ { \begin{array}{*{20}c } { \sqrt { r{{\mathcal i}_1 } } - \sqrt { t{{\mathcal i}_2 } } \cos { \phi _ 0 } } \\ { - \sqrt { t{{\mathcal i}_2 } } \sin { \phi _ 0 } } \\ \end{array } } \right]\,,\quad { \rm{and}}\quad { f_2 } = - { { 2\sqrt { 2\hbar { \omega _ p}r } } \over c}\left [ { \begin{array}{*{20}c } { \sqrt { t{{\mathcal i}_1 } } + \sqrt { r{{\mathcal i}_2 } } \cos { \phi _ 0 } } \\ { \sqrt { r{{\mathcal i}_2 } } \sin { \phi _ 0 } } \\ \end{array } } \right]\,,$$\end{document } and the term \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$- k\hat x(\omega)$\end{document } represents the dynamical back action with 258\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$k = { { 8{\omega _ p}\sqrt { rt{{\mathcal i}_1}{{\mathcal i}_2 } } \sin { \phi _ 0 } } \over { { c^2}}}$$\end{document } being a ponderomotive rigidity that arises in the potential created by the two counter propagating light waves . we can reduce both our readout quantities to the units of the signal force g according to eq . ( 140 ) : 259\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat o_1^f = { { \hat { \mathcal x}(\omega ) } \over { \chi _ { xx}^{{\rm{eff}}}(\omega ) } } + \hat { \mathcal f}(\omega ) + g,\qquad \hat o_2^f = { { { { \hat { \mathcal x}}_2}(\omega ) } \over { \chi _ { xx}^{{\rm{eff}}}(\omega ) } } + \hat { \mathcal f}(\omega ) + g$$\end{document } and define the two effective measurement noise sources as 260\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\hat{\mathcal x}_1}(\omega ) = { { \hat o_1^{(0 ) } } \over { { \chi _ { { o_1}f}}(\omega)}}\,,\quad { \rm{and}}\quad { \hat{\mathcal x}_2}(\omega ) = { { \hat o_2^{(0 ) } } \over { { \chi _ { { o_2}f}}(\omega)}}$$\end{document } and an effective force noise as 261\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat{\mathcal f}(\omega ) = \hat f_{{\rm{b.}}{\rm{a.}}}^{(0)},$$\end{document } absorbing optical rigidity k into the effective mechanical susceptibility : 262\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\chi _ { xx}^{{\rm{eff}}}(\omega ) = { { { \chi _ { xx}}(\omega ) } \over { 1 + k{\chi _ { xx}}(\omega ) } } = { 1 \over { k - m{\omega ^2}}}.$$\end{document } one can then easily calculate their power ( double - sided ) spectral densities according to eq . ( 144 ) : 263\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}{c } } { s_{11}^f = \frac{{{s_{{\mathcal{x}_1}{\mathcal{x}_1}}}(\omega ) } } { { |\chi _ { xx}^{{\text{eff}}}(\omega ) { |^2 } } } + { s_{\mathcal{f}\mathcal{f}}}(\omega ) + 2{\text{re}}\left [ { \frac{{{s_{{\mathcal{x}_1}\mathcal{f}}}(\omega ) } } { { \chi _ { xx}^{{\text{eff}}}(\omega ) } } } \right]{\mkern 1mu } , } \\ { s_{22}^f = \frac{{{s_{{\mathcal{x}_2}{\mathcal{x}_2}}}(\omega ) } } { { |\chi _ { xx}^{{\text{eff}}}(\omega ) { |^2 } } } + { s_{\mathcal{f}\mathcal{f}}}(\omega ) + 2{\text{re}}\left [ { \frac{{{s_{{\mathcal{x}_2}\mathcal{f}}}(\omega ) } } { { \chi _ { xx}^{{\text{eff}}}(\omega ) } } } \right ] , } \\ { s_{12}^f = s_{21}^{f * } = \frac{{{s_{{\mathcal{x}_1}{\mathcal{x}_2}}}(\omega ) } } { { |\chi _ { xx}^{{\text{eff}}}(\omega ) { |^2 } } } + { s_{\mathcal{f}\mathcal{f}}}(\omega ) + \left [ { \frac{{{s_{{\mathcal{x}_1}\mathcal{f}}}(\omega ) } } { { \chi _ { xx}^{{\text{eff}}}(\omega ) } } + \frac{{s_{{\mathcal{x}_2}\mathcal{f}}^*(\omega ) } } { { \chi _ { xx}^{{\text{eff*}}}(\omega ) } } } \right ] , } \end{array}$$\end{document } where , if both lights are in coherent states that implies \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\langle { \hat a_i}(\omega ) \circ \hat a_j^\dagger ( \omega \prime)\rangle = 2\pi { \mathbb s}_{{\rm{vac}}}{\delta _ { ij}}\delta ( \omega - \omega \prime)\,,\qquad ( i , j ) = ( 1,2)$$\end{document } , one can get : 264\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { s_{{{\mathcal x}_1}{{\mathcal x}_1}}}(\omega ) = { { \hbar { c^2 } } \over { 16{\omega _ p}{{\mathcal i}_1}r{{\sin}^2}{\phi _ 1}}},\qquad { s_{{{\mathcal x}_2}{{\mathcal x}_2}}}(\omega ) = { { \hbar { c^2 } } \over { 16{\omega _ p}{{\mathcal i}_2}r{{\sin}^2}({\phi _ 2 } - { \phi _ 0})}},\qquad { s_{{{\mathcal x}_1}{{\mathcal x}_2}}}(\omega ) = 0 , } \\ { { s_{{\mathcal f}{\mathcal f}}}(\omega ) = { { 4\hbar { \omega _ p}r({{\mathcal i}_1 } + { { \mathcal i}_2 } ) } \over { { c^2}}},\qquad { s_{{{\mathcal x}_1}{\mathcal f}}}(\omega ) = { \hbar \over 2}\cot { \phi _ 1},\qquad { s_{{{\mathcal x}_2}{\mathcal f}}}(\omega ) = { \hbar \over 2}\cot ( { \phi _ 2 } - { \phi _ 0}).\quad \quad } \\ \end{array}$$\end{document } comparison of these expressions with the eqs . ( 195 ) shows that we have obtained results similar to that of the toy example in section 4.4 . if we switched one of the pumping carriers off , say the right one , the resulting spectral densities for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat o_1^f(\omega)$\end{document } would be exactly the same as in the simple case of eqs . ( 195 ) , except for the substitution of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${f^2}{{\mathcal i}_0 } \to r{{\mathcal i}_1}$\end{document } and 1 lo . it was easy to calculate the above spectral densities by parts , distinguishing the effective measurement and back - action noise sources and making separate calculations for them . had we considered a bit more complicated situation with the incident fields in the squeezed states with arbitrary squeezing angles , the calculation of all six of the above individual spectral densities ( 264 ) and subsequent substitution to the sum spectral densities expressions ( 263 ) would be more difficult . thus , it would be beneficial to have a tool to do all these operations at once numerically . we start with 1 and rewrite it as follows : 265\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { { \hat o}_1 } = { h^{\rm{t}}}[{\phi _ 1}]\left({- \sqrt r { { \hat a}_1 } + \chi _ { xx}^{eff}{r_1}f_1^{\rm{t}}{{\hat a}_1 } + \sqrt t { { \hat a}_2 } + \chi _ { xx}^{eff}{r_1}f_2^{\rm{t}}{{\hat a}_2 } } \right ) + \chi _ { xx}^{eff}{h^{\rm{t}}}[{\phi _ 1}]{r_1}g } \\ { = { h^{\rm{t}}}[{\phi _ 1}]\,\cdot{\mathbb m_1}\cdot\left [ { \begin{array}{*{20}c } { { { \hat a}_1 } } \\ { { { \hat a}_2 } } \\ \end{array } } \right ] + \chi _ { xx}^{eff}{h^{\rm{t}}}[{\phi _ 1}]{r_1}g,\quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad } \\ \end{array}$$\end{document } where we omitted the frequency dependence of the constituents for the sake of brevity and introduced a 2 4 full transfer matrix \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathbb m}_1}$\end{document } for the first readout observable defined as 266\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathbb m_1 } = \left [ { - \sqrt r\mathbb i + \chi _ { xx}^{eff}{r_1}f_2^{\rm{t } } } \right]$$\end{document } with outer product of two arbitrary vectors = { 1 , 2 } and = { 1 , 2 } written in short notation as : 267\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\alpha { \beta ^t } \equiv \left [ { \begin{array}{*{20}c } { { \alpha _ 1}{\beta _ 1 } } & { { \alpha _ 1}{\beta _ 2 } } \\ { { \alpha _ 2}{\beta _ 1 } } & { { \alpha _ 2}{\beta _ 2 } } \\ \end{array } } \right]\,.$$\end{document } in a similar manner , the full transfer matrix for the second readout can be defined as : 268\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathbb m_2 } = \left [ { \sqrt t\mathbb i + \chi _ { xx}^{eff}{r_2}f_1^{\rm{t}}\quad \sqrt r\mathbb i + \chi _ { xx}^{eff}{r_2}f_2^{\rm{t } } } \right].$$\end{document } having accomplished this , one is prepared to calculate all the spectral densities ( 263 ) at once using the following matrix formulas : 269\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$s_{ij}^f(\omega ) = { 1 \over { 2|\chi _ { xx}^{eff}{|^2}}}{{{h^{\rm{t}}}[{\phi _ i}]\,\cdot\,{\mathbb m_i}{\mathbb s_{{\rm{in}}}}\mathbb m_j^{\dagger}\,\cdot\,h[{\phi _ j } ] + { h^{\rm{t}}}[{\phi _ i}]\,\cdot\,\mathbb m_j^*{\mathbb s_{{\rm{in}}}}\mathbb m_i^{\rm{t}}\,\cdot\,h[{\phi _ j } ] } \over { { h^{\rm{t}}}[{\phi _ i}]{r_i}r_j^{\dagger}h[{\phi _ j}]}}\,,\qquad ( i , j ) = ( 1,2)$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathbb m}^ * } \equiv { ( { { \mathbb m}^\dagger})^{\rm{t}}}$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathbb s}_{{\rm{in}}}}$\end{document } is the 4 4-matrix of spectral densities for the two input fields : 270\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathbb s_{{\rm{in } } } } = \left [ { \begin{array}{*{20}c } { { \mathbb s_{{\rm{sqz}}}}[{r_1},{\theta _ 1 } ] } & 0 \\ 0 & { { \mathbb s_{{\rm{sqz}}}}[{r_2},{\theta _ 2 } ] } \\ \end{array } } \right]$$\end{document } with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathbb s}_{{\rm{sqz}}}}[{r_i},{\theta _ i}]$\end{document } defined by eq . ( 83 ) . thus , we obtain the formula that can be ( and , actually , is ) used for the calculation of quantum noise spectral densities of any , however complicated , interferometer given the full transfer matrix of this interferometer . thus far we have assumed that there is no dissipation in the transition from the outgoing light to the readout photocurrent of the homodyne detector . this is , unfortunately , not the case since any real photodetector has the finite quantum efficiency d < 1 that indicates how many photons absorbed by the detector give birth to photoelectrons , i.e. , it is the measure of the probability for the photon to be transformed into the photoelectron . as with any other dissipation , this loss of photons gives rise to an additional noise according to the fdt that we should factor in . we have shown in section 2.2.4 that this kind of loss can be taken into account by means of a virtual asymmetric beamsplitter with transmission coefficients \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\sqrt { { \eta _ d}}$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\sqrt { 1 - { \eta _ d}}$\end{document } for the signal light and for the additional noise , respectively . this beamsplitter is set into the readout optical train as shown in figure 8 and the i - th readout quantity needs to be modified in the following way : 271\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat o_i^{{\rm{loss}}}(\omega ) = \sqrt { { \eta _ d } } { \hat o_i}(\omega ) + \sqrt { 1 - { \eta _ d } } { \hat n_i}(\omega),$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\hat n_i}(\omega ) = { h^{\mathsf t}}[{\phi _ i}]{\hat n_i}(\omega ) = { \hat n_{i , c}}(\omega)\cos { \phi _ i } + { \hat n_{i , s}}(\omega)\sin { \phi _ i}$$\end{document } is the additional noise that is assumed to be in a vacuum state . since the overall factor in front of the readout quantity does not matter for the final noise spectral density , one can redefine \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat o_i^{{\rm{loss}}}(\omega)$\end{document } in the following way : 272\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat o_i^{{\rm{loss}}}(\omega ) = { \hat o_i}(\omega ) + { \epsilon _ d}{\hat n_i}(\omega)\,,\qquad { \rm{where}}\qquad { \epsilon _ d } \equiv \sqrt { { 1 \over { { \eta _ d } } } - 1 } .$$\end{document } the influence of this loss on the final sum spectral densities ( 269 ) is straightforward to calculate if one assumes the additional noise sources in different readout trains to be uncorrelated . if it is so , then eq . ( 269 ) modifies as follows : 273\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { s_{ij}^{f,{\rm{loss}}}(\omega ) = { 1 \over { 2|\chi _ { xx}^{{\rm eff}}{|^2}}}\left\{{{{{h^{\rm{t}}}[{\phi _ i}]\,\cdot\,{\mathbb m_i}{\mathbb s_{{\rm{in } } } } \mathbb m_j^{\dagger}\,\cdot\,h[{\phi _ j } ] + { h^{\rm{t}}}[{\phi _ i}]\,\cdot\,\mathbb m_j^*{\mathbb s_{{\rm{in } } } } \mathbb m_i^{\rm{t}}\,\cdot\,h[{\phi _ j } ] } \over { { h^t}[{\phi _ i}]{r_i}r_j^{\dagger}h[{\phi _ j } ] } } + } \right . } \\ { \left . { \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad { { \epsilon _ d^2{\delta _ { ij } } } \over { { h^{\rm{t}}}[{\phi _ i}]{r_i}r_i^{\dagger}h[{\phi _ i } ] } } } \right\}},\qquad ( i , j ) = ( 1,2)\ , . \\ \end{array}$$\end{document } now , when we have considered all the stages of the quantum noise spectral densities calculation on a simple example of a single movable mirror , we are ready to consider more complicated systems . our next target is a fabry - prot cavity . the schematic view of a fabry - prot cavity with two movable mirrors is drawn in figure 29 . this simple scheme is important for at least two reasons : ( i ) it is the most common element for more sophisticated interferometer configurations , which are considered below ; and ( ii ) the analysis of real - life high - sensitivity interferometers devoted , in particular , to detection of gws , can be reduced to a single fabry - prot cavity by virtue of the scaling law theorem , see section 5.3 . figure 29fabry - prot cavity a fabry - prot cavity consists of two movable mirrors that are separated by a distance l + x1 + x2 , where l = c is the distance at rest with standing for a single pass light travel time , and x1 and x2 are the small deviations of the mirrors position from the equilibrium . each of the mirrors is described by the transfer matrix \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathbb m}_{1,2}}$\end{document } with real coefficients of reflection \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\sqrt { { r_{1,2}}}$\end{document } and transmission \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\sqrt { { t_{1,2}}}$\end{document } according to eq . as indicated on the scheme , the outer faces of the mirrors are assumed to have negative reflectivities . while the intermediate equations depend on this choice , the final results are invariant to it . the cavity is pumped from both sides by two laser sources with the same optical frequency p . the coupling equations for the ingoing and outgoing fields at each of the mirrors are absolutely the same as in section 2.2.5 . the only new thing is the free propagation of light between the mirrors that adds two more field continuity equations to the full set , describing the transformation of light in the fabry - prot cavity . it is illuminating to write down input - output relations first in the time domain : 274\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { { \hat e}_{{b_1}}}(t ) = - \sqrt { { r_1 } } { { \hat e}_{{a_1}}}(t + 2{x_1}/c ) + \sqrt { { t_1 } } { { \hat e}_{{e_1}}}(t ) , } & { { { \hat e}_{{b_2}}}(t ) = - \sqrt { { r_2 } } { { \hat e}_{{a_2}}}(t + 2{x_2}/c ) + \sqrt { { t_2 } } { { \hat e}_{{e_2}}}(t ) , } \\ { { { \hat e}_{{f_1}}}(t ) = \sqrt { { r_1 } } { { \hat e}_{{e_1}}}(t - 2{x_1}/c ) + \sqrt { { t_1 } } { { \hat e}_{{a_1}}}(t)\,,\,\,\ , } & { { { \hat e}_{{f_2}}}(t ) = \sqrt { { r_2 } } { { \hat e}_{{a_2}}}(t - 2{x_2}/c ) + \sqrt { { t_2 } } { { \hat e}_{{e_2}}}(t),\,\,\,\ , } \\ { { { \hat e}_{{e_1}}}(t ) = { { \hat e}_{{f_2}}}(t - l / c),\quad \quad \quad \quad \quad \quad \quad \quad } & { { { \hat e}_{{e_2}}}(t ) = { { \hat e}_{{f_1}}}(t - l / c).\quad \quad \quad \quad \quad \quad \quad \quad } \\ \end{array}$$\end{document } further we use notation = l / c for the light travel time between the mirrors . the frequency domain version of the above equations and their solutions are derived in appendix a.1 . ( 545 ) in terms of complex amplitudes instead of 2 photon amplitudes , for the expressions look much more compact in that representation . however , one can simplify them even more using the single - mode approximation . single - mode approximation . we note that : ( i)in gw detection , rather high - finesse cavities are used , which implies low transmittance coefficients for the mirrors 275\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${t_{1,2 } } \ll 1;$$\end{document}(ii)the cavities are relatively short , so their free spectral range ( fsr ) ffsr = ( 2 ) is much larger than the characteristic frequencies of the mirrors motion : 276\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\vert\omega \vert\tau \ll 1;$$\end{document } and ( iii ) the detuning of the pump frequency from one of the cavity eigenfrequencies : 277\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta = { \omega _ p } - { { \pi n } \over \tau}\quad ( n\;{\rm{is\ , an\ , integer}})$$\end{document } is also small in comparison with the fsr : 278\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\vert\delta \vert\tau \ll 1.$$\end{document } in this case , only this mode of the cavity can be taken into account , and the cavity can be treated as a single - mode lumped resonator . note also that , while our intermediate equations below depend on whether n is even or odd , the final results do not . . in gw detection , rather high - finesse cavities are used , which implies low transmittance coefficients for the mirrors 275\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${t_{1,2 } } \ll 1;$$\end{document } the cavities are relatively short , so their free spectral range ( fsr ) ffsr = ( 2 ) is much larger than the characteristic frequencies of the mirrors motion : 276\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\vert\omega \vert\tau \ll 1;$$\end{document } and ( iii ) the detuning of the pump frequency from one of the cavity eigenfrequencies : 277\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta = { \omega _ p } - { { \pi n } \over \tau}\quad ( n\;{\rm{is\ , an\ , integer}})$$\end{document } is also small in comparison with the fsr : 278\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\vert\delta \vert\tau \ll 1.$$\end{document } expanding the numerators and denominators of eqs . ( 540 , 545 ) into taylor series in and keeping only the first non - vanishing terms , we obtain that 279\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { { \rm{b}}_{1,2 } } = { { \mathcal r}_{1,2}}(0){{\rm{a}}_{1,2 } } + { \mathcal t}(0){{\rm{a}}_{2,1}},\quad \quad } \\ { { { \rm{e}}_{1,2 } } = { { \rm{f}}_{1,2 } } = { \rm{e } } = { { \sqrt { { \gamma _ 1 } } { { \rm{a}}_1 } + \sqrt { { \gamma _ 2 } } { { \rm{a}}_2 } } \over { \ell ( 0)\sqrt \tau } } , } \\ \end{array}$$\end{document } and 280\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{{\hat{\rm{b}}}_{1,2}}(\omega ) = { { \mathcal r}_{1,2}}(\omega){{{\rm{\hat a}}}_{1,2}}(\omega ) + { \mathcal t}(\omega){{{\rm{\hat a}}}_{2,1}}(\omega ) + { { 2\sqrt { { \gamma _ { 1,2 } } } x(\omega ) } \over { \ell ( \omega)}},}$$\end{document } 281\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{{{\rm{\hat e}}}_{1,2}}(\omega ) = { { { \rm{\hat f}}}_{1,2}}(\omega ) = { \rm{\hat e}}(\omega ) = { { \sqrt { { \gamma _ 1 } } { { { \rm{\hat a}}}_1}(\omega ) + \sqrt { { \gamma _ 2 } } { { { \rm{\hat a}}}_2}(\omega ) + \hat x(\omega ) } \over { \ell ( \omega)\sqrt \tau}},}$$\end{document } where 282\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\gamma _ { 1,2 } } = { { { t_{1,2 } } } \over { 4\tau}},$$\end{document } 283\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\gamma = { \gamma _ 1 } + { \gamma _ 2}$$\end{document } is the cavity half - bandwidth , 284\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\ell ( \omega ) = \gamma - i(\delta + \omega)\,,$$\end{document } 285\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\mathcal r}_{1,2}}(\omega ) = { { 2{\gamma _ { 1,2 } } } \over { \ell ( \omega ) } } - 1\,,\qquad { \mathcal t}(\omega ) = { { 2\sqrt { { \gamma _ 1}{\gamma _ 2 } } } \over { \ell ( \omega)}}$$\end{document } are the cavity left and right reflectivities and its transmittance , 286\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat x(\omega ) = { { i{k_p}{\rm{e}}\hat x(\omega ) } \over { \sqrt \tau}}\,,$$\end{document } and 287\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat x = { \hat x_1 } + { \hat x_2}$$\end{document } is the sum variation of the cavity length . the above optical i / o - relations are obtained in terms of the complex amplitudes . in order to transform them to two - photon quadrature notations , one needs to employ the following linear transformations : change frequency p and rewrite the relations between the input \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat \alpha ( \omega)$\end{document } and output operators \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat \beta ( \omega)$\end{document } in the form : 288\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { \hat \beta ( \omega ) = f(\omega)\hat \alpha ( \omega)\ ; \rightarrow \;{{\hat \beta } _ + } \equiv \hat \beta ( { \omega _ p } + \omega ) = f({\omega _ p } + \omega)\hat \alpha ( { \omega _ p } + \omega ) \equiv { f _ + } { { \hat \alpha } _ + } \;{\rm{and}}\quad \quad \quad \quad \quad \quad \quad \quad \quad } \\ { \hat \beta _ - ^\dagger \equiv { { \hat \beta}^\dagger}({\omega _ p } - \omega ) = { f^{\ast}}({\omega _ p } - \omega){{\hat \alpha}^\dagger}({\omega _ p } - \omega ) \equiv f _ - ^{\ast}\hat \alpha _ - ^\dagger \ , ; } \\ \end{array}$$\end{document } where f( ) is an arbitrary complex - valued function of sideband frequency ;use the definition ( 57 ) to get the following relations for two - photon quadrature operators : 289\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\left [ { \begin{array}{*{20}c } { { { \hat \beta}_c}(\omega ) } \\ { { { \hat \beta}_s}(\omega ) } \\ \end{array } } \right ] = { 1 \over 2}\left [ { \begin{array}{*{20}c } { ( { f _ + } + f _ - ^{\ast } ) } & { i({f _ + } - f _ - ^{\ast } ) } \\ { - i({f _ + } - f _ - ^{\ast } ) } & { ( { f _ + } + f _ - ^{\ast } ) } \\ \end{array } } \right ] \cdot \left [ { \begin{array}{*{20}c } { { { \hat \alpha}_c}(\omega ) } \\ { { { \hat \alpha}_s}(\omega ) } \\ \end{array } } \right].$$\end{document } change frequency p and rewrite the relations between the input \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat \alpha ( \omega)$\end{document } and output operators \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat \beta ( \omega)$\end{document } in the form : 288\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { \hat \beta ( \omega ) = f(\omega)\hat \alpha ( \omega)\ ; \rightarrow \;{{\hat \beta } _ + } \equiv \hat \beta ( { \omega _ p } + \omega ) = f({\omega _ p } + \omega)\hat \alpha ( { \omega _ p } + \omega ) \equiv { f _ + } { { \hat \alpha } _ + } \;{\rm{and}}\quad \quad \quad \quad \quad \quad \quad \quad \quad } \\ { \hat \beta _ - ^\dagger \equiv { { \hat \beta}^\dagger}({\omega _ p } - \omega ) = { f^{\ast}}({\omega _ p } - \omega){{\hat \alpha}^\dagger}({\omega _ p } - \omega ) \equiv f _ - ^{\ast}\hat \alpha _ - ^\dagger \ , ; } \\ \end{array}$$\end{document } where f( ) is an arbitrary complex - valued function of sideband frequency ; use the definition ( 57 ) to get the following relations for two - photon quadrature operators : 289\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\left [ { \begin{array}{*{20}c } { { { \hat \beta}_c}(\omega ) } \\ { { { \hat \beta}_s}(\omega ) } \\ \end{array } } \right ] = { 1 \over 2}\left [ { \begin{array}{*{20}c } { ( { f _ + } + f _ - ^{\ast } ) } & { i({f _ + } - f _ - ^{\ast } ) } \\ { - i({f _ + } - f _ - ^{\ast } ) } & { ( { f _ + } + f _ - ^{\ast } ) } \\ \end{array } } \right ] \cdot \left [ { \begin{array}{*{20}c } { { { \hat \alpha}_c}(\omega ) } \\ { { { \hat \alpha}_s}(\omega ) } \\ \end{array } } \right].$$\end{document } applying transformations ( 289 ) to eqs . ( 280 ) , we rewrite the i / o - relations for a fabry - prot cavity in the two - photon quadratures notations : 290\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\hat b_{1,2}}(\omega ) = { { \mathbb r}_{1,2}}(\omega){\hat a_{1,2}}(\omega ) + { \mathbb t } ( \omega){\hat a_{2,1}}(\omega ) + 2\sqrt { { \gamma _ { 1,2}}}{\mathbb l } ( \omega)\hat{\bf{x}}(\omega)\,,$$\end{document } 291\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat e(\omega ) = { 1 \over { \sqrt \tau}}{\mathbb l}(\omega)\left [ { \sqrt { { \gamma _ { 1,2 } } } { { \hat a}_{1,2}}(\omega ) + \sqrt { { \gamma _ { 2,1 } } } { { \hat a}_{2,1}}(\omega ) + \hat{\bf{x}}(\omega ) } \right],$$\end{document } where 292\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat{\bf{x}}(\omega ) = \left [ { \begin{array}{*{20}c } { - { { \rm{e}}_s } } \\ { { { \rm{e}}_c } } \\ \end{array } } \right]\;\;{{{k_p}\hat x(\omega ) } \over { \sqrt \tau}},$$\end{document } 293\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\rm{e}}_c } = \sqrt 2 { \rm re}\;{\rm{e}}\,,\qquad { { \rm{e}}_s } = \sqrt 2{\rm im}\;{\rm{e}}\,,$$\end{document } 294\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathbb l}(\omega ) = { 1 \over { { \mathcal d}(\omega)}}\;\;\left [ { \begin{array}{*{20}c } { \gamma - i\omega } & { - \delta } \\ \delta & { \gamma - i\omega } \\ \end{array } } \right],$$\end{document } 295\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal d}(\omega ) = \ell ( \omega){\ell ^{\ast}}(- \omega ) = { ( \gamma - i\omega)^2 } + { \delta ^2}\,,$$\end{document } 296\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\mathbb r}_{1,2}}(\omega ) = 2{\gamma _ { 1,2}}{\mathbb l}(\omega ) -{\mathbb i } \,,\qquad { \mathbb t}(\omega ) = 2\sqrt { { \gamma _ 1}{\gamma _ 2}}{\mathbb l } ( \omega)\,.$$\end{document } therefore , the i / o - relations in standard form read : 297\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\left [ { \begin{array}{*{20}c } { { { \hat b}_1}(\omega ) } \\ { { { \hat b}_2}(\omega ) } \\ \end{array } } \right ] = { \mathbb m}_{{\rm{fp}}}^{(0)}\cdot\;\left [ { \begin{array}{*{20}c } { { { \hat a}_1}(\omega ) } \\ { { { \hat a}_2}(\omega ) } \\ \end{array } } \right]\ ; + \;\left [ { \begin{array}{*{20}c } { r_1^{{\rm{fp}}}(\omega ) } \\ { r_2^{{\rm{fp}}}(\omega ) } \\ \end{array } } \right]\hat x(\omega)$$\end{document } with optical transfer matrix defined as : 298\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathbb m}_{{\rm{fp}}}^{(0)}(\omega ) = \left [ { \begin{array}{*{20}c } { { { \mathbb r}_1}(\omega ) } & { \mathbb t}{(\omega ) } \\ { { \mathbb t}(\omega ) } & { { { \mathbb r}_2}(\omega ) } \\ \end{array } } \right]$$\end{document } and the response to the cavity elongation \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat x(\omega)$\end{document } defined as : 299\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$r_1^{{\rm{fp}}}(\omega ) = 2{k_p}\sqrt { { { { \gamma _ 1 } } \over \tau } } { \mathbb l}(\omega)\;\cdot\;\left [ { \begin{array}{*{20}c } { - { { \rm{e}}_s } } \\ { { { \rm{e}}_c } } \\ \end{array } } \right]\quad { \rm{and}}\quad r_2^{{\rm{fp}}}(\omega ) = 2{k_p}\sqrt { { { { \gamma _ 2 } } \over \tau}}{\mathbb l } ( \omega)\;\cdot\;\left [ { \begin{array}{*{20}c } { -{{\rm{e}}_s } } \\ { { { \rm{e}}_c } } \\ \end{array } } \right].$$\end{document } note that due to the fact that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${({\mathbb m}_{{\rm{fp}}}^{(0)})^\dagger } = { ( { \mathbb m}_{{\rm{fp}}}^{(0)})^{- 1}}$\end{document } the reflectivity and the transmission matrices 1,2 and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathbb t}$\end{document } satisfy the following unitarity relations : 300\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\mathbb r}_1}{\mathbb r}_1^{\dagger } + { \mathbb t}{\mathbb t}^{\dagger } = { { \mathbb r}_2}{\mathbb r}_2^{\dagger } + { \mathbb t}{\mathbb t}^{\dagger } = { \mathbb i } \,,\qquad { { \mathbb r}_1}{{\mathbb t}^{\dagger } } + { \mathbb t}{\mathbb r}_2^{\dagger } = 0\,.$$\end{document } the mechanical equations of motion of the fabry - prot cavity mirrors , in spectral representation , are the following : 301\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\rm{f}}\chi _ { xx , i}^{- 1}(\omega){\hat x_i}(\omega ) = { \hat f_i}(\omega ) + { g_i}(\omega)\qquad i = 1,2\,,$$\end{document } where xx , i are the mechanical susceptibilities of the mirrors , gi stand for any external classical forces that could act on the mirrors ( for example , a signal force to be detected ) , 302\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\hat f_i } = { { { { \hat { \mathcal i}}_{{\rm{e}}\,i } } + { { \hat { \mathcal i}}_{{\rm{f}}\,i } } - { { \hat { \mathcal i}}_{{\rm{a}}\,i } } - { { \hat { \mathcal i}}_{{\rm{b}}\,i } } } \over c}$$\end{document } are the radiation pressure forces acting on the mirrors , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat { \mathcal i}_{{\rm{e}}i}},\,{\hat { \mathcal i}_{{\rm{f}}i}},{\hat { \mathcal i}_{{\rm{a}}i}},{\hat { \mathcal i}_{{\rm{b}}i}}$\end{document } are the powers of the waves ei , fi , etc . the signs for all forces are chosen in such a way that the positive forces are oriented outwards from the cavity , increasing the corresponding mirror displacement x1,2 . in the spectral representation , using the quadrature amplitudes notation , the radiation pressure forces read : 303\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { { \hat f}_{1,2}}(\omega ) = { { \hbar { k_p } } \over 2}\left({{\bf{e}}_{1,2}^ \top { { \bf{e}}_{1,2 } } + { \bf{f}}_{1,2}^ \top { { \bf{f}}_{1,2 } } - { \bf{a}}_{1,2}^ \top { { \bf{a}}_{1,2 } } - { \bf{b}}_{1,2}^ \top { { \bf{b}}_{1,2 } } } \right)\quad \quad \quad \quad \quad \quad \quad \quad \quad \quad } \\ { + \hbar { k_p}\left [ { { \bf{e}}_{1,2}^ \top { { \hat e}_{1,2}}(\omega ) + { \bf{f}}_{1,2}^ \top { { \hat f}_{1,2}}(\omega ) - { \bf{a}}_{1,2}^ \top { { \hat a}_{1,2}}(\omega ) - { \bf{b}}_{1,2}^ \top { { \hat b}_{1,2}}(\omega ) } \right ] . } \\ \end{array}$$\end{document } the first group , as we have already seen , describes the regular constant force ; therefore , we omit it henceforth . in the single - mode approximation , the radiation pressure forces acting on both mirrors are equal to each other : 304\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\hat f_{1,2}}(\omega ) \equiv { \hat f_{{\rm{b}}{.}{\rm{a}}{.}}}(\omega ) = 2\hbar { k_p}{{\bf{e}}^ \top}\hat e(\omega)\,,$$\end{document } and the optical field in the cavity is sensitive only to the elongation mechanical mode described by the coordinate x. therefore , combining eqs . ( 301 ) , we obtain for this mode : 305\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\chi _ { xx}^{- 1}(\omega)\hat x(\omega ) = { \hat f_{{\rm{b}}{.}{\rm{a}}{.}}}(\omega ) + g(\omega)\,,$$\end{document } where 306\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\chi _ { xx}}(\omega ) = [ { \chi _ { xx,1}}(\omega ) + { \chi _ { xx,2}}(\omega)]$$\end{document } is the reduced mechanical susceptibility and 307\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g(\omega ) = { { { \chi _ { xx,1}}(\omega){g_1}(\omega ) + { \chi _ { xx,2}}(\omega){g_2}(\omega ) } \over { { \chi _ { xx}}(\omega)}}$$\end{document } is the effective external force . in the simplest and at the same time the most important particular case of free mirrors : 308\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\chi _ { xx , i}}(\omega ) = - { 1 \over { { m_i}{\omega ^2}}}\qquad i = 1,2\,,$$\end{document } the reduced mechanical susceptibility and the effective external force are equal to 309\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\chi _ { xx}}(\omega ) = - { 1 \over { \mu { \omega ^2}}}\,,$$\end{document } and 310\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g(\omega ) = \mu \left [ { { { { g_1}(\omega ) } \over { { m_1 } } } + { { { g_2}(\omega ) } \over { { m_2 } } } } \right],$$\end{document } where 311\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mu = { \left({{1 \over { { m_1 } } } + { 1 \over { { m_2 } } } } \right)^{- 1}}$$\end{document } is the effective mass of the elongation mechanical mode . ( 291 ) and ( 304 ) that the radiation pressure force can be written as a sum of the random and dynamical back - action terms , similarly to the single mirror case : 312\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\hat f_{{\rm{b}}{.}{\rm{a}}{.}}}(\omega ) = \hat f_{{\rm{b}}{.}{\rm{a}}{.}}^{(0)}(\omega ) - k(\omega)\hat x(\omega)\,,$$\end{document } with the random component equal to 313\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat f_{{\rm{b}}{.}{\rm{a}}{.}}^{(0)}(\omega ) = { { 2\hbar { k_p}{{\bf{e}}^ \top } } \over { \sqrt \tau}}{\mathbb l}(\omega)\left [ { \sqrt { { \gamma _ 1 } } { { \hat a}_1}(\omega ) + \sqrt { { \gamma _ 2 } } { { \hat a}_2}(\omega ) } \right]$$\end{document } and the ponderomotive rigidity that reads 314\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$k(\omega ) = { { mj\delta } \over { { \mathcal d}(\omega)}}\,.$$\end{document } we introduced here the normalized optical power 315\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$j = { { 4\hbar k_p^2\vert e\vert ^{2 } } \over { m\tau } } = { { 4{\omega _ p}{{\mathcal i}_c } } \over { mcl}}$$\end{document } with 316\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\mathcal i}_c } = \hbar { \omega _ p}\vert e\vert ^{2}$$\end{document } standing for the mean optical power circulating inside the cavity , and m is some ( in general , arbitrary ) mass . typically , it is convenient to make it equal to the reduced mass . substitution of the force ( 312 ) into eq . ( 305 ) gives the following final equation of motion : 317\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[\chi _ { xx}^{- 1}(\omega ) + k(\omega)]\hat x(\omega ) = \hat f_{{\rm{b}}{.}{\rm{a}}{.}}^{(0)}(\omega ) + g(\omega)\,.$$\end{document } thus , the effective mechanical susceptibility \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal x}_{xx}^{{\rm{eff , fp}}}(\omega)$\end{document } for a fabry - prot cavity reads : 318\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\chi _ { xx}^{{\rm{eff}},\,{\rm{fp}}}(\omega ) = \chi _ { xx}^{- 1}(\omega ) + k(\omega ) = { 1 \over { k(\omega ) - \mu { \omega ^2}}}\,.$$\end{document } in real - life high - precision experiments with mechanical test objects , interferometer schemes that are much more sophisticated than the simple fabry - prot cavity are used . in particular , the best sensitivity in mechanical displacement measurements is achieved by the laser gw detectors . it is this scheme which is planned to be used for the next generation advanced ligo [ 143 , 64 , 137 ] , advanced virgo , and lcgt gw detectors , and its simplified versions are ( or were ) in use in the contemporary first generation detectors : initial ligo [ 3 , 98 ] , virgo , geo 600 [ 168 , 66 ] , and tama [ 8 , 142 ] . the beams , reflected off the fabry - prot cavities are recombined on the beamsplitter in such a way that , in the ideal case of perfect symmetry of the arms , all the light goes back to the laser , i.e. , keeping the signal ( south ) port dark . any imbalance of the interferometer arms , caused by signal forces acting on the end test masses ( etms ) makes part of the pumping light leak into the dark port where it is monitored by a photodetector . the fabry - prot cavities in the arms , formed by the input test masses ( itms ) and the end test masses , provide the increase of the optomechanical coupling , thus making photons bounce many times in the cavity and therefore carry away a proportionally - amplified mirror displacement signal in their phase ( cf . with the f factor in the toy systems considered in section 4 ) . the two auxiliary recycling mirrors : the prm and the signal recycling ( srm ) allow one to increase the power , circulating inside the fabry - prot cavities , for a given laser power , and provide the means for fine - tuning of the quantum noise spectral density [ 103 , 155 ] , respectively . it was shown in that quantum noise of this dual ( power and signal ) recycled interferometer is equivalent to that of a single fabry - prot cavity with some effective parameters ( the analysis in that paper was based on earlier works [ 112 , 128 ] , where the classical regime had been considered ) . here we reproduce this scaling law theorem , extending it in two aspects : ( i ) we factor in optical losses in the arm cavities by virtue of modeling it by the finite transmissivity of the etms , and ( ii ) we do not assume the arm cavities tuned in resonance ( the detuned arm cavities could be used , in particular , to create optical rigidity in non - signal - recycled configurations ) . ( ( 279 ) ) and ( ( 280 ) ) for the arm cavities . the fields referring to the interferometer arms are marked with the subscripts ( northern ) and ( eastern ) following the convention of labeling the gw interferometer parts in accordance with the cardinal directions they are located at with respect to the drawing ( up - direction coincides with north ) . in order to avoid subscripts , we rename some of the field amplitudes as follows : 319\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\rm{a}}_{1\,n , e } } \rightarrow { { \rm{a}}_{n , e}}\,,\qquad { { \rm{b}}_{1\,n , e } } \rightarrow { { \rm{b}}_{n , e}}\,,\qquad { { \rm{a}}_{2\,n , e } } \rightarrow { { \rm{g}}_{n , e}}\,;$$\end{document } see also figure 30 . note that the fields gn , e now describe the noise sources due to optical losses in the arm cavities . ( 279 ) , ( 280 ) , ( 281 ) that are relevant to our consideration , in these notations : 320\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { { \rm{b}}_{n , e } } = { { \mathcal r}_{{\rm{arm}}}}(0){{\rm{a}}_{n , e}}\,,\quad \quad \;\ ; } \\ { { { \rm{e}}_{n , e } } = { 1 \over { { \ell _ { { \rm{arm}}}}(0)}}\sqrt { { { { \gamma _ { 1{\rm{arm } } } } } \over \tau } } { { \rm{a}}_{n , e}}\ , , } \\ \end{array}$$\end{document } 321\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { { \hat { \rm{b}}}_{n , e}}(\omega ) = { { \mathcal r}_{{\rm{arm}}}}(\omega){{\hat { \rm{a}}}_{n , e}}(\omega ) + { { \mathcal t}_{{\rm{arm}}}}(\omega){{\hat { \rm{g}}}_{n , e}}(\omega ) + { { 2\sqrt { { \gamma _ { 1{\rm{arm } } } } } { { \hat x}_{n , e}}(\omega ) } \over { { \ell _ { { \rm{arm}}}}(\omega)}}\ , , } \\ { { { \hat { \rm{e}}}_{n , e}}(\omega ) = { { \sqrt { { \gamma _ { 1{\rm{arm } } } } } { { \hat { \rm{a}}}_{n , e}}(\omega ) + \sqrt { { \gamma _ 2 } } { { \hat { \rm{g}}}_{n , e}}(\omega ) + { { \hat x}_{n , e}}(\omega ) } \over { { \ell _ { { \rm{arm}}}}(\omega)\sqrt \tau}}\,,\quad \quad \quad \quad \;\ ; } \\ \end{array}$$\end{document } 322\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\gamma _ { 1{\rm{arm } } } } = { { { t_{{\rm{arm } } } } } \over { 4\tau}}\,,\qquad { \gamma _ 2 } = { { { a_{{\rm{arm } } } } } \over { 4\tau}}\,,$$\end{document } tarm is the input mirrors power transmittance , aarm is the arm cavities power losses per bounce , 323\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\gamma _ { { \rm{arm } } } } = { \gamma _ { 1{\rm{arm } } } } + { \gamma _ 2}$$\end{document } is the arm cavities half - bandwidth , 324\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\ell _ { { \rm{arm}}}}(\omega ) = { \gamma _ { { \rm{arm } } } } - i({\delta _ { { \rm{arm } } } } + \omega)\,,$$\end{document } arm is the arm cavities detuning , 325\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\mathcal r}_{{\rm{arm}}}}(\omega ) = { { 2{\gamma _ { 1{\rm{arm } } } } } \over { { \ell _ { { \rm{arm}}}}(\omega ) } } - 1\,,\qquad { { \mathcal t}_{{\rm{arm}}}}(\omega ) = { { 2\sqrt { { \gamma _ { 1{\rm{arm}}}}{\gamma _ 2 } } } \over { { \ell _ { { \rm{arm}}}}(\omega)}}$$\end{document } and 326\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\hat x_{n , e } } = { { i{k_p}{{\rm{e}}_{n , e}}{{\hat x}_{n , e}}(\omega ) } \over { \sqrt \tau}}\,.$$\end{document } assume then that the beamsplitter is described by the matrix ( 32 ) , with r = t = 1/2 . let lw = cw be the power recycling cavity length ( the optical distance between the power recycling mirror and the input test masses ) and ls = cs power recycling cavity length ( the optical distance between the signal recycling mirror and the input test masses ) . in this case , the light propagation between the recycling mirrors and the input test masses is described by the following equations for the classical field amplitudes : 327\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { { \rm{a}}_{n , e } } = { { { { \rm{d}}_w}{e^{i{\phi _ w } } } \pm { { \rm{d}}_s}{e^{i{\phi _ s } } } } \over { \sqrt 2}}\ , , } \\ { { { \rm{c}}_{w , s } } = { { { { \rm{b}}_n } \pm { { \rm{b}}_e } } \over { \sqrt 2}}{e^{i{\phi _ { w , s}}}}\ , , } \\ \end{array}$$\end{document } where 328\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\phi _ { w , s } } = { \omega _ p}{\tau _ { w , s}}\,,$$\end{document } are the phase shifts gained by the carrier light with frequency p passing through the power and signal recycling cavities , and the similar equations : 329\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { { \hat{\rm{a}}}_{n , e}}(\omega ) = { { { { \hat{\rm{d}}}_w}(\omega){e^{i\omega { \tau _ w } } } \pm { { \hat{\rm{d}}}_s}(\omega){e^{i\omega { \tau _ s } } } } \over { \sqrt 2}}\ , , } \\ { { { \hat{\rm{c}}}_{w , s}}(\omega ) = { { { { \hat{\rm{b}}}_n}(\omega ) \pm { { \hat{\rm{b}}}_e}(\omega ) } \over { \sqrt 2}}{e^{i\omega { \tau _ { w , s}}}}\quad } \\ the last group of equations that completes our equations set is for the coupling of the light fields at the recycling mirrors : 330\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { { \hat{\rm{b}}}_w } = - \sqrt { { r_w } } { { \hat{\rm{a}}}_w } + \sqrt { { t_{\rm{w } } } } { { \hat{\rm{c}}}_w}\ , , } & { { { \hat{\rm{d}}}_w } = \sqrt { { t_w } } { { \hat{\rm{a}}}_w } + \sqrt { { r_{\rm{w } } } } { { \hat{\rm{c}}}_w}\ , , } \\ { { { \hat{\rm{b}}}_s } = - \sqrt { { r_s } } { { \hat{\rm{a}}}_s } + \sqrt { { t_{\rm{s } } } } { { \hat{\rm{c}}}_s}\,,\quad } & { { { \hat{\rm{d}}}_s } = \sqrt { { t_s } } { { \hat{\rm{a}}}_s } + \sqrt { { r_{\rm{w } } } } { { \hat{\rm{c}}}_s}\,,\ ; } \\ \end{array}$$\end{document } where rw , tw and rs , ts are the reflectivities and transmissivities of the power and signal recycling mirrors , respectively . these equations , being linear and frequency independent , are valid both for the zeroth - order classical amplitudes and for the first - order quantum ones . the striking symmetry of the above equations suggests that the convenient way to describe this system is to decompose all the optical fields in the interferometer arms into the superposition of the symmetric ( common ) and antisymmetric ( differential ) modes , which we shall denote by the subscripts + and , respectively : 331\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\hat{\rm{a } } _ \pm } = { { { { \hat{\rm{a}}}_n } \pm { { \hat{\rm{a}}}_e } } \over { \sqrt 2}}\,,\qquad { \hat{\rm{b } } _ \pm } = { { { { \hat{\rm{b}}}_n } \pm { { \hat{\rm{b}}}_e } } \over { \sqrt 2}}\,,\qquad { \hat{\rm{e } } _ \pm } = { { { { \hat{\rm{e}}}_n } \pm { { \hat{\rm{e}}}_e } } \over { \sqrt 2}}\,,\qquad { \hat{\rm{g } } _ \pm } = { { { { \hat{\rm{g}}}_n } \pm { { \hat{\rm{g}}}_e } } \over { \sqrt 2}}\,.$$\end{document } it follows from eqs . ( 329 ) that the symmetric mode is coupled solely to the western ( bright ) port , while the antisymmetric one couples exclusively to the southern ( dark ) port of the interferometer . it is easy to see that the classical field amplitudes of the antisymmetric mode are equal to zero . for the common mode , combining eqs . ( 320 ) , ( 327 ) , ( 330 ) , ( 331 ) , it is easy to obtain the following set of equation : 332\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { { \rm{b } } _ + } = { { \mathcal r}_{{\rm{arm}}}}(0){{\rm{a } } _ + } \,,\quad \quad \quad \quad } \\ { { { \rm{e } } _ + } = { 1 \over { { \ell _ { { \rm{arm}}}}(0)}}\sqrt { { { { \gamma _ { 1{\rm{arm } } } } } \over \tau } } { { \rm{a } } _ + } \,,\quad } \\ { { { \rm{a } } _ + } = { { \rm{d}}_w}{e^{i{\phi _ w}}}\,,\quad \quad \quad \quad \quad \ ; } \\ { { { \rm{c}}_w } = { { \rm{b } } _ + } { e^{i{\phi _ w}}}\,,\quad \quad \quad \quad \quad \ ; } \\ { { { \rm{b}}_w } = - \sqrt { { r_w } } { { \rm{a}}_w } + \sqrt { { t_w } } { { \rm{c}}_w}\ , , } \\ { { { \rm{d}}_w } = \sqrt { { t_w } } { { \rm{a}}_w } + \sqrt { { r_w } } { { \rm{c}}_w}\,.\quad } \\ \end{array}$$\end{document } its solution is equal to ( only those amplitudes are provided that we shall need below ) : 333a\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\rm{b}}_w } = { { { { \mathcal r}_{{\rm{arm}}}}(0){e^{2i{\phi _ w } } } - \sqrt { { r_w } } } \over { 1 - { { \mathcal r}_{{\rm{arm}}}}(0)\sqrt { { r_w } } { e^{2i{\phi _ w}}}}}\,{{\rm{a}}_w}\,,$$\end{document } 333b\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\rm{e } } _ + } = { 1 \over { { \ell _ { { \rm{arm}}}}(0)}}\sqrt { { { { \gamma _ { 1{\rm{arm } } } } } \over \tau } } { { \sqrt { { t_w } } { e^{i{\phi _ w } } } } \over { 1 - { { \mathcal r}_{{\rm{arm}}}}(0)\sqrt { { r_w } } { e^{2i{\phi _ w}}}}}\,{{\rm{a}}_w}\,.$$\end{document } in the differential mode , the first non - vanishing terms are the first - order quantum - field amplitudes . in this case , using eqs . ( 321 ) , ( 329 ) , ( 330 ) , ( 331 ) , and taking into account that 334\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\rm{e}}_n } = { { \rm{e}}_e } = { { { { \rm{e } } _ + } } \over { \sqrt 2}}\,,$$\end{document } we obtain : 335\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { { \hat{\rm{b } } } _ -}(\omega ) = { { \mathcal r}_{1{\rm{arm}}}}(\omega){{\hat{\rm{a } } } _ -}(\omega ) + { { \mathcal t}_{{\rm{arm}}}}(\omega){{\hat{\rm{g } } } _ -}(\omega ) + { { 2\sqrt { { \gamma _ { 1{\rm{arm } } } } } { { \hat x } _ -}(\omega ) } \over { { \ell _ { { \rm{arm}}}}(\omega)}}\,,}\\ { { { \hat{\rm{e } } } _ -}(\omega ) = { { \sqrt { { \gamma _ { 1{\rm{arm } } } } } { { \hat{\rm{a } } } _ -}(\omega ) + \sqrt { { \gamma _ 2 } } { { \hat{\rm{g } } } _ -}(\omega ) + { { \hat x } _ -}(\omega ) } \over { { \ell _ { { \rm{arm}}}}(\omega)\sqrt \tau}}\,,\quad \quad \quad \quad \quad}\\ \end{array}$$\end{document } 336\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { { \hat{\rm{a } } } _ -}(\omega ) = { { \hat{\rm{d}}}_s}(\omega){e^{i\omega { \tau _ s}}}\,,\quad \quad \quad \quad } \\ { { { \hat{\rm{c}}}_s}(\omega ) = { { \hat{\rm{b } } } _ -}(\omega){e^{i\omega { \tau _ s}}}\,,\quad \quad \quad \quad } \\ { { { \hat{\rm{b}}}_s}(\omega ) = - \sqrt { { r_s } } { { \hat{\rm{a}}}_s } + \sqrt { { t_s } } { { \hat{\rm{c}}}_s}(\omega)\ , , } \\ { { { \hat{\rm{d}}}_s}(\omega ) = \sqrt { { t_s } } { { \hat{\rm{a}}}_s } + \sqrt { { r_s } } { { \hat{\rm{c}}}_s}(\omega)\,,\quad } \\ \end{array}$$\end{document } where 337\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\hat x _ -}(\omega ) = { { i{k_p}{{\rm{e } } _ + } { { \hat x } _ -}(\omega ) } \over { \sqrt \tau}}\,,$$\end{document } 338\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\hat x _ - } = { { { { \hat x}_n } - { { \hat x}_e } } \over 2}\,.$$\end{document } the solution of this equation set is the following : 339\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { { \hat{\rm{b}}}_s}(\omega ) = { { [ { { \mathcal r}_{{\rm{arm}}}}(\omega){e^{2i\omega { \tau _ s } } } - \sqrt { { r_s } } ] { { \hat{\rm{a}}}_s}(\omega ) + \sqrt { { t_s } } { e^{i\omega { \tau _ s}}}\left [ { { { \mathcal t}_{{\rm{arm}}}}(\omega){{\hat{\rm{g } } } _ -}(\omega ) + { { 2\sqrt { { \gamma _ { 1{\rm{arm } } } } } { { \hat x } _ -}(\omega ) } \over { { \ell _ { { \rm{arm}}}}(\omega ) } } } \right ] } \over { 1 - { { \mathcal r}_{{\rm{arm}}}}(\omega)\sqrt { { r_s } } { e^{2i\omega { \tau _ s}}}}}\,,}\\ { { { \hat{\rm{e } } } _ -}(\omega ) = { { \sqrt { { t_s } } { e^{i\omega { \tau _ s}}}\sqrt { { \gamma _ { 1{\rm{arm } } } } } { { \hat{\rm{a}}}_s}(\omega ) + [ 1 + \sqrt { { r_s } } { e^{2i\omega { \tau _ s}}}][\sqrt { { \gamma _ 2 } } { { \hat{\rm{g } } } _ -}(\omega ) + { { \hat x } _ -}(\omega ) ] } \over { { \ell _ { { \rm{arm}}}}(\omega)\sqrt \tau [ 1 - { { \mathcal r}_{{\rm{arm}}}}(\omega)\sqrt { { r_s } } { e^{2i\omega { \tau _ s}}}]}}\,.\quad \quad \quad \quad \quad}\\ \end{array}$$\end{document } eqs . ( 332 ) and ( 333 ) , on the one hand , and ( 335 ) and ( 339 ) , on the other , describe two almost independent optical configurations each consisting of the two coupled fabry - prot cavities as featured in figure 31 . almost independent means that they do not couple in an ordinary linear way ( and , thus , indeed represent two optical modes ) . however , any variation of the differential mechanical coordinate x makes part of the pumping carrier energy stored in the common mode pour into the differential mode , which means a non - linear parametric coupling between these modes . figure 31effective model of the dual - recycled fabry - prot - michelson interferometer , consisting of the common ( a ) and the differential ( b ) modes , coupled only through the mirrors displacements . effective model of the dual - recycled fabry - prot - michelson interferometer , consisting of the common ( a ) and the differential ( b ) modes , coupled only through the mirrors displacements . the mechanical elongation modes of the two fabry - prot cavities are described by the following equations of motion [ see eq . ( 305 ) ] : 340\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$- \mu { \omega ^2}{\hat x_{n , e}}(\omega ) = 2\hbar { k_p}{\bf{e}}_{n , e}^ \top { \hat e_{n , e}}(\omega ) + { { { g_{n , e}}(\omega ) } \over 2}\,,$$\end{document } where = m/2 is the effective mass of these modes and gn , e are the external classical forces acting on the cavities end mirrors . ( 334 ) reads : 341\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$- 2\mu { \omega ^2}{\hat x _ -}(\omega ) = \hat f _ - ^{{\rm{r}}{.}{\rm{p}}{.}}(\omega ) + { { { g _ -}(\omega ) } \over 2}\,,$$\end{document } where 342\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$f _ - ^{{\rm{r}}{.}{\rm{p}}{.}}(\omega ) = 2\hbar { k_p}{\bf{e } } -}(\omega)$$\end{document } is the differential radiation - pressure force and 343\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${g _ - } = { g_n } - { g_e}$$\end{document } is the differential external force . equations ( 339 ) and ( 341 ) together form a complete set of equations describing the differential optomechanical mode of the interferometer featured in figure 31(b ) . ( 341 ) implies that the effective mass of the differential mechanical degree of freedom coincides with the single mirror mass : 344\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$2\mu = m,$$\end{document } which prescribes the mirrors of the effective cavity to be twice as heavy as the real mirrors , i.e. , 2 m . ( 334 ) implies for the effective optical power a value twice as high as the power of light , circulating in the arm cavities : 345\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\mathcal i}_c } = \hbar { \omega _ p}{\rm{e } } _ + ^2 = 2{{\mathcal i}_{{\rm{arm } } } } = 2\hbar { \omega _ p}{\rm{e}}_{n , e}^2\,.$$\end{document } return to eqs . introduce the following notations : 346\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\gamma _ { 1w } } = { \gamma _ { 1{\rm{arm}}}}{\rm re } { { 1 - \sqrt { { r_w } } { e^{2i{\phi _ w } } } } \over { 1 + \sqrt { { r_w } } { e^{2i{\phi _ w } } } } } = { { { \gamma _ { 1{\rm{arm}}}}{t_w } } \over { 1 + 2\sqrt { { r_w } } \cos 2{\phi _ w } + { r_w}}}\,,$$\end{document } 347\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\delta _ w } = { \delta _ { { \rm{arm } } } } - { \gamma _ { 1{\rm{arm}}}}{\rm im}{{1 - \sqrt { { r_w } } { e^{2i{\phi _ w } } } } \over { 1 + \sqrt { { r_w } } { e^{2i{\phi _ w } } } } } = { \delta _ { { \rm{arm } } } } + { { 2{\gamma _ { 1{\rm{arm}}}}\sqrt { { r_w } } \sin 2{\phi _ w } } \over { 1 + 2\sqrt { { r_w } } \cos 2{\phi _ w } + { r_w}}}\,,$$\end{document } 348\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\gamma _ w } = { \gamma _ { 1w } } + { \gamma _ 2}\,,$$\end{document } 349\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\ell _ w}(0 ) = { \gamma _ w } - i{\delta _ w}\,.$$\end{document } in these notation , eqs . ( 333 ) have the following form : 350\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { { \rm{b}}_w } = { { \mathcal r}_w}{{\rm{a}}_w}{e^{2i{\alpha _ w}}}\,,\quad \quad \ ; } \\ { { { \rm{e } } _ + } = { { \sqrt { { \gamma _ { 1w } } } } \over { { \ell _ w}(0)\sqrt \tau}}{{\rm{a}}_w}{e^{i{\alpha _ w}}}\ , , } \\ \end{array}$$\end{document } where 351\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\mathcal r}_w } = { { 2{\gamma _ { 1w } } } \over { { \ell _ w}(0 ) } } - 1\,,$$\end{document } 352\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\alpha _ w } = \arg { { { e^{i\phi w } } } \over { 1 + \sqrt { { r_w } } { e^{2i\phi w}}}}\,.$$\end{document } it is easy to see that these equations have the same form as eqs . ( 279 ) for the single fabry - prot cavity , with the evident replacement of 1 and with the effective parameters 1w and w . thus , we have shown that the power recycling cavity formed by the prm and the itms can be treated as a single mirror with some effective parameters defined implicitly by eqs . note also that the phase shift w can be absorbed into the field amplitudes simply by renaming 353\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\rm{a}}_w}{e^{i\alpha w } } \rightarrow { { \rm{a}}_w}\,,\qquad { { \rm{b}}_w}{e^{- i\alpha w } } \rightarrow { { \rm{b}}_w}\,,$$\end{document } which yields : 354\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { { \rm{b}}_w } = { { \mathcal r}_w}{{\rm{a}}_w}\,,\quad \quad \;\ , } \\ { { { \rm{e } } _ + } = { { \sqrt { { \gamma _ { 1w } } } } \over { { \ell _ w}(0)\sqrt \tau}}{{\rm{a}}_w}\ , . } \\ \end{array}$$\end{document } the corresponding equivalent model of the common mode is shown in figure 32(a ) . taking into account that the main goal of power recycling is the increase of the power \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}_c } = \hbar { \omega _ p}|{e _ + } { |^2}$\end{document } circulating in the arm cavities , for a given laser power \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}_0 } = \hbar { \omega _ p}|{a_w}{|^2}$\end{document } , the optimal tuning of the power recycling cavity corresponds to the critical coupling of the common mode with the laser : 355\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\gamma _ { 1w } } = { \gamma _ 2}\,,\qquad { \delta _ w } = 0\,.$$\end{document } in this case , 356\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\rm{e } } _ + } = { { { { \rm{a}}_w } } \over { 2\sqrt { { \gamma _ 2}\tau } } } \rightarrow { { \mathcal i}_c } = 2{{\mathcal i}_{{\rm{arm } } } } = { { { { \mathcal i}_0 } } \over { 4{\gamma _ 2}\tau}}\,.$$\end{document } note that this regime can be achieved even with the detuned arm cavities , 0 . note a factor e that describes a frequency - dependent phase shift the sideband fields acquire on their pass through the signal recycling cavity . it is due to this frequency - dependent phase shift that the differential mode can not be reduced , strictly speaking , to a single effective cavity mode , and a more complicated two - cavity model of figure 31 should be used instead . the reduction to a single mode is nevertheless possible in the special case of a short signal - recycling cavity , i.e. , such that : 357\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\vert \omega \vert { \tau _ s } \ll 1\,.$$\end{document } the above condition is satisfied in a vast majority of the proposed schemes of advanced gw interferometers and in all current interferometers that make use of the recycling techniques [ 156 , 66 ] . in this case , the phase shift s can be approximated by the frequency - independent value : 358\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\omega { \tau _ s } \approx { \omega _ p}{\tau _ s } \equiv { \phi _ s}\,.$$\end{document } it allows one to introduce the following effective parameters : 359\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { \gamma _ 1 } = { \gamma _ { 1{\rm{arm}}}}{\rm re } { { 1 - \sqrt { { r_s } } { e^{2i{\phi _ s } } } } \over { 1 + \sqrt { { r_s } } { e^{2i{\phi _ s } } } } } = { { { \gamma _ { 1{\rm{arm}}}}{t_s } } \over { 1 + 2\sqrt { { r_s } } \cos 2{\phi _ s } + { r_s}}}\,,\quad \quad \quad \;\ ; } \\ { \delta = { \delta _ { { \rm{arm } } } } - { \gamma _ { 1{\rm{arm}}}}{\rm i m } { { 1 - \sqrt { { r_s } } { e^{2i{\phi _ s } } } } \over { 1 + \sqrt { { r_s } } { e^{2i{\phi _ s } } } } } = { \delta _ { { \rm{arm } } } } + { { 2{\gamma _ { 1{\rm{arm}}}}\sqrt { { r_s } } \sin 2{\phi _ s } } \over { 1 + 2\sqrt { { r_s } } \cos 2{\phi _ s } + { r_s}}}\,,\quad \quad \quad \ , } \\ { \gamma = { \gamma _ 1 } + { \gamma _ 2}\,,\ ; } \\ { \ell ( \omega ) = \gamma - i(\delta + \omega ) } \\ \end{array}$$\end{document } and to rewrite eqs . ( 339 ) as follows : 360\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\hat{\rm{b}}_s}(\omega ) = \left [ { { { \mathcal r}_1}(\omega){{\hat{\rm{a}}}_s}(\omega){e^{i{\alpha _ s } } } + { \mathcal t}(\omega){{\hat{\rm{g } } } _ -}(\omega ) + { { 2\sqrt { { \gamma _ 1 } } { { \hat x } _ -}(\omega ) } \over { \ell ( \omega ) } } } \right]\;{e^{i{\alpha _ s}}}\,,$$\end{document } 361\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\hat{\rm{e } } _ -}(\omega ) = { { \sqrt { { \gamma _ 1 } } { { \hat{\rm{a}}}_s}(\omega){e^{i{\alpha _ s } } } + \sqrt { { \gamma _ 2 } } { { \hat{\rm{g } } } _ -}(\omega ) + { { \hat x } _ -}(\omega ) } \over { \ell ( \omega)\sqrt \tau}}\,,$$\end{document } where the reflectivity and transmittance of the equivalent fabry - prot cavity are still defined by the eqs . ( 285 ) , but with new effective parameters ( 359 ) , and 362\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\alpha _ s } = \arg { { { e^{i{\phi _ s } } } } \over { 1 + \sqrt { { r_s } } { e^{2i{\phi _ s}}}}}$$\end{document } is the phase shift introduced by the signal recycled cavity . along similar lines as in the common mode case , we make the following change of variables 363\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\hat{\rm{a}}_s}{e^{i{\alpha _ s } } } \rightarrow { \hat{\rm{a}}_s}\,,\qquad { \hat{\rm{b}}_s}{e^{- i{\alpha _ s } } } \rightarrow { \hat{\rm{b}}_s}\,.$$\end{document } eqs . ( 360 ) and ( 361 ) have exactly the same form as the corresponding equations for the fabry - prot cavity ( 280 ) . thus , we have successfully built a single cavity model for the differential mode , see figure 32(b ) . figure 32common ( top ) and differential ( bottom ) modes of the dual - recycled fabry - prot - michelson interferometer , reduced to the single cavities using the scaling law model . common ( top ) and differential ( bottom ) modes of the dual - recycled fabry - prot - michelson interferometer , reduced to the single cavities using the scaling law model . the mechanical equations of motion for the effective cavity are absolutely the same as for an ordinary fabry - prot cavity considered in section 5.2 except for the new values of the effective mirrors mass 2 m and effective circulating power \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}_c } = 2{{\mathcal i}_{{\rm{arm}}}}$\end{document}. bearing this in mind , we can procede to the quantum noise spectral density calculation for this interferometer . the scaling law we have derived above enables us to calculate spectral densities of quantum noise for a dual - recycled fabry - prot - michelson featured in figure 30 as if it were a bare fabry - prot cavity with movable mirrors pumped from one side , similar to that shown in figure 32 . we remove some of the subscripts in our notations , for the sake of notational brevity : 364\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { { \hat{\rm{a}}}_s } \rightarrow \hat{\rm{a}}\ , , } & { { { \hat{\rm{b}}}_s } \rightarrow \hat{\rm{b}}\,,\quad \ ; } & { { { \hat{\rm{e } } } _ - } \rightarrow \hat{\rm{e}}\,,\;\ , } & { { { \rm{e } } _ - } \rightarrow { \rm{e}}\,,\quad \ , } \\ { { x _ - } \rightarrow x\ , , } & { f _ - ^{{\rm{r.p . } } } \rightarrow { f_{{\rm{r.p.}}}}\ , , } & { { g _ - } \rightarrow g\ , , } & { f _ - ^{{\rm{b.a . } } } \rightarrow { f_{{\rm{b.a . } } } } , } \\ we also choose the phase of the classical field e amplitude inside the arm cavities to be zero : 365\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\rm im}\;{\rm{e } } = 0 \rightarrow { \bf{e } } = \sqrt 2 { \rm{e}}\;\left [ { \begin{array}{*{20}c } 1 \\ 0 \\ \end{array } } \right]$$\end{document } that obviously does not limit the generality of our consideration , yet sets the reference point for all the classical and quantum fields phases . figure 33the differential mode of the dual - recycled fabry - prot - michelson interferometer in simplified notation ( 364 ) . the differential mode of the dual - recycled fabry - prot - michelson interferometer in simplified notation ( 364 ) . with this in mind , we rewrite i / o - relations ( 360 ) and ( 361 ) in the two - photon quadratures notation : 366\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat{b}(\omega ) = { { \mathbb r}_1}(\omega)\hat a(\omega ) + { \mathbb t}(\omega)\hat g(\omega ) + \sqrt { { { 2m\,j{\gamma _ 1 } } \over \hbar } } \,{{{\bf{d}}(\omega)\hat x(\omega ) } \over { { \mathcal d}(\omega)}},$$\end{document } 367\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat e(\omega ) = { 1 \over { \sqrt \tau}}\left\{{\mathbb l } { ( \omega)[\sqrt { { \gamma _ 1 } } \hat a(\omega ) + \sqrt { { \gamma _ 2 } } \hat g(\omega ) ] + \sqrt { { { m\,j } \over { 2\hbar } } } \,{{{\bf{d}}(\omega)\hat x(\omega ) } \over { { \mathcal d}(\omega ) } } } \right\},$$\end{document } where the matrices \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathbb l}$\end{document } , 1 , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathbb t}$\end{document } are defined by eqs . ( 294 ) and ( 296 ) , 368\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\bf{d}}(\omega ) = { \mathcal d}(\omega){\mathbb l}(\omega)\;\left [ { \begin{array}{*{20}c } 0 \\ 1 \\ \end{array } } \right ] = \left [ { \begin{array}{*{20}c } { - \delta } \\ { \gamma - i\omega } \\ \end{array } } \right]$$\end{document } and 369\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$j = { { 4\hbar k_p^2{{\rm{e}}^2 } } \over { m\tau } } = { { 4{\omega _ p}{{\mathcal i}_c } } \over { mcl } } = { { 8{\omega _ p}{{\mathcal i}_{{\rm{arm } } } } } \over { mcl}}$$\end{document } is the normalized optical power , circulating in the interferometer arms . suppose that the output beam is registered by the homodyne detector ; see section 2.3.1 . ( 366 ) and ( 272 ) , we obtain for the homodyne detector a readout expressed in units of signal force : 370\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\hat o^{f,{\rm{loss}}}}(\omega ) = { { { { \hat{\mathcal x}}^{{\rm{loss } } } } } \over { \chi _ { xx}^{{\rm{eff}},\,{\rm{fp}}}(\omega ) } } + \hat{\mathcal f}(\omega ) + { { g(\omega ) } \over 2}\,,$$\end{document } where 371\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\hat{\mathcal x}^{{\rm{loss}}}}(\omega ) = { { { { \hat o}^{(0),\,{\rm{loss}}}}(\omega ) } \over { { \chi _ { of}}(\omega ) } } = \sqrt { { \hbar \over { 2m\,j{\gamma _ 1 } } } } \,{{{\mathcal d}(\omega ) } \over { { h^{\mathsf t}}[{\phi _ { { \rm{lo}}}}]{\bf{d}}(\omega)}}{h^{\mathsf t}}[{\phi _ { { \rm{lo}}}}][{{\mathbb r}_1}(\omega)\hat a(\omega ) + { \mathbb t}(\omega)\hat g(\omega ) + { \epsilon_d}\hat n(\omega)]$$\end{document } stands for the measurement noise , which is typically referred to as shot noise in optomechanical measurement with the interferometer opto - mechanical response function defined as 372\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\chi _ { of}}(\omega ) = \sqrt { { { 2m\,j{\gamma _ 1 } } \over \hbar } } { { { h^{\mathsf t}}[{\phi _ { { \rm{lo}}}}]{\bf{d}}(\omega ) } \over { { \mathcal d}(\omega)}}$$\end{document } and the back - action noise caused by the radiation pressure fluctuations equal to 373\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat{\mathcal f}(\omega ) \equiv { \hat f_{{\rm{ba}}}}(\omega ) = \sqrt { 2\hbar mj } \;{\left [ { \begin{array}{*{20}c } 1 \\ 0 \\ \end{array } } \right]^{\mathsf t}}{\mathbb l}(\omega)[\sqrt { { \gamma _ 1 } } \hat a(\omega ) + \sqrt { { \gamma _ 2 } } \hat g(\omega)]$$\end{document } the dynamics of the interferometer is described by the effective susceptibility \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal x}_{xx}^{{\rm{eff , fp}}}(\omega)$\end{document } that is the same as the one given by eq . ( 318 ) where 374\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$k(\omega ) = { { m\,j\delta } \over { { \mathcal d}(\omega)}}$$\end{document } is the frequency - dependent optical rigidity that has absolutely the same form as that of a single fabry - prot cavity given by eq . ( 314 ) . suppose then that the input field of the interferometer is in the squeezed quantum state that is equivalent to the following transformation of the input fields : 375\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat a = { { \mathbb s}_{{\rm{sqz}}}}[r,\theta ] { \hat a^{{\rm{vac}}}}\,,$$\end{document } where the squeezing matrix is defined by eq . ( 74 ) , and the quadrature vector corresponds to the vacuum state . using the rules of spectral densities computation given in eqs . ( 89 ) and ( 92 ) , taking into account unitarity conditions ( 300 ) , one can get the following expressions for the power ( double - sided ) spectral densities of the dual - recycled fabry - prot - michelson interferometer measurement and back - action noise sources as well as their cross - correlation spectral density : 376\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { s_{{\mathcal x}{\mathcal x}}}(\omega ) = { \hbar \over { 4m\,j{\gamma _ 1}}}{{\vert { \mathcal d}(\omega){\vert ^2 } } \over { { { \left\vert { { h^{\mathsf t}}[{\phi _ { { \rm{lo}}}}]{\bf{d}}(\omega ) } \right\vert}^2}}}\quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad } \\ { \times \,\{{h^{\mathsf t}}({\phi _ { { \rm{lo}}}}){{\mathbb r}_1}(\omega)[{{\mathbb s}_{{\rm{sqz}}}}(2r,\theta ) - { \mathbb i}]{\mathbb r}_1^\dagger ( \omega)h[{\phi _ { { \rm{lo } } } } ] + 1 + \epsilon_d^2\ } , } \\ \end{array}$$\end{document } 377\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s_{{\mathcal f}{\mathcal f}}}(\omega ) = \hbar m\,j\;\,{\left [ { \begin{array}{*{20}c } 1 \\ 0 \\ \end{array } } \right]^{\mathsf t}}{\mathbb l}(\omega)[{\gamma _ 1}{{\mathbb s}_{{\rm{sqz}}}}(2r,\theta ) + { \gamma _ 2}]{{\mathbb l}^\dagger}(\omega)\,\;\left [ { \begin{array}{*{20}c } 1 \\ 0 \\ \end{array } } \right]\;\,,$$\end{document } 378\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s_{{\mathcal x}{\mathcal f}}}(\omega ) = { \hbar \over 2}{{{\mathcal d}(\omega ) } \over { { h^{\mathsf t}}[{\phi _ { { \rm{lo}}}}]{\bf{d}}(\omega)}}{h^{\mathsf t}}({\phi _ { { \rm{lo}}}})[{{\mathbb r}_1}(\omega){{\mathbb s}_{{\rm{sqz}}}}(2r,\theta ) + \sqrt { { \gamma _ 2}/{\gamma _ 1 } } { \mathbb t}(\omega)]{{\mathbb l}^\dagger}(\omega)\;\,\left [ { \begin{array}{*{20}c } 1 \\ 0 \\ \end{array } } \right]\;\,.$$\end{document } these spectral densities satisfy the schrdinger - robertson uncertainty relation : 379\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s_{{\mathcal x}{\mathcal x}}}(\omega){s_{{\mathcal f}{\mathcal f}}}(\omega)\ ; - \vert { s_{{\mathcal x}{\mathcal f}}}(\omega){\vert ^2 } \geq { { { \hbar ^2 } } \over 4}$$\end{document } of the same form as in the general linear measurement case considered in section 4.2 , see eq . ( 148 ) , with the exact equality in the ideal lossless case : 380\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\gamma _ 2 } = 0\,,\qquad { \eta _ d } = 1\,.$$\end{document } see appendix a.2 in order to compute the sum quantum noise spectral density one has to first calculate s( ) , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${s_{{\mathcal f}{\mathcal f}}}(\omega)$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${s_{{\mathcal x}{\mathcal f}}}(\omega)$\end{document } using eqs . ( 376 ) , ( 377 ) , and ( 378 ) and then insert them into the general formula ( 144 ) . however , there is another option that is more convenient from the computational point of view . one can compute the full quantum noise transfer matrix of the fabry - prot - michelson interferometer in the same manner as for a single mirror in section 5.1.4 . write down the readout observable of the homodyne detector in units of signal force : 381\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { { \hat o}^f}(\omega ) = { { { { \hat{\mathcal x}}^{{\rm{loss}}}}(\omega ) } \over { \chi _ { xx}^{{\rm{eff}},\,{\rm{fp}}}(\omega ) } } + \hat{\mathcal f}(\omega ) + { { g(\omega ) } \over 2 } = { { g(\omega ) } \over 2 } + \sqrt { { \hbar \over { 2m\,j{\gamma _ 1 } } } } { { - m{\omega ^2 } } \over { { h^{\mathsf t}}[{\phi _ { { \rm{lo}}}}]{\bf{d}}(\omega)}}{h^{\mathsf t}}[{\phi _ { { \rm{lo}}}}]\quad \quad \quad \quad \quad \quad \quad \;\ ; } \\ { \times \left\{{{{\mathbb c}_1}(\omega){{\mathbb s}_{{\rm{sqz}}}}[r,\theta ] { { \hat a}^{{\rm{vac}}}}(\omega ) + { { \mathbb c}_2}(\omega)\hat g(\omega ) + \left [ { { \mathcal d}(\omega ) - { { j\delta } \over { { \omega ^2 } } } } \right]\;\,{\epsilon_d}\hat n(\omega ) } \right\}\ ; , } \\ \end{array}$$\end{document } where matrices 1,2( ) can be computed using the fact that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[\delta + { \bf{d}}(\omega)\;\,{\left [ { \begin{array}{*{20}c } 1 \\ 0 \\ \end{array } } \right]^{\mathsf t}}]{\mathbb l}(\omega ) = \left [ { \begin{array}{*{20}c } 0 & 0 \\ 1 & 0 \\ \end{array } } \right]\;\,,$$\end{document } which yields : 382\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\mathbb c}_1}(\omega ) = \left [ { \begin{array}{*{20}c } { 2{\gamma _ 1}(\gamma - i\omega ) - { \mathcal d}(\omega ) + j\delta /{\omega ^2 } } & { - 2{\gamma _ 1}\delta } \\ { 2{\gamma _ 1}\delta - 2j{\gamma _ 1}/{\omega ^2 } } & { 2{\gamma _ 1}(\gamma - i\omega ) - { \mathcal d}(\omega ) + j\delta /{\omega ^2 } } \\ \end{array } } \right]\;\,,$$\end{document } 383\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\mathbb c}_2}(\omega ) = 2\sqrt { { \gamma _ 1}{\gamma _ 2 } } \;\left [ { \begin{array}{*{20}c } { \gamma - i\omega } & { - \delta } \\ { \delta - j/{\omega ^2 } } & { \gamma - i\omega } \\ \end{array } } \right]\;\,.$$\end{document } in the gw community , it is more common to normalize the signal of the interferometer in units of gw amplitude spectrum h( ) . ( 138 ) and taking into account that in our case g( ) g()/2 : 384\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { { \hat o}^h}(\omega ) = { h_{{\rm{gw}}}}(\omega ) + { { \hat h}_n}(\omega ) = { h_{{\rm{gw}}}}(\omega ) + { 2 \over l}\sqrt { { \hbar \over { 2m\,j{\gamma _ 1 } } } } { 1 \over { { h^{\mathsf t}}[{\phi _ { { \rm{lo}}}}]{\bf{d}}(\omega)}}{h^{\mathsf t}}[{\phi _ { { \rm{lo}}}}]\quad \quad \quad \quad \quad \quad } \\ { \times \left\{{{{\mathbb c}_1}(\omega){{\mathbb s}_{{\rm{sqz}}}}[r,\theta ] { { \hat a}^{{\rm{vac}}}}(\omega ) + { { \mathbb c}_2}(\omega)\hat g(\omega ) + \;\,\left [ { { \mathcal d}(\omega ) - { { j\delta } \over { { \omega ^2 } } } } \right]{\epsilon_d}\hat n(\omega ) } \right\}\ ; , } \\ \end{array}$$\end{document } where hgw( ) is the spectrum of the gw signal and the second term n( ) stands for the sum quantum noise expressed in terms of metrics variation spectrum units , i.e. , in hz . the power ( double - sided ) spectral density of the sum quantum noise then reads : 385\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { s^h}(\omega ) = { { 4{s^f}(\omega ) } \over { { m^2}{l^2}{\omega ^4 } } } = { \hbar \over { m\,j{\gamma _ 1}{l^2}}}{1 \over { \vert { h^{\mathsf t}}[{\phi _ { { \rm{lo}}}}]{\bf{d}}(\omega){\vert ^2 } } } } \\ { \times \left\{{{h^{\mathsf t}}[{\phi _ { { \rm{lo}}}}]\;[{{\mathbb c}_1}(\omega){{\mathbb s}_{{\rm{sqz}}}}[2r,\theta ] { \mathbb c}_1^\dagger ( \omega ) + { { \mathbb c}_2}(\omega){\mathbb c}_2^\dagger ( \omega)]h[{\phi _ { { \rm{lo } } } } ] + { { \left\vert { { \mathcal d}(\omega ) - { { j\delta } \over { { \omega ^2 } } } } \right\vert}^2}\epsilon_d^2 } \right\}\;. } \\ \end{array}$$\end{document } in conclusion , we should say that the quantum noise of the fabry - pichelson interferometer has been calculated in many papers , starting from the seminal work by kimble et al . where a resonance - tuned case with = 0 was analyzed , and then by buonanno and chen in [ 32 , 34 ] , who considered a more general detuned case . thus , treading their steps , we have shown that the quantum noise of the fabry - prot - michelson interferometer ( as well as the single cavity fabry - prot one ) has the following distinctive features : it comprises two effective noise sources as in any quantum linear measurement device . these are measurement noise \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat { \mathcal x}^{{\rm{loss}}}}$\end{document } , more frequently called quantum shot noise in the gw community , and the back - action noise \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat { \mathcal f}$\end{document } , often referred to as quantum radiation - pressure noise.these noise sources are correlated and this correlation depends not only on the homodyne angle lo or the correlations in the input light ( e.g. , squeezing angle in case of squeezed input ) , but also on the interferometer effective detuning , which , according to the scaling law theorem , can be changed by varying signal - recycling cavity parameters.the scaling law theorem also shows that changing the arm cavities detuning is equivalent to the modification of the signal recycling cavity parameters in terms of effective detuning and bandwidth of the interferometer.another important corollary of the scaling law is that the effective bandwidths and detunings for the common and differential optical modes can be chosen independently , thus making it possible to tune the former in resonance with the pumping laser to keep as high a value of the circulating optical power in the arms as possible , and to detune the latter one to modify the test masses dynamical response by virtue of the introduction of optical rigidity that arises in the detuned cavity as we have shown . these are measurement noise \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat { \mathcal x}^{{\rm{loss}}}}$\end{document } , more frequently called quantum shot noise in the gw community , and the back - action noise \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat { \mathcal f}$\end{document } , often referred to as quantum radiation - pressure noise . these noise sources are correlated and this correlation depends not only on the homodyne angle lo or the correlations in the input light ( e.g. , squeezing angle in case of squeezed input ) , but also on the interferometer effective detuning , which , according to the scaling law theorem , can be changed by varying signal - recycling cavity parameters . the scaling law theorem also shows that changing the arm cavities detuning is equivalent to the modification of the signal recycling cavity parameters in terms of effective detuning and bandwidth of the interferometer . another important corollary of the scaling law is that the effective bandwidths and detunings for the common and differential optical modes can be chosen independently , thus making it possible to tune the former in resonance with the pumping laser to keep as high a value of the circulating optical power in the arms as possible , and to detune the latter one to modify the test masses dynamical response by virtue of the introduction of optical rigidity that arises in the detuned cavity as we have shown . all of these features can be used to decrease the quantum noise of the interferometer and reach a sensitivity below the sql in a decent range of frequencies as we show in section 6 . in this section , we consider the interferometer configurations that use the idea of the cross - correlation of the shot and the radiation pressure noise sources discussed in section 4.4 . this cross - correlation allows the measurement and the back - action noise to partially cancel each other out and thus effectively reduce the sum quantum noise to below the sql . as we noted above , eq . ( 378 ) tells us that this cross - correlation can be created by tuning either the homodyne angle lo , the squeezing angle , or the detuning . in section 4.4 , the simplest particular case of the frequency - independent correlation created by means of measurement of linear combination of the phase and amplitude quadratures , that is , by using the homodyne angle lo /2 , has been considered . we were able to obtain a narrow - band sensitivity gain at some given frequency that was similar to the one achievable by introducing a constant rigidity to the system , therefore such correlation was called effective rigidity . however , the broadband gain requires a frequency - dependent correlation , as it was first demonstrated for optical interferometric position meters , and then for general position measurement case . later , this idea was developed in different contexts by several authors [ 81 , 118 , 159 , 90 , 70 , 69 , 149 , 9 ] . in particular , in , a practical method of creation of the frequency - dependent correlation was proposed , based on the use of additional filter cavities , which were proposed to be placed either between the squeeze light source and the main interferometer , creating the frequency - dependent squeezing angle ( called pre - filtering ) , or between the main interferometer and the homodyne detector , creating the effective frequency - dependent squeezing angle ( post - filtering ) . as we show below , in principle , both pre- and post - filtering can be used together , providing some additional sensitivity gain . it is necessary to note also an interesting method of noise cancellation proposed by tsang and caves recently . the idea was to use matched squeezing ; that is , to place an additional cavity inside the main interferometer and couple the light inside this additional cavity with the differential mode of the interferometer by means of an optical parametric amplifier ( opa ) . the squeezed light created by the opa should compensate for the ponderomotive squeezing created by back - action at all frequencies and thus decrease the quantum noise below the sql at a very broad frequency band . however , the thorough analysis of the optical losses influence , that as we show later , are ruinous for the subtle quantum correlations this scheme is based on , was not performed . coming back to the filter - cavities - based interferometer topologies , we limit ourselves here by the case of the resonance - tuned interferometer , = 0 . this assumption simplifies all the equations considerably , and allows one to clearly separate the sensitivity gain provided by the quantum noise cancellation due to cross - correlation from the one provided by the optical rigidity , which will be considered in section 6.3 . we also neglect optical losses inside the interferometer , assuming that 2 = 0 . in broadband interferometer configurations considered here , with typical values of 10 s , the influence of these losses is negligible compared to those of the photodetector inefficiency and the losses in the filter cavities . indeed , taking into account the fact that with modern high - reflectivity mirrors , the losses per bounce do not exceed aarm 10 , and the arms lengths of the large - scale gw detectors are equal to several kilometers , the values of 2 1 s , and , correspondingly , 2/ 10 , are feasible . at the same time , the value of photodetector quantum inefficiency \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\epsilon _ d^2 \approx 1 - { \eta _ d } \approx 0.05$\end{document } ( factoring in the losses in the interferometer output optical elements as well ) is considered quite optimistic . note , however , that in narrow - band regimes considered in section 6.3 , the bandwidth can be much smaller and influence of 2 could be significant ; therefore , we take these losses into account in section 6.3 . using these assumptions , the quantum noises power ( double - sided ) spectral densities ( 376 ) , ( 377 ) and ( 378 ) can be rewritten in the following explicit form : 386\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s_{{\mathcal x}{\mathcal x}}}(\omega ) = { \hbar \over { 2m{\omega ^2}{\mathcal k}(\omega){{\sin}^2}{\phi _ { { \rm{lo}}}}}}[\cosh 2r + \sinh 2r\cos 2(\theta - { \phi _ { { \rm{lo } } } } ) + \epsilon_d^2],$$\end{document } 387\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s_{{\mathcal f}{\mathcal f}}}(\omega ) = { { \hbar m{\omega ^2}{\mathcal k}(\omega ) } \over 2}(\cosh 2r + \sinh 2r\;\cos 2\theta),$$\end{document } 388\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s_{{\mathcal x}{\mathcal f}}}(\omega ) = { \hbar \over { 2\sin { \phi _ { { \rm{lo}}}}}}[\cosh 2r\cos { \phi _ { { \rm{lo } } } } + \sinh 2r\cos ( 2\theta - { \phi _ { { \rm{lo}}}})],$$\end{document } where 389\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal k}(\omega ) = { { 2j\gamma } \over { { \omega ^2}({\gamma ^2 } + { \omega ^2})}}$$\end{document } is the convenient optomechanical coupling factor introduced in . ( 381 ) and ( 385 ) for the sum quantum noise and its power ( double - sided ) spectral density in this case can also be simplified significantly : 390\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\hat h_n}(\omega ) = { 2 \over l}\sqrt { { \hbar \over { 2m\,j\gamma } } } \,{1 \over { \sin { \phi _ { { \rm{lo}}}}}}{h^{\mathsf t}}[{\phi _ { { \rm{lo}}}}]\;\left [ { ( \gamma + i\omega)\,{\mathbb k}(\omega){{\mathbb s}_{{\rm{sqz}}}}[r,\theta ] { { \hat a}^{{\rm{vac}}}}(\omega ) + ( \gamma - i\omega){\epsilon_d}\hat n(\omega ) } \right],$$\end{document } 391\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s^h}(\omega ) = { { 2\hbar } \over { m\,{l^2}{\omega ^2}{\mathcal k}(\omega){{\sin}^2}{\phi _ { { \rm{lo}}}}}}\left [ { { h^{\mathsf t}}[{\phi _ { { \rm{lo}}}}]\,{\mathbb k}\,(\omega){{\mathbb s}_{{\rm{sqz}}}}[2r,\theta ] { { \mathbb k}^\dagger}(\omega)\,h\,[{\phi _ { { \rm{lo } } } } ] + \epsilon_d^2 } \right]\;,$$\end{document } where 392\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathbb k}(\omega ) = \left [ { \begin{array}{*{20}c } 1 & 0 \\ { - { \mathcal k}(\omega ) } & 1 \\ \end{array } } \right]\;\,.$$\end{document } in section 6.1.2 we consider the optimization of the spectral density ( 391 ) , assuming that the arbitrary frequency dependence of the homodyne and/or squeezing angles can be implemented . as we see below , this case corresponds to the ideal lossless filter cavities . in section 6.1.3 , we consider two realistic schemes , taking into account the losses in the filter cavities . classical optimization . as a reference point , consider first the simplest case of frequency independent homodyne and squeezing angles . we choose the specific values of these parameters following the classical optimization , which minimizes the shot noise ( 386 ) without taking into account the back action . because , the shot noise dominates at high frequencies , therefore , this optimization gives a smooth broadband shape of the sum noise spectral density . it is evident that this minimum is provided by 393\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\phi _ { { \rm{lo } } } } = { \pi \over 2}\,,\qquad \theta = 0\,.$$\end{document } in this case , the sum quantum noise power ( double - sided ) spectral density is equal to 394\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s^h}(\omega ) = { { 2\hbar } \over { m\,{l^2}{\omega ^2}}}\;\left [ { { { { e^{- 2r } } + \epsilon_d^2 } \over { { \mathcal k}(\omega ) } } + { \mathcal k}(\omega){e^{2r } } } \right]\;.$$\end{document } it is easy to note the similarity of this spectral density with the ones of the toy position meter considered above , see eq . ( 173 ) . the only significant differences introduced here are the optical losses and the decrease of the optomechanical coupling at high frequencies due to the finite bandwidth of the interferometer . ( 173 ) and ( 394 ) become identical , with the evident correspondence 395\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\mathcal i}_0}{\digamma ^2 } \leftrightarrow { { { { \mathcal i}_c } } \over { \gamma \tau}}\,.$$\end{document } in particular , the spectral density ( 394 ) can never be smaller than the free mass sql \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$s_{{\rm{sql}}\,{\rm{f}}{\rm{.m}}.}^h(\omega)$\end{document } ( see ( 174 ) ) . indeed , it can be minimized at any given frequency by setting 396\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal k}(\omega ) = { e^{- r}}\sqrt { { e^{- 2r } } + \epsilon_d^2 } \,,$$\end{document } and in this case , 397\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\xi ^2}(\omega ) \equiv { { { s^h}(\omega ) } \over { s_{{\rm{sql}}\,{\rm{fm}}}^h(\omega ) } } = \sqrt { 1 + \epsilon_d^2{e^{2r } } } \geq 1\,.$$\end{document } the spectral density ( 394 ) was first calculated in the pioneering work of , where the existence of two kinds of quantum noise in optical interferometric devices , namely the measurement ( shot ) noise and the back action ( radiation pressure ) noise , were identified for the first time , and it was shown that the injection of squeezed light with = 0 into the interferometer dark port is equivalent to the increase of the optical pumping power . however , it should be noted that in the presence of optical losses this equivalence holds unless squeezing is not too strong , e > d . the noise spectral density curves for the resonance - tuned interferometer are drawn in figure 34 . the default parameters for this and all subsequent similar plots are chosen to be close to those planned for the advanced ligo interferometer : the value of j = jaligo ( 2 100 ) s corresponds to the circulating power of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}_{{\rm{arm } } } } = 840\,{\rm{kw,}}\,l = 4{\rm{km}}$\end{document } , and m = 40 kg ; the interferometer bandwidth = 2 500 s is close to the one providing the best sensitivity for advanced ligo in the presence of technical noise ; 10 db squeezing ( e = 10 ) , which corresponds to the best squeezing available at the moment ( 2011 ) in the low - frequency band [ 102 , 150 , 151 ] ) ; d = 0.95 can be considered a reasonably optimistic estimate for the real interferometer quantum efficiency . figure 34examples of the sum quantum noise spectral densities of the classically - optimized ( lo = /2 , = 0 ) resonance - tuned interferometer . increased power : j = 10jaligo , no squeezing . squeezed : j = jaligo , 10 db squeezing . examples of the sum quantum noise spectral densities of the classically - optimized ( lo = /2 , = 0 ) resonance - tuned interferometer . noteworthy is the proximity of the plots for the interferometer with 10 db input squeezing and the one with 10-fold increased optical power . now suppose that the homodyne angle can be frequency dependent , and calculate the corresponding minimum of the sum noise spectral density ( 391 ) . the first term in square brackets in this equation can be rewritten as : 398\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${v^{\mathsf t}}{\mathbb p}[\theta ] { { \mathbb s}_{{\rm{sqz}}}}[2r,0]{{\mathbb p}^\dagger}[\theta ] v = { e^{2r}}{({v_c}\cos \theta + { v_s}\sin \theta)^2 } + { e^{- 2r}}{(- { v_c}\sin \theta + { v_s}\cos \theta)^2}\,,$$\end{document } where 399\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\bf{v } } \equiv \left [ { \begin{array}{*{20}c } { { v_c } } \\ { { v_s } } \\ \end{array } } \right ] = { { \mathbb k}^\dagger}(\omega)h[{\phi _ { { \rm{lo}}}}]\,.$$\end{document } it is evident that the minimum of ( 398 ) is provided by 400\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tan \theta = - { { { v_c } } \over { { v_s } } } = - \cot { \phi _ { { \rm{lo } } } } + { \mathcal k}(\omega)$$\end{document } and is equal to vve . therefore , 401\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { s^h}(\omega ) = { { 2\hbar } \over { m\,{l^2}{\omega ^2}{\mathcal k}(\omega){{\sin}^2}{\phi _ { { \rm{lo}}}}}}\left [ { { h^{\mathsf t}}[{\phi _ { { \rm{lo}}}}]{\mathbb k}(\omega){{\mathbb k}^\dagger}(\omega)h[{\phi _ { { \rm{lo}}}}]{e^{- 2r } } + \epsilon_d^2 } \right]\quad \quad \quad \quad \quad \quad } \\ { = { { 2\hbar } \over { m\,{l^2}{\omega ^2}}}\left\{{\left [ { { 1 \over { { \mathcal k}(\omega){{\sin}^2}{\phi _ { { \rm{lo } } } } } } - 2\cot { \phi _ { { \rm{lo } } } } + { \mathcal k}(\omega ) } \right]{e^{- 2r } } + { { \epsilon_d^2 } \over { { \mathcal k}(\omega){{\sin}^2}{\phi _ { { \rm{lo } } } } } } } \right\}\;\ , . } \\ \end{array}$$\end{document } thus , we obtaine a well - known result that , using an optimal squeezing angle , the quantum noise spectral density can be reduced by the squeezing factor e in comparison with the vacuum input case . concerning the homodyne angle lo , we use again the classical optimization , setting 402\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\phi _ { { \rm{lo } } } } = { \pi \over 2}\,.$$\end{document } in this case , the sum noise power ( double - sided ) spectral density and the optimal squeezing angle are equal to 403\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s^h}(\omega ) = { { 2\hbar } \over { m\,{l^2}{\omega ^2}}}\;\left [ { { { { e^{- 2r } } + \epsilon_d^2 } \over { { \mathcal k}(\omega ) } } + { \mathcal k}(\omega){e^{- 2r } } } \right]$$\end{document } and 404\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tan \theta = { \mathcal k}(\omega)\,.$$\end{document } the sum quantum noise power ( double - sided ) spectral density ( 403 ) is plotted in figure 35 for the ideal lossless case and for d = 0.95 ( dotted line ) . in both cases , the optical power and the squeezing factor are equal to j = jaligo and e = 10 , respectively . figure 35examples of the sum quantum noise power ( double - sided ) spectral densities of the resonance - tuned interferometers with frequency - dependent squeezing and/or homodyne angles . left : no optical losses , right : with optical losses , d = 0.95 . squeezed : 10 db squeezing , = 0 , lo = /2 ( these two plots are provided for comparison ) . ( 403 ) ] : 10 db squeezing , lo = /2 , frequency - dependent squeezing angle . dashes [ post - filtering , eq . ( 408 ) ] : 10 db squeezing , = 0 , frequency - dependent homodyne angle . ( 410 ) ] : 10 db squeezing , frequency - dependent squeeze and homodyne angles . for all plots , j = jaligo and = 2 500 s. examples of the sum quantum noise power ( double - sided ) spectral densities of the resonance - tuned interferometers with frequency - dependent squeezing and/or homodyne angles . left : no optical losses , right : with optical losses , d = 0.95 . ordinary : no squeezing , lo = /2 . squeezed : 10 db squeezing , = 0 , lo = /2 ( these two plots are provided for comparison ) . ( 403 ) ] : 10 db squeezing , lo = /2 , frequency - dependent squeezing angle . dashes [ post - filtering , eq . ( 408 ) ] : 10 db squeezing , = 0 , frequency - dependent homodyne angle . ( 410 ) ] : 10 db squeezing , frequency - dependent squeeze and homodyne angles . for all plots , suppose now that the squeezing angle corresponds to the classical optimization : 405\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\theta = 0$$\end{document } and minimize the resulting sum noise spectral density : 406\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s^h}(\omega ) = { { 2\hbar } \over { m\,{l^2}{\omega ^2}}}\;\left [ { { { \cosh 2r + \sinh 2r\cos 2{\phi _ { { \rm{lo } } } } + \epsilon_d^2 } \over { { \mathcal k}(\omega){{\sin}^2}{\phi _ { { \rm{lo } } } } } } - 2{e^{2r}}\cot { \phi _ { { \rm{lo } } } } + { \mathcal k}(\omega){e^{2r } } } \right]$$\end{document } with respect to lo . the minimum is provided by the following dependence 407\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\cot { \phi _ { { \rm{lo } } } } = { { { \mathcal k}(\omega ) } \over { 1 + \epsilon_d^2{e^{- 2r}}}}\,,$$\end{document } and is equal to 408\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s^h}(\omega ) = { { 2\hbar } \over { m\,{l^2}{\omega ^2}}}\;\left [ { { { { e^{- 2r } } + \epsilon_d^2 } \over { { \mathcal k}(\omega ) } } + { { \epsilon_d^2 } \over { 1 + \epsilon_d^2{e^{- 2r}}}}\,{\mathcal k}(\omega ) } \right]\;.$$\end{document } the sum quantum noise spectral density ( 408 ) is plotted in figure 35 for the ideal lossless case and for d = 0.95 ( dashed lines ) . compare this spectral density with the one for the frequency - dependent squeezing angle ( pre - filtering ) case , see eq . the shot noise components in both cases are exactly equal to each other . concerning the residual back - action noise , in the pre - filtering case it is limited by the available squeezing , while in the post - filtering case , were there no optical losses , the back - action noise could be removed completely , as shown in figure 35 ( left ) . for the parameters of the noise curves presented in figure 35 ( right ) , the post - filtering still has some advantage of about 40% in the back - action noise amplitude \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\sqrt s$\end{document}. note that the required frequency dependences ( 404 ) and ( 407 ) in both cases are similar to each other ( and become exactly equal to each other in the lossless case d = 0 ) . therefore , similar setups can be used in both cases in order to create the necessary frequency dependences with about the same implementation cost . from this simple consideration , it is possible to conclude that pre - filtering is preferable if good squeezing is available , and the optical losses are relatively large , and vice versa . in particular , post - filtering can be used even without squeezing , r = 0 . finally , consider the most sophisticated configuration : double - filtering with both the homodyne angle lo and the squeezing angle being frequency dependent . concerning the squeezing angle , we can reuse eqs . the minimum of the spectral density ( 401 ) in lo corresponds to 409\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\cot { \phi _ { { \rm{lo } } } } = { { { \mathcal k}(\omega ) } \over { 1 + \epsilon_d^2{e^{2r}}}}\,,$$\end{document } and is equal to 410\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s^h}(\omega ) = { { 2\hbar } \over { m\,{l^2}{\omega ^2}}}\;\left [ { { { { e^{- 2r } } + \epsilon_d^2 } \over { { \mathcal k}(\omega ) } } + { { \epsilon_d^2 } \over { 1 + \epsilon_d^2{e^{2r}}}}\,{\mathcal k}(\omega ) } \right]\;.$$\end{document } it also follows from eqs.(400 ) and ( 409 ) that the optimal squeezing angle in this case is given by 411\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tan \theta = { { \epsilon_d^2 } \over { { e^{- 2r } } + \epsilon_d^2}}\,{\mathcal k}(\omega)\,.$$\end{document } it is easy to see that in the ideal lossless case the double - filtering configuration reduces to a post - filtering one . really , if d = 0 , the spectral density ( 410 ) becomes exactly equal to that for the post - filtering case ( 408 ) , and the frequency dependent squeezing angle ( 411 ) degenerates into a constant value ( 405 ) . however , if d > 0 , then the additional pre - filtering allows one to decrease more the residual back - action term . for example , if e = 10 and d = 0.95 then the gain in the back - action noise amplitude \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\sqrt s$\end{document } is equal to about 25% . we have plotted the sum quantum noise spectral density ( 410 ) in figure 34 , right ( dash - dots ) . this plot demonstrates the best sensitivity gain of about 3 in signal amplitude , which can be provided employing squeezing and filter cavities at the contemporary technological level . due to the presence of the residual back - action term in the spectral density ( 410 ) , there exists an optimal value of the coupling factor \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal k}(\omega)$\end{document } ( that is , the optical power ) which provides the minimum to the sum quantum noise spectral density at any given frequency 412\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal k}(\omega ) = { 1 \over { { \epsilon _ d}{e^r } } } + { \epsilon _ d}{e^r}\,,$$\end{document } the minimum is equal to 413\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$s_{{\rm{min}}}^h the reasonably optimistic value of quantum efficiency d = 0.95 that we use for our estimates corresponds to d 0.23 . it means that without squeezing ( r = 0 ) one is only able to beat the sql in amplitude by 414\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\xi _ { { \rm{min } } } } \equiv \sqrt { { { s_{{\rm{min}}}^h } \over { s_{{\rm{sql}}}^h } } } = \sqrt { { \epsilon _ d } } \approx 0.5\,.$$\end{document } the gain can be improved using squeezing and , if r then , in principle , arbitrarily high sensitivity can be reached . but depends on r only as e , and for the 10 db squeezing , only a modest value of 415\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\xi _ { { \rm{min } } } } \approx 0.27$$\end{document } can be obtained . in our particular case , the fact that the additional noise associated with the photodetector quantum inefficiency d > 0 does not correlate with the quantum fluctuations of the light in the interferometer gives rise to this limit . this effect is universal for any kind of optical loss in the system , impairing the cross - correlation of the measurement and back - action noises and thus limiting the performance of the quantum measurement schemes , which rely on this cross - correlation . ( 410 ) does not take into account optical losses in the filter cavities . as we shall see below , the sensitivity degradation thereby depends on the ratio of the light absorption per bounce to the filter cavities length , af / lf . therefore , this method calls for long filter cavities . in particular , in the original paper , filter cavities with the same length as the main interferometer arm cavities ( 4 km ) , placed side by side with them in the same vacuum tubes , were proposed . for such long and expensive filter cavities , the influence of their losses indeed can be small . however , as we show below , in section 6.1.3 , for the more practical short ( up to tens of meters ) filter cavities , optical losses thereof could be the main limiting factor in terms of sensitivity . the optimization performed above can be viewed also in a different way , namely , as the minimization of the sum quantum noise spectral density of an ordinary interferometer with frequency - independent homodyne and squeezing angles , yet at some given frequency 0 . in section 4.4 , this kind of optimization was considered for a simple lossless system . it was shown capable of the narrow - band gain in sensitivity , similar to the one provided by the harmonic oscillator ( thus the term virtual rigidity ) . this narrow - band gain could be more interesting not for the full - scale gw detectors ( where broadband optimization of the sensitivity is required in most cases ) but for smaller devices like the 10-m hannover prototype interferometer , designed for the development of the measurement methods with sub - sql sensitivity . due to shorter arm length , the bandwidth in those devices is typically much larger than the mechanical frequencies . if one takes , e.g. , the power transmissivity value of t 10 for the itms and length of arms equal to l 10 m , then \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\gamma \gtrsim { 10 ^ 5}\,{{\rm{s}}^{- 1}}$\end{document } , which is above the typical working frequencies band of such devices . in the literature , this particular case is usually referred to as a bad cavity approximation . in this case , the coupling factor \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal k}(\omega)$\end{document } can be approximated as : 416\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal k}(\omega ) \approx { { \omega _ q^2 } \over { { \omega ^2}}}\,,$$\end{document } where 417\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\omega _ q^2 = { { 2j } \over \gamma}\,.$$\end{document } note that in this approximation , the noise spectral densities ( 386 ) , ( 387 ) and ( 388 ) turn out to be frequency independent . in figure 36 , the sql beating factor 418\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\xi ( \omega ) = \sqrt { { { { s^h}(\omega ) } \over { s_{{\rm{sql}}\,{\rm{f.m.}}}^h(\omega)}}}$$\end{document } is plotted for the sum quantum noise spectral density s()with the following values of homodyne and squeezing angles 419\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\cot { \phi _ { { \rm{lo } } } } = { { { \mathcal k}({\omega _ 0 } ) } \over { 1 + \epsilon _ d^2{e^{2r}}}}\,,\qquad \tan \theta = { { \epsilon _ d^2 } \over { { e^{- 2r } } + \epsilon _ the four panes correspond to the following four combinations : ( upper left ) no losses ( d = 1 ) and no squeezing ( r = 0 ) ; ( lower left ) no losses ( d = 1 ) and 10 db squeezing ( e = 10 ) ; ( upper right ) with losses ( d = 0.95 ) and no squeezing ( r = 0 ) ; ( lower right ) with losses ( d = 0.95 ) and 10 db squeezing(e = 10 ) . in each pane , the family of plots is shown that corresponds to different values of the ratio 0/q , ranging from 0.1 to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\sqrt { 10}$\end{document}. figure 36plots of the locally - optimized sql beating factor ( ) ( 418 ) of the interferometer with cross - correlated noises for the bad cavity case 0 , for several different values of the optimization frequency 0 within the range \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$0.1 \times { \omega _ q } \le { \omega _ 0 } \le \sqrt { 10 } \times { \omega _ q}$\end{document}. thick solid lines : the common envelopes of these plots ; see eq . ( 420 ) . left column : d = 1 ; top row : no squeezing , r = 0 ; bottom row : 10 db squeezing , e = 10 . plots of the locally - optimized sql beating factor ( ) ( 418 ) of the interferometer with cross - correlated noises for the bad cavity case 0 , for several different values of the optimization frequency 0 within the range \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$0.1 \times { \omega _ q } \le { \omega _ 0 } \le \sqrt { 10 } \times { \omega _ q}$\end{document}. thick solid lines : the common envelopes of these plots ; see eq . ( 420 ) . left column : d = 1 ; top row : no squeezing , r = 0 ; bottom row : 10 db squeezing , e = 10 . ( 410 ) and ( 416 ) : 420\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\xi _ { { \rm{min}}}^2({\omega _ 0 } ) = { 1 \over 2}\;\left [ { ( { e^{- 2r } } + \epsilon _ d^2){{\omega _ 0 ^ 2 } \over { \omega _ q^2 } } + { { \epsilon _ d^2 } \over { 1 + \epsilon _ d^2{e^{2r}}}}\,{{\omega _ q^2 } \over { \omega _ 0 ^ 2 } } } \right]\;\,,$$\end{document } which is also plotted in figure 36 . it is easy to see that in the ideal case of d = 0 , there is no limitation on the sql beating factor , provided a sufficiently small ratio of 0/q : 421\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\xi _ { { \rm{min}}}^2(\omega ) = { { { e^{- 2r } } } \over 2}\,{{\omega _ 0 ^ 2 } \over { \omega _ q^2}}\,.$$\end{document } however , if d > 0 , then function ( 420 ) has a minimum in q at 422\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\omega _ q } = { \omega _ 0}\sqrt { { 1 \over { { \epsilon _ d}{e^r } } } + { \epsilon _ d}{e^r } } \,,$$\end{document } [ compare with eq . in essence , a filter cavity is an ordinary fabry - prot cavity with one partly transparent input / output mirror . the technical problem of how to spatially separate the input and output beam can be solved in different ways . in the original paper the triangular cavities were considered . however , in this case , an additional mirror in each cavity is required , which adds to the optical loss per bounce . another option is an ordinary linear cavity with additional optical circulator , which can be implemented , for example , by means of the polarization beamsplitter and faraday rotator ( note that while the typical polarization optics elements have much higher losses than the modern high - quality mirrors , the mirrors losses appear in the final expressions inflated by the filter cavity finesse ) . in both cases , the filter cavity can be described by the input / output relation , which can be easily obtained from eqs . ( 290 ) and ( 291 ) by setting ec , s = 0 ( there is no classical pumping in the filter cavity and , therefore , there is no displacement sensitivity ) and by some changes in the notations : 423\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\bf{{\hat o}}}(\omega ) = { { \mathbb r}_f}(\omega){\bf{\hat{i}}}(\omega ) + { { \mathbb t}_f}(\omega){\bf{\hat{q}}}(\omega)\,,$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\rm{\hat i}}$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\rm{\hat o}}$\end{document } are the two - photon quadrature amplitude vectors of the input and output beams , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\rm{\hat q}}$\end{document } stands for noise fields entering the cavity due to optical losses ( which are assumed to be in a vacuum state ) , 424\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\mathbb r}_f}(\omega ) = { 1 \over { { { \mathcal d}_f}(\omega)}}\;\left [ { \begin{array}{*{20}c } { \gamma _ { f1}^2 - \gamma _ { f2}^2 - \delta _ f^2 + { \omega ^2 } + 2i\omega { \gamma _ { f2 } } } & { - 2{\gamma _ { f1}}{\delta _ f } } \\ { 2{\gamma _ { f1}}{\delta _ f } } & { \gamma _ { f1}^2 - \gamma _ { f2}^2 - \delta _ f^2 + { \omega ^2 } + 2i\omega { \gamma _ { f2 } } } \\ \end{array } } \right]\;\,,$$\end{document } 425\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\mathbb r}_f}(\omega ) = { { 2\sqrt { { \gamma _ { f1}}{\gamma _ { f2 } } } } \over { { { \mathcal d}_f}(\omega)}}\;\left [ { \begin{array}{*{20}c } { { \gamma _ f } - i\omega } & { - { \delta _ f } } \\ { { \delta _ f } } & { { \gamma _ f } - i\omega } \\ \end{array } } \right]\;\,,$$\end{document } 426\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\mathcal d}_f}(\omega ) = { ( { \gamma _ f } - i\omega)^2 } + \delta _ f^2\,,$$\end{document } 427\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\gamma _ { f1 } } = { { c{t_f } } \over { 4{l_f}}}$$\end{document } 428\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\gamma _ { f2 } } = { { c{a_f } } \over { 4{l_f}}}\,,$$\end{document } where tf is the power transmittance of the input / output mirror , af is the factor of power loss per bounce , lf is the filter cavity length , 429\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\gamma _ f } = { \gamma _ { f1 } } + { \gamma _ { f2}}$$\end{document } is its half - bandwidth , and f is its detuning . in order to demonstrate how the filter cavity works , consider the particular case of the lossless cavity . in this case , 430\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\bf{\hat{o}}}(\omega ) = { { \mathbb r}_f}(\omega){\bf{\hat{i}}}(\omega)\,,$$\end{document } and the reflection matrix describes field amplitude rotations with the frequency - dependent rotation angle : 431\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\mathbb r}_f}(\omega ) = { \mathbb p}[{\theta _ f}(\omega)]{e^{i{\beta _ f}(\omega)}}\,,$$\end{document } where 432\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\theta _ f}(\omega ) = \arctan { { 2{\gamma _ f}{\delta _ f } } \over { \gamma _ f^2 - \delta _ f^2 + { \omega ^2}}}\,,$$\end{document } 433\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${e^{i{\beta _ f}(\omega ) } } = { { \vert { { \mathcal d}_f}(\omega)\vert } \over { { { \mathcal d}_f}(\omega)}}\,.$$\end{document } the phase factor \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${e^{i{\beta _ f}(\omega)}}$\end{document } is irrelevant , for it does not appear in the final equations for the spectral densities . let us now analyze the influence of the filter cavities on the interferometer sensitivity in postand pre - filtering variational schemes suppose that the light , entering the interferometer from the signal port is in the squeezed state with fixed squeezing angle and squeezing factor r and thus can be described by the following two - photon quadrature vector 434\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\bf{\hat{a } } } = { { \mathbb s}_{{\rm{sqz}}}}[r,\theta ] { { \bf{\hat{a}}}^{{\rm{vac}}}}\,,$$\end{document } where the quadratures vector \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat a^{{\rm{vac}}}}$\end{document } describes the vacuum state . after reflecting off the filter cavity , this light will be described with the following expression 435\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\bf{\hat{o}}}(\omega ) = { \mathbb p}[{\theta _ f}(\omega)]{{\mathbb s}_{{\rm{sqz}}}}[r,\theta ] { { { \hat a}}^{{\rm{vac}}}}{e^{i{\beta _ f}(\omega ) } } = { { \mathbb s}_{{\rm{sqz}}}}[r,\theta + { \theta _ f}(\omega)]{{\hat{a}}^{{\rm{va}}{{\rm{c}}\prime}}}\,,$$\end{document } [ see eq . ( 75 ) ] , where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat a^{va{c\prime } } } = { \mathbb p}[{\theta _ thus , the pre - filtering indeed rotates the squeezing angle by a frequency - dependent angle f( ) . in a similar manner suppose that prior to detection , the light described by the quadrature vector \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat b$\end{document } , reflects from the filter cavity . in this case , the photocurrent ( in fourier representation ) is proportional to 436\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { i _ -}(\omega ) \propto { h^{\mathsf t}}[{\phi _ { { \rm{lo}}}}]\left [ { { \mathbb p}\left [ { { \theta _ f}(\omega ) } \right]\hat b(\omega){e^{i{\beta _ f}(\omega ) } } + { \epsilon _ d}\hat n(\omega ) } \right]\quad \quad \;\ ; } \\ { = { h^{\mathsf t}}\left [ { { \phi _ { { \rm{lo } } } } - { \theta _ f}(\omega ) } \right]\;\left [ { \hat b(\omega ) + { \epsilon _ d}{{\hat n}\prime}(\omega ) } \right]{e^{i{\beta _ f}(\omega)}}\ , , } \\ \end{array}$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat n\prime } = { \mathbb p } [ - { \theta _ this formula demonstrates that post - filtering is equivalent to the introduction of a frequency - dependent shift of the homodyne angle by f ( ) . it is easy to see that the necessary frequency dependencies of the homodyne and squeezing angles ( 404 ) or ( 407 ) ( with the second - order polynomials in in the r.h.s . denominators ) can not be implemented by the rotation angle ( 432 ) ( with its first order in polynomial in the r.h.s . denominator ) . as was shown in the paper , two filter cavities are required in both these cases . in the double pre- and post - filtering case , later it was also shown that , in principle , arbitrary frequency dependence of the homodyne and/or squeezing angle can be implemented , providing a sufficient number of filter cavities . however , in most cases , a more simple setup consisting of a single filter cavity might suffice . really , the goal of the filter cavities is to compensate the back - action noise , which contributes significantly in the sum quantum noise only at low frequencies \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\omega \lesssim { \omega _ q } = \sqrt { 2j/\gamma}$\end{document}. however , when 437\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\gamma > { j^{1/3}},$$\end{document } which is actually the case for the planned second and third generation gw detectors , the factor \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal k}(\omega)$\end{document } can be well approximated by eq . . moreover , the second filter cavity could actually degrade sensitivity due to the additional optical losses it superinduces to the system . following this reasoning , we consider below two schemes , each based on a single filter cavity that realize pre - filtering and post - filtering , respectively . the schemes under consideration are shown in figure 37 . in the pre - filtering scheme drawn in the left panel of figure 37 , a squeezed light source emits frequency - independent squeezed vacuum towards the filter cavity , where it gets reflected , gaining a frequency - dependent phase shift f ( ) , and the light going out of the dark port is detected by the homodyne detector with fixed homodyne angle lo in the usual way . figure 37schemes of interferometer with the single filter cavity . left : in the pre - filtering scheme , squeezed vacuum from the squeezor is injected into the signal port of the interferometer after the reflection from the filter cavity ; right : in the post - filtering scheme , a squeezed vacuum first passes through the interferometer and , coming out , gets reflected from the filter cavity . in both cases the readout is performed by an ordinary homodyne detector with frequency independent homodyne angle lo . schemes of interferometer with the single filter cavity . left : in the pre - filtering scheme , squeezed vacuum from the squeezor is injected into the signal port of the interferometer after the reflection from the filter cavity ; right : in the post - filtering scheme , a squeezed vacuum first passes through the interferometer and , coming out , gets reflected from the filter cavity . in both cases the readout is performed by an ordinary homodyne detector with frequency independent homodyne angle lo . following the prescriptions of section 6.1.2 the optimal squeezing angle should then be equal to zero at higher frequencies , see ( 404 ) . taking into account that the phase shift introduced by the filter cavity goes to zero at high frequencies , we obtain that the squeezing angle of the input squeezed vacuum must be zero . combining eqs . ( 390 ) and ( 423 ) taking these assumptions into account , we obtain the following equation for the sum quantum noise of the pre - filtering scheme : 438\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\hat h_{{\rm{sum}}}}(\omega ) = - { 2 \over l}\sqrt { { \hbar \over { 2m\,j\gamma } } } \,{h^ { \mathsf t}}[\pi /2]\{(\gamma + i\omega){\mathbb k}(\omega)\,[{{\mathbb r}_f}(\omega){{\mathbb s}_{{\rm{sqz}}}}[r,0]{\hat a^{{\rm{vac}}}}(\omega ) + { { \mathbb t}_f}(\omega)\hat q(\omega ) ] + ( \gamma - i\omega){\epsilon _ d}\hat n(\omega)\ } \,,$$\end{document } which yields the following expression for a power ( double - sided ) spectral density 439\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s^h}(\omega ) = { { 2\hbar } \over { m\,{l^2}{\omega ^2}{\mathcal k}(\omega)}}\{{h^ { \mathsf t}}[\pi /2]\,{\mathbb k}(\omega)\left [ { { { \mathbb r}_f}(\omega){{\mathbb s}_{{\rm{sqz}}}}[2r,0]{\mathbb r}_f^\dagger ( \omega ) + { { \mathbb t}_f}(\omega){\mathbb t}_f^\dagger ( \omega ) } \right]\,{{\mathbb k}^\dagger}(\omega)h[\pi /2 ] + \epsilon _ d^2\ } \;.$$\end{document } in the ideal lossless filter cavity case , taking into account eq . ( 431 ) , this spectral density can be simplified as follows : 440\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s^h}(\omega ) = { { 2\hbar } \over { m\,{l^2}{\omega ^2}{\mathcal k}(\omega)}}\;\left [ { { h^ { \mathsf t}}[\pi /2]\,{\mathbb k}(\omega){\mathbb s}\left({2r,{\theta _ f}(\omega ) } \right){{\mathbb k}^\dagger}(\omega)h[\pi /2 ] + \epsilon _ d^2 } \right]$$\end{document } [ compare with eq . ( 391 ) ] . in this case , the necessary frequency dependence of the squeezing angle ( 404 ) can be implemented by the following filter cavity parameters : 441\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\gamma _ f } = { \delta _ f } = { \gamma _ { f0}}\,,$$\end{document } where 442\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\gamma _ { f0 } } = \sqrt { j/\gamma } \,.$$\end{document } along similar lines , the post - filtering scheme drawn in the right panel of figure 37 can be considered . here , the squeezed - vacuum produced by the squeezor first passes through the interferometer and then , coming out , gets reflected from the filter cavity , gaining a frequency - dependent phase shift , which is equivalent to introducing a frequency dependence into the homodyne angle , and then goes to the fixed angle homodyne detector . taking into account that this equivalent homodyne angle at high frequencies has to be /2 , and that the phase shift introduced by the filter cavity goes to zero at high frequencies , we obtain that the real homodyne angle must also be /2 . assuming that the squeezing angle is defined by eq . ( 405 ) and again using eqs . ( 390 ) and ( 423 ) , we obtain that the sum quantum noise and its power ( double - sided ) spectral density are equal to 443\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { { \hat h}_{{\rm{sum}}}}(\omega ) = - { 2 \over l}\sqrt { { \hbar \over { 2mj\gamma } } } \,{1 \over { { h^\mathsf { t}}[\pi /2]{{{\mathbb r}}_f}(\omega)\left(\begin{array}{*{20}c } 0 \\ 1 \\\end{array } \right)}}\quad \quad \quad \quad \quad \quad \quad \quad \quad \quad } \\ { \times { h^\mathsf { t}}[\pi /2]\{(\gamma + i\omega){{{\mathbb r}}_f}(\omega){{\mathbb k}}(\omega){{{\mathbb s}}_{{\rm{sqz}}}}[r,0]{{\hat a}^{{\rm{vac}}}}(\omega ) + ( \gamma - i\omega)[{{{\mathbb t}}_f}(\omega)\hat q(\omega ) + { \epsilon _ d}\hat n(\omega)]\ } } \\ \end{array}$$\end{document } and 444\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { s^h}(\omega ) = { { 2\hbar } \over { m{l^2}{\omega ^2}{\mathcal k}(\omega)}}{1 \over { { { \left\vert { { h^\mathsf { t}}[\pi /2]{{\mathbb r}_f}(\omega)\left(\begin{array}{*{20}c } 0 \\ 1 \\\end{array } \right ) } \right\vert}^2}}}\quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad } \\ { \{\times { h^\mathsf { t}}[\pi /2][{{{\mathbb r}}_f}(\omega){\mathbb k}(\omega){{{\mathbb s}}_{{\rm{sqz}}}}[2r,0]{{{\mathbb k}}^\dagger}(\omega){\mathbb r}_f^\dagger ( \omega ) + { { \mathbb t}_f}(\omega){\mathbb t}_f^\dagger ( \omega)]h[\pi /2 ] + \epsilon _ d^2\ } { . } } \\ \end{array}$$\end{document } in the ideal lossless filter cavity case , factoring in eq . ( 431 ) , this spectral density takes a form similar to ( 391 ) , but with the frequency - dependent homodyne angle : 445\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s^h}(\omega ) = { { 2\hbar } \over { m{l^2}{\omega ^2}{\mathcal k}(\omega){{\sin}^2}{\phi _ { { \rm{lo}}}}(\omega)}}[{h^\mathsf { t}}[{\phi _ { { \rm{lo}}}}(\omega)]{\mathbb k}(\omega){{\mathbb s}_{{\rm{sqz}}}}[2r,0]{\mathbb k}^\dagger(\omega){h^\mathsf { t}}[{\phi _ { { \rm{lo}}}}(\omega ) ] + \epsilon _ d^2],$$\end{document } where 446\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\phi _ { { \rm{lo}}}}(\omega ) = \pi /2 - { \theta _ f}(\omega){.}$$\end{document } the necessary frequency dependence ( 407 ) of this effective homodyne angle can be implemented by the following parameters of the filter cavity : 447\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\gamma _ f } = { \delta _ f } = { { { \gamma _ { f0 } } } \over { \sqrt { 1 + \epsilon _ d^2{e^{- 2r}}}}}\,{.}$$\end{document } note that for reasonable values of loss and squeezing factors , these parameters differ only by a few percents from the ones for the pre - filtering . it is easy to show that substitution of the conditions ( 441 ) and ( 447 ) into eqs . ( 440 ) and ( 445 ) , respectively , taking condition ( 437 ) into account , results in spectral densities for the ideal frequency dependent squeezing and homodyne angle , see eqs . ( 403 ) and ( 408 ) . in the general case of lossy filter cavities , the conditions ( 404 ) and ( 407 ) therefore , the optimal filter cavity parameters should be determined using some integral sensitivity criterion , which will be considered at the end of this section . however , it would be a reasonable assumption that the above consideration holds with good precision , if losses in the filter cavity are low compared to other optical losses in the system : 448\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{{\gamma _ { f2 } } } \over { { \gamma _ f } } } \approx { { { \gamma _ { f2 } } } \over { { \gamma _ { f0 } } } } \ll \epsilon _ d^2\,{.}$$\end{document } this inequality can be rewritten as the following condition for the filter cavity specific losses : 449\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{{a_f } } \over { { l_f } } } \lesssim { { 4{\gamma _ { f0 } } } \over c}\,\epsilon _ d^2\,.$$\end{document } in particular , for our standard parameters used for numerical estimates ( j = jaligo , = 2 500 s , d= 0.95 ) , we obtain f0 280 s , and there should be 450\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{{a_f } } \over { { l_f } } } \lesssim 2 \times { 10^{- 7}}\;{{\rm{m}}^{- 1}},$$\end{document } ( the r.h.s . corresponds , in particular , to a 50 m filter cavity with the losses per bounce af = 10 ) . another more crude limitation can be obtained from the condition that f2 should be small compared to the filter cavity bandwidth f : 451\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{{a_f } } \over { { l_f } } } \lesssim { { 4{\gamma _ { f0 } } } \over c}\,.$$\end{document } apparently , were it not the case , the filter cavity would just cease to work properly . for the same numerical values of j and as above , we obtain : 452\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{{a_f } } \over { { l_f } } } \lesssim 4 \times { 10^{- 6}}\;{{\rm{m}}^{- 1}},$$\end{document } ( for example , a very short 2.5 m filter cavity with af = 10 or 25 m cavity with af = 10 ) . numerical optimization of filter cavities . in the experiments devoted to detection of small forces , and , in particular , in the gw detection experiments , the main integral sensitivity measure is the probability to detect some calibrated signal . this probability , in turn , depends on the matched filtered snr defined as 453\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\rho ^2 } = \int\nolimits_{- \infty}^\infty { { { \vert { h_s}(\omega){\vert ^2 } } \over { { s^h}(\omega)}}\,{{d\omega } \over { 2\pi}}}$$\end{document } with hs ( ) the spectrum of this calibrated signal . in the low and medium frequency range , where back - action noise dominates , and wherein our interest is focused , the most probable source of signal is the gravitational radiation of the inspiraling binary systems of compact objects such as neutron stars and/or black holes [ 131 , 124 ] . in this case , the snr is equal to ( see ) 454\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\rho ^2 } = { k_0}\int\nolimits_0^{2\pi { f_{{\rm{max } } } } } { { { { \omega ^{- 7/3 } } } \over { { s^h}(\omega ) } } } \,{{d\omega } \over { 2\pi}}\,,$$\end{document } where k0 is a factor that does not depend on the interferometer parameters , and fmax is the cut - off frequency that depends on the binary system components masses . in particular , for neutron stars with masses equal to 1.4 solar mass , fmax 1.5 khz . since our goal here is not the maximal value of the snr itself , but rather the relative sensitivity gain offered by the filter cavity , and the corresponding optimal parameters f1 and f , providing this gain , we choose to normalize the snr by the value corresponding to the ordinary interferometer ( without the filter cavities ) : 455\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\rho _ 0 ^ 2 = { k_0}\int\nolimits_0^{2\pi { f_{{\rm{max } } } } } { { { { \omega ^{- 7/3 } } } \over { s_0^h(\omega ) } } } \,{{d\omega } \over { 2\pi}}\,,$$\end{document } with power ( double - sided ) spectral density 456\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$s_0^h(\omega ) = { { 2\hbar } \over { m{l^2}{\omega ^2}}}\left [ { { { 1 + \epsilon _ d^2 } \over { { \mathcal k}(\omega ) } } + { \mathcal k}(\omega ) } \right]$$\end{document } [ see eq . we optimized numerically the ratio \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$^{{\rho ^2}\rho _ 0 ^ 2}$\end{document } , with filter cavity half - bandwidth f1 and detuning f as the optimization parameters , for the values of the specific loss factor af / lf ranging from 10 ( e.g. , very long 10 km filter cavity with af = 10 ) to 10 ( e.g. , 10 m filter cavity with af = 10 ) . concerning the main interferometer parameters , we used the same values as in all our previous examples , namely , j = jaligo , = 2 500 s , and d = 0.95 . the results of the optimization are shown in figure 38 . in the left pane , the optimal values of the filter cavity parameters f1 and f are plotted , and in the right one the corresponding optimized values of the snr are . it follows from these plots that the optimal values of f1 and f are virtually the same as f0 , while the specific loss factor af / lf satisfies the condition ( 448 ) , and starts to deviate sensibly from f0 only when af / lf approaches the limit ( 451 ) . actually , for such high values of specific losses , the filter cavities only degrade the sensitivity , and the optimization algorithm effectively turns them off , switching to the ordinary frequency - independent squeezing regime ( see the right - most part of the right pane ) . figure 38left : numerically - optimized filter - cavity parameters for a single cavity based pre- and post - filtering schemes : half - bandwidth f1 ( solid lines ) and detuning f ( dashed lines ) , normalized by f0 [ see eq . ( 442 ) ] , as functions of the filter cavity specific losses af / lf . right : the corresponding optimal snrs , normalized by the snr for the ordinary interferometer [ see eq . dashed lines : the normalized snrs for the ideal frequency - dependent squeeze and homodyne angle cases , see eqs . ordinary squeezing : frequency - independent 10 db squeezing with = 0 . in all cases , j = jaligo , = 2 500 s , and d = 0.95 . left : numerically - optimized filter - cavity parameters for a single cavity based pre- and post - filtering schemes : half - bandwidth f1 ( solid lines ) and detuning f ( dashed lines ) , normalized by f0 ( 442 ) ] , as functions of the filter cavity specific losses af / lf . right : the corresponding optimal snrs , normalized by the snr for the ordinary interferometer [ see eq . dashed lines : the normalized snrs for the ideal frequency - dependent squeeze and homodyne angle cases , see eqs . ordinary squeezing : frequency - independent 10 db squeezing with = 0 . in all cases , j = jaligo , = 2 500 s , and d = 0.95 . it also follows from these plots that post - filtering provides slightly better sensitivity , if the optical losses in the filter cavity are low , while the pre - filtering has some advantage in the high - losses scenario . the post - filtration effectively rotates the homodyne angle from lo = /2 ( phase quadrature ) at high frequencies to lo 0 ( amplitude quadrature ) at low frequencies , in order to measure the back - action noise , which dominates the low frequencies . as a result , the optomechanical transfer function reduces at low frequencies , emphasizing all noises introduced after the interferometer [ see the factor sin lo( ) in the denominator of eq . ( 445 ) ] . in the pre - filtering case there is no such effect , for the value of lo = /2 , corresponding to the maximum of the optomechanical transfer function , holds for all frequencies ( the squeezing angle got rotated instead ) . the optimized sum quantum noise power ( double - sided ) spectral densities are plotted in figure 39 for several typical values of the specific loss factor , and for the same values of the rest of the parameters , as in figure 38 . for comparison , the spectral densities for the ideal frequency - dependent squeezing angle eqs . these plots clearly demonstrate that providing sufficiently - low optical losses ( say , af / lf 10 ) , the single filter cavity based schemes can provide virtually the same result as the abstract ones with the ideal frequency dependence for squeezing or homodyne angles . figure 39examples of the sum quantum noise power ( double - sided ) spectral densities of the resonance - tuned interferometers with the single filter cavity based pre- and post - filtering . left : pre - filtering , see figure 37 ( left ) ; dashes 10 db squeezing , lo = /2 , ideal frequency - dependent squeezing angle ( 404 ) ; thin solid 10 db squeezing , lo = /2 , numerically - optimized lossy pre - filtering cavity with af / lf = 10 , 10 10 and 10 . right : post - filtering , see figure 37 ( right ) ; dashes : 10 db squeezing , = 0 , ideal frequency - dependent homodyne angle ( 407 ) ; thin solid 10 db squeezing , = 0 , numerically optimized lossy post - filtering cavity with af / lf = 10 , 10 and 10 . in both panes ( for the comparison ) : ordinary no squeezing , lo = /2 ; squeezed : 10 db squeezing , = 0 , lo = /2 examples of the sum quantum noise power ( double - sided ) spectral densities of the resonance - tuned interferometers with the single filter cavity based pre- and post - filtering . left : pre - filtering , see figure 37 ( left ) ; dashes 10 db squeezing , lo = /2 , ideal frequency - dependent squeezing angle ( 404 ) ; thin solid 10 db squeezing , lo = /2 , numerically - optimized lossy pre - filtering cavity with af / lf = 10 , 10 10 and 10 . right : post - filtering , see figure 37 ( right ) ; dashes : 10 db squeezing , = 0 , ideal frequency - dependent homodyne angle ( 407 ) ; thin solid 10 db squeezing , = 0 , numerically optimized lossy post - filtering cavity with af / lf = 10 , 10 and 10 . in both panes ( for the comparison ) : ordinary no squeezing , lo = /2 ; squeezed : 10 db squeezing , = 0 , lo = /2 a quantum speed meter epitomizes the approach to the broadband sql beating , in some sense , opposite to the one based on the quantum noises cross - correlation tailoring with filter cavities , considered above . here , instead of fitting the quantum noise spectral dependence to the fabry - prot - michelson interferometer optomechanical coupling factor ( 389 ) , the interferometer topology is modified in such a way as to mold the new optomechanical coupling factor \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal k}_{{\rm{sm}}}}(\omega)$\end{document } so that it turns out frequency - independent in the low- and medium - frequency range , thus making the frequency - dependent cross - correlation not necessary . the general approach to speed measurement is to use pairs of position measurements separated by a time delay 1/ , where is the characteristic signal frequency ( cf . the simplified consideration in section 4.5 ) . ideally , the successive measurements should be coherent , i.e. , they should be performed by the same photons . in effect , the velocity of the test mass is measured in this way , which gives the necessary frequency dependence of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal k}_{{\rm{sm}}}}(\omega)$\end{document}. in section 4.5 , we have considered the simplest toy scheme that implements this principle and which was first proposed by braginsky and khalili in . also in this paper , a modified version of this scheme , called the sloshing - cavity speed meter , was proposed . this version uses two coupled resonators ( e.g. , microwave ones ) , as shown in figure 40 ( left ) , one of which ( 2 ) , the sloshing cavity , is pumped on resonance through the input waveguide , so that another one ( 1 ) becomes excited at its eigenfrequency e . the eigenfrequency of resonator 1 is modulated by the position x of the test mass and puts a voltage signal proportional to position x into resonator 2 , and a voltage signal proportional to velocity dx / dt into resonator 1 . the velocity signal flows from resonator 1 into an output waveguide , from which it is monitored . the coupling between the resonators causes voltage signals to slosh periodically from one resonator to the other at frequency . after each cycle of sloshing , the sign of the signal is reversed , so the net signal in resonator 1 is proportional to the difference of the position at times t and t + 2/ , thus implementing the same principle of the double position measurement . figure 40left : schematic diagram of the microwave speed meter on coupled cavities as given in . right : optical version of coupled - cavities speed meter proposed in . left : schematic diagram of the microwave speed meter on coupled cavities as given in . later , the optical version of the sloshing - cavity speed - meter scheme suitable for large - scale laser gw detectors was developed [ 20 , 126 , 127 ] . the most elaborated variant proposed in here , the differential mode of a michelson interferometer serves as the resonator 1 of the initial scheme of , and an additional kilometer - scale fabry - prot cavity as the resonator 2 , thus making a practical interferometer configuration . in parallel , it was realized by chen and khalili [ 42 , 85 ] that the zero area sagnac interferometer [ 140 , 11 , 145 ] actually implements the initial double - measurement variant of the quantum speed meter , shown in figure 26 . further analysis with account for optical losses was performed in and with detuned signal - recycling in . the core idea is that light from the laser gets split by the beamsplitter ( bs ) and directed to fabry - prot cavities in the arms , exactly as in conventional fabry - prot - michelson interferometers . however , after it leaves the cavity , it does not go back to the beamsplitter , but rather enters the cavity in the other arm , and only afterwards returns to the beamsplitter , and finally to the photo detector at the dark port . the scheme of uses ring fabry - prot cavities in the arms to spatially separate ingoing and outgoing light beams to redirect the light leaving the first arm to the second one evading the output beamsplitter . the variant analyzed in [ 85 , 50 ] uses polarized optics for the same purposes : light beams after ordinary beamsplitter , having linear ( e.g. , vertical ) polarization , pass through the polarized beamsplitter ( pbs ) , then meet the /4 plates that transform their linear polarization into a circular one , and then enter the fabry - prot cavity . after reflection from the fabry - prot cavity , light passes through a /4-plate again , changing its polarization again to linear , but orthogonal to the initial one . as a result , the pbs reflects it and redirects to another arm of the interferometer where it passes through the same stages , restoring finally the initial polarization and comes out of the interferometer . with the exception of the implementation method for this round - robin pass of the light through the interferometer , both schemes have the same performance , and the same appellation left panel : the ring cavities can be used to spatially separate the ingoing fields from the outgoing ones , in order to redirect output light from one arm to another . right panel : the same goal can be achieved using an optical circulator consisting of the polarization beamsplitter ( pbs ) and two /4-plates [ 85 , 50 ] . two possible optical realizations of zero area sagnac speed meter . left panel : the ring cavities can be used to spatially separate the ingoing fields from the outgoing ones , in order to redirect output light from one arm to another . right panel : the same goal can be achieved using an optical circulator consisting of the polarization beamsplitter ( pbs ) and two /4-plates [ 85 , 50 ] . visiting both arms , counter propagating light beams acquire phase shifts proportional to a sum of end mirrors displacements of both cavities taken with time delay equal to average single cavity storage time arm : 457\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta { \phi _ r } \propto { x_n}(t ) + { x_e}(t + { \tau _ { { \rm{arm}}}})\,,\quad \delta { \phi _ l } \propto { x_e}(t ) + { x_n}(t + { \tau _ { { \rm{arm}}}})\,.$$\end{document } after recombining at the beamsplitter and photo detection the output signal will be proportional to the phase difference of clockwise ( r ) and counter clockwise ( l ) propagating light beams : 458\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta { \phi _ r } - \delta { \phi _ l } \propto [ { x_n}(t ) - { x_n}(t + { \tau _ { { \rm{arm } } } } ) ] - [ { x_e}(t ) - { x_e}(t + { \tau _ { { \rm{arm } } } } ) ] \propto { \dot x_n}(t ) - { \dot x_e}(t ) + o({\tau _ { { \rm{arm}}}})$$\end{document } that , for frequencies \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\ll \tau _ { { \rm{arm}}}^{- 1}$\end{document } , are proportional to the relative velocity of the interferometer end test masses . both versions of the optical speed meter , the sloshing cavity and the sagnac ones , promise about the same sensitivity , and the choice between them depends mostly on the relative implementation cost of these schemes . below we consider in more detail the sagnac speed meter , which does not require the additional long sloshing cavity . we will not present here the full analysis of the sagnac topology similar to the one we have provided for the fabry - prot - michelson one . we limit ourselves by the particular case of the resonance tuned interferometer ( that is , no signal recycling and resonance tuned arm cavities ) . it seems that the detuned sagnac interferometer can provide a quite interesting regime , in particular , the negative inertia one . however , for now ( 2011 ) the exhaustive analysis of these regimes is yet to be done . we assume that the squeezed light can be injected into the interferometer dark port , but consider only the particular case of the classical optimization , = 0 , which gives the best broadband sensitivity for a given optical power . in order to reveal the main properties of the quantum speed meter , start with the simplified case of the lossless interferometer and the ideal photodetector . in this case , the sum quantum noise power ( double - sided ) spectral density of the speed meter can be written in a form similar to the one for the fabry - prot - michelson interferometer [ see , e.g. , eqs . ( 386 ) , ( 387 ) and ( 388 ) ] : 459\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s^h}(\omega ) = { { s_{{\rm{sql}}}^h(\omega ) } \over 2}\left [ { { { { e^{- 2r } } + { e^{2r}}{{\cot}^2}{\phi _ { { \rm{lo } } } } } \over { { { \mathcal k}_{{\rm{sm}}}}(\omega ) } } - 2{e^{2r}}\cot { \phi _ { { \rm{lo } } } } + { { \mathcal k}_{{\rm{sm}}}}(\omega){e^{2r } } } \right],$$\end{document } but with a different form of the optomechanical coupling factor , see : 460\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\mathcal k}_{{\rm{sm}}}}(\omega ) = { { 4j\gamma } \over { { { ( { \gamma ^2 } + { \omega ^2})}^2}}}\,.$$\end{document } the factor j here is still defined by eq . ( 369 ) , but the circulating power is now twice as high as that of the position meter , for the given input power , because after leaving the beamsplitter , here each of the north and east beams visit both arms sequentially . the key advantage of speed meters over position meters is that at low frequencies , < , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal k}_{{\rm{sm}}}}$\end{document } is approximately constant and reaches the maximum there : 461\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\mathcal k}_{{\rm{sm}}}}(\omega \ll \gamma ) \approx { \rm{arccot}}\,{{\mathcal k}_{{\rm{sm}}}}(0 ) = { \rm{arccot}}{{4j } \over { { \gamma ^3}}}\,.$$\end{document } as a consequence , a frequency - independent readout quadrature optimized for low frequencies can be used : 462\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\phi _ { { \rm{lo } } } } = { \rm{arccot}}\,{{\mathcal k}_{{\rm{sm}}}}(0 ) = { \rm{arccot}}{{4j } \over { { \gamma ^3}}}\,,$$\end{document } which gives the following power ( double - sided ) spectral density 463\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s^h}(\omega ) = { { s_{{\rm{sql}}}^h(\omega ) } \over 2}\left [ { { { { e^{- 2r } } } \over { { { \mathcal k}_{{\rm{sm}}}}(\omega ) } } + { { { \omega ^4}{{(2{\gamma ^2 } + { \omega ^2})}^2 } } \over { { \gamma ^8}}}{{\mathcal k}_{{\rm{sm}}}}(\omega){e^{2r } } } \right].$$\end{document } here , the radiation - pressure noise ( the second term in brackets ) is significantly suppressed in low frequencies ( ) , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$s_{{\rm{smlf}}}^h$\end{document } can beat the sql in a broad frequency band . this spectral density is plotted in figure 42 ( left ) . for comparison , spectral densities for the lossless ordinary fabry - prot - michelson interferometer without and with squeezing , as well as for the ideal post - filtering configuration one might conclude from these plots that the fabry - prot - michelson interferometer with the additional filter cavities is clearly better than the speed meter . figure 42examples of the sum quantum noise power ( double - sided ) spectral densities of the sagnac speed - meter interferometer ( thick solid line ) in comparison with the fabry - prot - michelson based topologies considerd above ( dashed lines ) . left : no optical losses , right : with optical losses , d = 0.95 , the losses part of the bandwidth 2 = 1.875 s ( which corresponds to the losses aarm = 10 per bounce in the 4 km length arms ) . squeezed : 10 db squeezing , = 0 , lo = /2 . post - filtering : 10 db squeezing , = 0 , ideal frequency - dependent homodyne angle [ see eq . ( 408 ) ] . for the fabry - prot - michelson - based topologies , j = jaligo and = 2 500 s. in the speed - meter case , j = 2jaligo and the bandwidth is set to provide the same high - frequency noise as in the other plots ( = 2 385 s in the lossless case and = 2 360 s in the lossy one ) . examples of the sum quantum noise power ( double - sided ) spectral densities of the sagnac speed - meter interferometer ( thick solid line ) in comparison with the fabry - prot - michelson based topologies considerd above ( dashed lines ) . left : no optical losses , right : with optical losses , d = 0.95 , the losses part of the bandwidth 2 = 1.875 s ( which corresponds to the losses aarm = 10 per bounce in the 4 km length arms ) . squeezed : 10 db squeezing , = 0 , lo = /2 . post - filtering : 10 db squeezing , = 0 , ideal frequency - dependent homodyne angle [ see eq . ( 408 ) ] . for the fabry - prot - michelson - based topologies , j = jaligo and = 2 500 s. in the speed - meter case , j = 2jaligo and the bandwidth is set to provide the same high - frequency noise as in the other plots ( = 2 385 s in the lossless case and = 2 360 s in the lossy one ) . in speed meters , optical losses in the arm cavities could noticeably affect the sum noise at low frequencies , even if 464\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\epsilon _ { { \rm{arm}}}^2 \equiv { { { \gamma _ 2 } } \over { { \gamma _ 1 } } } \ll \epsilon _ d^2\,,$$\end{document } because the radiation pressure noise component created by the arm cavity losses has a frequency dependence similar to the one for position meters ( remember that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal k}{\rm{/}}{{\mathcal k}_{{\rm{sm } } } } \to \infty$\end{document } if 0 ; see eqs . ( , we will use the following expression for the lossy speed - meter sum noise , which takes these losses into account ( more detailed treatment of the lossy speed meter can be found in papers [ 127 , 50 ] ) : 465\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { s^h}(\omega ) = { { s_{{\rm{sql}}}^h(\omega ) } \over 2}\left\{{{1 \over { { { \mathcal k}_{{\rm{sm}}}}(\omega)}}\left [ { { e^{- 2r } } + { e^{2r}}{{\cot}^2}{\phi _ { { \rm{lo } } } } + { { \epsilon _ d^2 } \over { { { \sin}^2}{\phi _ { { \rm{lo } } } } } } } \right]\quad \quad } \right . } \\ { \quad \quad \quad \quad \quad \quad \quad \left . { - 2{e^{2r}}\cot { \phi _ { { \rm{lo } } } } + { { \mathcal k}_{{\rm{sm}}}}(\omega){e^{2r } } + \epsilon _ { { \rm{arm}}}^2{\mathcal k}(\omega ) } \right\}. } \\ \end{array}$$\end{document } the low - frequency optimized detection angle , in presence of loss , is 466\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\phi _ { { \rm{lo } } } } = { \rm{arccot}}{{{{\mathcal k}_{{\rm{sm}}}}(0 ) } \over { 1 + \epsilon _ d^2{e^{- 2r } } } } = { \rm{arccot}}{{4j/{\gamma ^3 } } \over { 1 + \epsilon _ d^2{e^{- 2r}}}}\,,$$\end{document } which gives 467\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s^h}(\omega ) = { { s_{{\rm{sql}}}^h(\omega ) } \over 2}\left\{{{{{e^{- 2r } } + \epsilon _ d^2 } \over { { { \mathcal k}_{{\rm{sm}}}}(\omega ) } } + { { { { \mathcal k}_{{\rm{sm}}}}(\omega ) } \over { 1 + \epsilon _ d^2{e^{- 2r}}}}\left [ { { { { \omega ^4}{{(2{\gamma ^2 } + { \omega ^2})}^2 } } \over { { \gamma ^8}}}\,{e^{2r } } + \epsilon _ d^2 } \right ] + \epsilon _ { { \rm{arm}}}^2{\mathcal k}(\omega ) } \right\}$$\end{document } [ compare with eq . ( 463 ) and note the additional residual back - action term similar to one in eq . this spectral density is plotted in figure 42 ( right ) , together with the lossy variants of the same configurations as in figure 42 ( left ) , for the same moderately optimistic value of d = 0.95 , the losses part of the bandwidth and for 2 = 1.875 s [ which corresponds to the losses aarm = 10 per bounce in the 4 km length arms , see eq . these plots demonstrate that the speed meter in more robust with respect to optical losses than the filter cavities based configuration and is able to provide better sensitivity at very low frequencies . it should also be noted that we have not taken into account here optical losses in the filter cavity . comparison of figure 42 with figure 39 , where the noise spectral density for the more realistic lossy - filter - cavity cases are plotted , shows that the speed meter has advantage over , at least , the short and medium length ( tens or hundred of meters ) filter cavities . in the choice between very long ( and hence expensive ) kilometer scale filter cavities and the speed meter we have seen in section 4.3 that the harmonic oscillator , due to its strong response on near - resonance force , is characterized by the reduced values of the effective quantum noise and , therefore , by the sql around the resonance frequency , see eqs . . however , practical implementation of this gain is limited by the following two shortcomings : ( i ) the stronger the sensitivity gain , the more narrow the frequency band in which it is achieved ; see eq . ( 171 ) ; ( ii ) in many cases , and , in particular , in a gw detection scenario with its low signal frequencies and heavy test masses separated by the kilometers - scale distances , ordinary solid - state springs can not be used due to unacceptably high levels of mechanical loss and the associated thermal noise . at the same time , in detuned fabry - prot cavities , as well as in the detuned configurations of the fabry - prot - michelson interferometer , the radiation pressure force depends on the mirror displacement ( see eqs . ( 312 ) ) , which is equivalent to the additional rigidity , called the optical spring , inserted between the cavity mirrors . it does not introduce any additional thermal noise , except for the radiation pressure noise \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat f_{{\rm{b}}{\rm{.a}}{\rm{.}}}}$\end{document } , and , therefore , is free from the latter of the above mentioned shortcomings . moreover , as we shall show below , spectral dependence of the optical rigidity k( ) alleviates , to some extent , the former shortcoming of the ordinary rigidity and provides some limited sensitivity gain in a relatively broad band . the electromagnetic rigidity was first discovered experimentally in radio - frequency systems . then its existence was predicted for the optical fabry - prot cavities . much later it was shown that the excellent noise properties of the optical rigidity allows its use in quantum experiments with macroscopic mechanical objects [ 17 , 23 , 24 ] . the frequency dependence of the optical rigidity was explored in papers [ 32 , 83 , 33 ] . it was shown that depending on the interferometer tuning , either two resonances can exist in the system , mechanical and optical ones , or a single broader second - order resonance will exist . in the last decade , the optical rigidity has been observed experimentally both in the table - top setup and in the larger prototype interferometer . in detuned interferometer configurations , where the optical rigidity arises , the phase shifts between the input and output fields , as well as between the input fields and the field , circulating inside the interferometer , depend in sophisticated way on the frequency . therefore , in order to draw full advantage from the squeezing , the squeezing angle of the input field should follow this frequency dependence , which is problematic from the implementation point of view . due to this reason , considering the optical - rigidity - based regimes , we limit ourselves to the vacuum - input case only , setting \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathbb s}_{{\rm{sqz}}}}[r,\theta ] = { \mathbb i}$\end{document } in eq . ( 375 ) . in this case , it is convenient to redefine the input noise operators as follows : 468\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { \sqrt \gamma { { \hat a}_{new } } = \sqrt { { \gamma _ 1 } } \hat a + \sqrt { { \gamma _ 2 } } \hat a\,,\quad \quad } \\ { \sqrt \gamma { { \hat g}_{{\rm{new } } } } = \sqrt { { \gamma _ 1 } } \hat g - \sqrt { { \gamma _ 2 } } \hat a\,,\quad \quad } \\ { \epsilon { { \hat n}_{{\rm{new } } } } = \sqrt { { { { \gamma _ 2 } } \over { { \gamma _ 1 } } } } \,\hat g + \sqrt { { \gamma \over { { \gamma _ 1 } } } } { \epsilon _ \end{array}$$\end{document } where 469\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\epsilon = \sqrt { { 1 \over \eta } - 1 } \qquad { \rm{and}}\qquad \eta = { { { \gamma _ 1 } } \over \gamma}{\eta _ d}$$\end{document } is the unified quantum efficiency , which accounts for optical losses both in the interferometer and in the homodyne detector . note that if the operators \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat a,\hat g$\end{document } , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat n$\end{document } describe mutually - uncorrelated vacuum noises , then the same is valid for the new \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat a_{{\rm{new}}}},\,{\hat g_{{\rm{new}}}}$\end{document } , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat n_{{\rm{new}}}}$\end{document}. expressing eqs . ( 371 and 373 ) in terms of new noises ( 468 ) and renaming them , for brevity , 470\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\hat a_{{\rm{new } } } } \rightarrow \hat a\,,\qquad { \hat n_{{\rm{new } } } } \rightarrow \hat n\,,$$\end{document } we obtain : 471\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\hat{\mathcal x}_{{\rm{meas}}}}(\omega ) = \sqrt { { \hbar \over { 2mj\gamma } } } \,{{{\mathcal d}(\omega ) } \over { { { \rm{h}}^\mathsf { t}}({\phi _ { { \rm{lo}}}}){\rm{d}}(\omega)}}{{\rm{h}}^\mathsf { t}}({\phi _ { { \rm{lo}}}})[{\mathbb r}(\omega)\hat a(\omega ) + \epsilon \hat n(\omega)]\,,$$\end{document } 472\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\hat { \mathcal f}_{{\rm{b}{.}{\rm a}{.}}}}(\omega ) = \sqrt { 2\hbar mj\gamma } { \left [ { \begin{array}{*{20}c } 1 \\ 0 \\ \end{array } } \right]^\mathsf { t}}{\mathbb l}(\omega)\hat a(\omega)\,,$$\end{document } where ( ) is the lossless cavity reflection factor ; see eq . ( 550 ) . thus , we have effectively reduced our lossy interferometer to the equivalent lossless one , but with less effective homodyne detector , described by the unified quantum efficiency < d . now we can write down explicit expressions for the interferometer quantum noises ( 376 ) , ( 377 ) and ( 378 ) , which can be calculated using eqs . ( 552 ) : 473\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { s_{{\mathcal x}{\mathcal x}}}(\omega ) = { \hbar \over { 4mj\gamma \eta}}\,{{\vert { \mathcal d}(\omega){\vert ^2 } } \over { { \gamma ^2}{{\sin}^2}\varphi + { \omega ^2}{{\sin}^2}{\phi _ { { \rm{lo}}}}}}\ , , } \\ { { s_{{\mathcal f}{\mathcal f}}}(\omega ) = { { \hbar mj\gamma ( { \gamma ^2 } + { \omega ^2 } ) } \over { \vert { \mathcal d}(\omega){\vert ^2}}}\,,\quad \quad \quad \quad \quad \quad } \\ { { s_{{\mathcal x}{\mathcal f}}}(\omega ) = { \hbar \over 2}\,{{\gamma \cos \varphi - i\omega \cos { \phi _ { { \rm{lo } } } } } \over { \gamma \sin \varphi - i\omega \sin { \phi _ { { \rm{lo}}}}}}\,,\quad \quad \quad \quad } \\ \end{array}$$\end{document } where 474\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\gamma = \sqrt { { \gamma ^2 } + { \delta ^2 } } \quad \quad \varphi = { \phi _ { { \rm{lo } } } } - \beta , \quad \quad \beta = \arctan { \delta \over \gamma}.$$\end{document } we start our treatment of the optical rigidity with the bad cavity approximation , discussed in section 6.1.2 for the resonance - tuned interferometer case . this approximation , in addition to its importance for the smaller - scale prototype interferometers , provides a bridge between our idealized harmonic oscillator consideration of section 4.3.2 and the frequency - dependent rigidity case specific to the large - scale gw detectors , which will be considered below , in section 6.3.4 . in the bad cavity ( 473 ) for the interferometer quantum noises , as well as the expression ( 374 ) for the optical rigidity can be significantly simplified : 475\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s_{{\mathcal x}{\mathcal x } } } = { { \hbar { \gamma ^2 } } \over { 4mj\gamma \eta { { \sin}^2}\varphi}}\,,\qquad { s_{{\mathcal f}{\mathcal f } } } = { { \hbar mj\gamma } \over { { \gamma ^2}}}\,,\qquad { s_{{\mathcal x}{\mathcal f } } } = { \hbar \over 2}\,\cot \varphi \,,$$\end{document } and 476\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$k = { { mj\delta } \over { { \gamma ^2}}}\,.$$\end{document } substituting these equations into the equation for the sum quantum noise ( cf . ( 144 ) ) : 477\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { s^f}(\omega ) = \vert k - m{\omega ^2}{\vert ^2}{s_{{\mathcal x}{\mathcal x } } } + 2{\rm re } \{[k - m{\omega ^2}]{s_{{\mathcal x}{\mathcal f}}}\ } + { s_{{\mathcal f}{\mathcal f } } } } \\ { = \vert { k_{{\rm{eff } } } } - m{\omega ^2}{\vert ^2}{s_{{\mathcal x}{\mathcal x } } } + s_{{\mathcal f}{\mathcal f}}^{{\rm{eff}}}\,,\quad \quad \quad \quad \quad \ , } \\ \end{array}$$\end{document } where 478\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$s_{{\mathcal f}{\mathcal f}}^{{\rm{eff } } } = { { { s_{{\mathcal x}{\mathcal x}}}{s_{{\mathcal f}{\mathcal f } } } - \vert { s_{{\mathcal x}{\mathcal f}}}{\vert ^2 } } \over { { s_{{\mathcal x}{\mathcal x}}}}}\qquad { \rm{and}}\qquad { k_{{\rm{eff } } } } = k - { { { s_{{\mathcal x}{\mathcal f } } } } \over { { s_{{\mathcal x}{\mathcal x}}}}}$$\end{document } stand for the effective back - action noise and effective optical rigidity , respectively , and dividing by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$s_{{\rm{sql,}}\,{\rm{f}}{\rm{.m}}{\rm{.}}}^f$\end{document } defined by eq . ( 161 ) , we obtain the sql beating factor ( 418 ) : 479\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\xi ^2}(\omega ) = { 1 \over { { \omega ^2}}}\left [ { { { ( \omega _ m^2 - { \omega ^2})}^2}{{{\gamma ^2 } } \over { 4j\gamma \eta { { \sin}^2}\varphi } } + { { j\gamma } \over { { \gamma ^2}}}(1 - \eta { { \cos}^2}\varphi ) } \right],$$\end{document } where 480\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\omega _ m^2 = { { { k_{{\rm{eff } } } } } \over m } = { j \over { { \gamma ^2}}}(\delta - \gamma \eta \sin 2\varphi)$$\end{document } is the effective resonance frequency ( which takes into account both real and virtual parts of the effective rigidity keff ) . following the reasoning of section 4.3.2 , it is easy to see that this spectral density allows for narrow - band sensitivity gain equal to 481\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\xi ^2}({\omega _ m } + \nu ) \leq { \xi ^2}({\omega _ m } \pm \delta \omega /2 ) \approx { { 2j\gamma } \over { { \gamma ^2}\omega _ m^2}}(1 - \eta { \cos ^2}\varphi)$$\end{document } within the bandwidth 482\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\delta \omega } \over { { \omega _ m } } } = { { 2j\gamma } \over { { \gamma ^2}\omega _ m^2}}\sqrt { \eta { { \sin}^2}\varphi ( 1 - \eta { { \cos}^2}\phi ) } = { \xi ^2}({\omega _ m } \pm \delta \omega /2)\sqrt { { { \eta { { \sin}^2}\varphi } \over { 1 - \eta { { \cos}^2}\varphi } } } \,.$$\end{document } in the ideal lossless case ( = 1 ) , 483\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\delta \omega } \over { { \omega _ m } } } = { \xi ^2}({\omega _ m } \pm \delta \omega /2)\,,$$\end{document } in accord with eq.(171 ) . however , if < 1 , then the bandwidth , for a given lessens gradually as the homodyne angle goes down . therefore , the optimal case of the broadest bandwidth , for a given , corresponds to = /2 , and , therefore , to sxf = 0 [ see eqs . ( 475 ) ] , that is , to the pure real rigidity case with non - correlated radiation - pressure and shot noises . this result naturally follows from the above conclusion concerning the amenability of the quantum noise sources cross - correlation to the influence of optical loss . ( 479 ) and taking into account that 484\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{j\gamma } \over { { \gamma ^2 } } } = { { \omega _ q^2 } \over 2}{\cos ^2}\beta \,,\qquad { k \over m } = { { j\delta } \over { { \gamma ^2 } } } = { { \omega _ q^2 } \over 4}\sin 2\beta \,,$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\omega _ q^2$\end{document } is the normalized optical power defined in eq . ( 417 ) , we obtain that 485\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\xi ^2}(\omega ) = { 1 \over { 2{\omega ^2}}}\left[{\left({{{\omega _ q^2 } \over 4}\sin 2\beta - { \omega ^2 } } \right)^2}{1 \over { \omega _ q^2\eta { { \cos}^2}\beta } } + \omega _ q^2{\cos ^2}\beta \right].$$\end{document } consider now the local minimization of this function at some given frequency 0 , similar to one discussed in section 6.1.2 . now , the optimization parameter is , that is , the detuning of the interferometer . it is easy to show that the optimal is given by the following equation : 486\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{4\omega _ 0 ^ 2 } \over { \omega _ q^2 } } - { 2 \over { \tan \beta } } - { { \omega _ q^2 } \over { \omega _ 0 ^ 2}}(4\eta - 1){\cos ^4}\beta = 0\,.$$\end{document } this fifth - order equation for tan can not be solved in radicals . however , in the most interesting case of 0 q , the following asymptotic solution can easily be obtained : 487\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta \approx { \pi \over 2 } - { { 2\omega _ 0 ^ 2 } \over { \omega _ q^2}}\,,$$\end{document } thus yielding 488\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\xi ^2}(\omega ) \approx { 1 \over 2}\left [ { { { \left({1 - { { { \omega ^2 } } \over { \omega _ 0 ^ 2 } } } \right)}^2}{{\omega _ q^2 } \over { \omega _ 0 ^ 2\eta } } + { { 4\omega _ 0 ^ 2 } \over { \omega _ q^2 } } } \right].$$\end{document } the function ( 485 ) , with optimal values of defined by the condition ( 486 ) , is plotted in figure 43 for several values of the normalized detuning . we assumed in these plots that the unified quantum efficiency is equal to = 0.95 . in the ideal lossless case = 1 it means that in the real rigidity case , contrary to the virtual one , the sensitivity is not affected significantly by optical loss . it stems from the fact that quantum noise sources cross - correlation , amenable to the optical loss , has not been used here . instead , the sensitivity gain is obtained by means of signal amplification using the resonance character of the effective harmonic oscillator response , provided by the optical rigidity . figure 43plots of the sql beating factor ( 485 ) of the detuned interferometer , for different values of the normalized detuning : 0 = arctan(/ ) < /2 , and for unified quantum efficiency = 0.95 . thick solid line : the common envelope of these plots . dashed lines : the common envelopes ( 420 ) of the sql beating factors for the virtual rigidity case , without squeezing , r = 0 , and with 10 db squeezing , e = 10 ( for comparison ) . plots of the sql beating factor ( 485 ) of the detuned interferometer , for different values of the normalized detuning : 0 = arctan(/ ) < /2 , and for unified quantum efficiency = 0.95 . thick solid line : the common envelope of these plots . dashed lines : the common envelopes ( 420 ) of the sql beating factors for the virtual rigidity case , without squeezing , r = 0 , and with 10 db squeezing , e = 10 ( for comparison ) . the common envelope of these plots ( that is , the optimal sql - beating factor ) , defined implicitly by eqs . note that at low frequencies , q , it can be approximated as follows : 489\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\xi _ { { \rm{env}}}^2(\omega ) = { { 2{\omega ^2 } } \over { \omega _ q^2 } } \approx { \gamma \over \delta}\,,$$\end{document } ( actually , this approximation works well starting from 0.3q ) . it follows from this equation that in order to obtain a sensitivity significantly better than the sql level , the interferometer should be detuned far from the resonance , . for comparison , we reproduce here the common envelopes of the plots of (q ) for the virtual rigidity case with = 0.95 ; see figure 36 ( the dashed lines ) . ( 489 ) and ( 420 ) that in absence of the optical loss , the sensitivity of the real rigidity case is inferior to that of the virtual rigidity one . however , even a very modest optical loss value changes the situation drastically . the noise cancellation ( virtual rigidity ) method proves to be advantageous only for rather moderate values of the sql beating factor of 0.5 in the absence of squeezing and 0.3 with 10 db squeezing . the conclusion is forced upon you that in order to dive really deep under the sql , the use of real rather than virtual rigidity is inevitable . noteworthy , however , is the fact that optical rigidity has an inherent feature that can complicate its experimental implementation . . really , the expansion of the optical rigidity ( 374 ) into a taylor series in gives 490\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$k(\omega ) \approx { { mj\delta } \over { { \gamma ^2 } } } + { { 2mj\gamma \delta } \over { { \gamma ^4}}}i\omega + \ldots$$\end{document } the second term , proportional to i , describes an optical friction , and the positive sign of this term ( if > 0 ) means that this friction is negative . the corresponding characteristic instability time is equal to 491\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\tau _ { { \rm{inst } } } } = { { { \gamma ^4 } } \over { 2j\gamma \delta}}\,.$$\end{document } in principle , this instability can be damped by some feedback control system as analyzed in [ 32 , 43 ] . however , it can be done without significantly affecting the system dynamics , only if the instability is slow in the timescale of the mechanical oscillations frequency : 492\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\omega _ m}{\tau _ { { \rm{inst } } } } = { { { \gamma ^2 } } \over { 2\gamma { \omega _ m } } } \approx { j \over { 2\omega _ m^3{\xi ^2 } } } \gg 1\,.$$\end{document } taking into account that in real - life experiments the normalized optical power j is limited for technological reasons , the only way to get a more stable configuration is to decrease , that is , to improve the sensitivity by means of increasing the detuning . another way to vanquish the instability is to create a stable optical spring by employing the second pumping carrier light with opposite detuning as proposed in [ 48 , 129 ] . the parameters of the second carrier should be chosen so that the total optical rigidity must have both positive real and imaginary parts in eq . ( 490 ) : 493\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${k_{{\rm{sum}}}}(\omega ) = { k_1}(\omega ) + { k_2}(\omega ) \approx \left({{{m{j_1}{\delta _ 1 } } \over { \gamma _ 1 ^ 2 } } + { { m{j_2}{\delta _ 2 } } \over { \gamma _ 2 ^ 2 } } } \right ) + i\omega \left({{{2m{j_1}{\gamma _ 1}{\delta _ 1 } } \over { \gamma _ 1 ^ 4 } } + { { 2m{j_2}{\gamma _ 2}{\delta _ 2 } } \over { \gamma _ 2 ^ 4 } } } \right ) + \ldots$$\end{document } that can always be achieved by a proper choice of the parameters j1,2 , 1,2 and 1,2 ( 12 < 0 ) . gw detectors with kilometer - scale arm cavities , the interferometer bandwidth can easily be made comparable or smaller than the gw signal frequency . in this case , frequency dependences of the quantum noise spectral densities ( 376 ) , ( 377 ) and ( 378 ) and of the optical rigidity ( 374 ) influence the shape of the sum quantum noise and , therefore , the detector sensitivity . most quantum noise spectral density is affected by the effective mechanical dynamics of the probe bodies , established by the frequency - dependent optical rigidity ( 374 ) . consider the characteristic equation for this system : 494\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$- { \omega ^2}[{(\gamma - i\omega)^2 } + { \delta ^2 } ] + j\delta = 0\,.$$\end{document } in the asymptotic case of = 0 , the roots of this equation are equal to 495\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\omega _ m^{(0 ) } = \sqrt { { { { \delta ^2 } } \over 2 } - \sqrt { { { { \delta ^4 } } \over 4 } - j\delta } } \,,\qquad \omega _ o^{(0 ) } = \sqrt { { { { \delta ^2 } } \over 2 } + \sqrt { { { { \delta ^4 } } \over 4 } - j\delta } } , $ $ \end{document } ( hereafter we omit the roots with negative - valued real parts ) . the corresponding maxima of the effective mechanical susceptibility : 496\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\chi _ { xx}^{{\rm{eff}}}(\omega ) = { 1 \over { k(\omega ) - m{\omega ^2}}}$$\end{document } are , respectively , called the mechanical resonance ( m ) and the optical resonance ( 0 ) of the interferometer . in order to clarify their origin , consider an asymptotic case of the weak optomechanical coupling , j . in this case , 497\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\omega _ m } \approx { j \over { { \delta ^3 } } } = \sqrt { { { k(0 ) } \over m } } \,,\qquad { \omega _ o } \approx \delta \,.$$\end{document } it is easy to see that m originates from the ordinary resonance of the mechanical oscillator consisting of the test mass m and the optical spring k [ compare with eq . ( 476 ) ] . at the same time , o , in this approximation , does not depend on the optomechanical coupling and , therefore , has a pure optical origin namely , sloshing of the optical energy between the carrier power and the differential optical mode of the interferometer , detuned from the carrier frequency by . in the realistic general case of 0 , the characteristic equation roots are complex . for small values of , keeping only linear in terms , they can be approximated as follows : 498\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\omega _ m } = \omega _ m^{(0)}\left [ { 1 + { { i\gamma \omega _ m^{(0 ) } } \over { \sqrt { { \delta ^4 } - 4j\gamma } } } } \right],\qquad { \omega _ o } = \omega _ o^{(0)}\left [ { 1 - { { i\gamma \omega _ o^{(0 ) } } \over { \sqrt { { \delta ^4 } - 4j\gamma } } } } \right].$$\end{document } note that the signs of the imaginary parts correspond to a positive dumping for the optical resonance , and to a negative one ( that is , to instability ) for the mechanical resonance ( compare with eq . ( 490 ) ) . in figure 44 , the numerically - calculated roots of eq . ( 494 ) are plotted as a function of the normalized optical power j/ , together with the analytical approximate solution ( 498 ) , for the particular case of / = 0.03 . these plots demonstrate the peculiar feature of the parametric optomechanical interaction , namely , the decrease of the separation between the eigenfrequencies of the system as the optomechanical coupling strength goes up . this behavior is opposite to that of the ordinary coupled linear oscillators , where the separation between the eigenfrequencies increases as the coupling strength grows ( the well - known avoided crossing feature ) . figure 44roots of the characteristic equation ( 494 ) as functions of the optical power , for / = 0.03 . ( 498 ) roots of the characteristic equation ( 494 ) as functions of the optical power , for / = 0.03 . ( 498 ) as a result , if the optomechanical coupling reaches the critical value : 499\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$j = { { { \delta ^3 } } \over 4}\,,$$\end{document } then , in the asymptotic case of = 0 , the eigenfrequencies become equal to each other : 500\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\omega _ m^{(0 ) } = \omega _ o^{(0 ) } = { \omega _ 0 } \equiv { \delta \over { \sqrt 2}}\,.$$\end{document } if > 0 , then some separation retains , but it gets smaller than 2 , which means that the corresponding resonance curves effectively merge , forming a single , broader resonance . this second - order pole regime , described for the first time in , promises some significant advantages for high - precision mechanical measurements , and we shall consider it in more detail below . if j < jcrit , then two resonances yield two more or less separated minima of the sum quantum noise spectral density , whose location on the frequency axis mostly depends on the detuning , and their depth ( inversely proportional to their width ) hinges on the bandwidth . the choice of the preferable configuration depends on the criterion of the optimization , and also on the level of the technical ( non - quantum ) noise in the interferometer . the first one features the sensitivity of a broadband configuration , which provides the best snr for the gw radiation from the inspiraling neutron - star - neutron - star binary and , at the same time , for broadband radiation from the gw burst sources , for the parameters planned for the advanced ligo interferometer ( in particular , the circulating optical power \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal i}_{{\rm{arm } } } } = 840\,{\rm{kw}}$\end{document } , l = 4 km , and m = 40 kg , which translates to j = jaligo = ( 2 100 ) s , and the planned technical noise ) . it is easy to notice two ( yet not discernible ) minima on this plot , which correspond to the mechanical and the optical resonances . figure 45examples of the sum noise power ( double - sided ) spectral densities of the detuned interferometer . broadband : double optimization of the advanced ligo interferometer for ns - ns inspiraling and burst sources in presence of the classical noises ( j = jaligo = ( 2 100 ) s , = 3100 s , = 0.80 , lo = /2 0.44 ) . high - frequency : low - power configuration suitable for detection of the gw signals from the millisecond pulsars , similar to one planned for geo hf [ j = 0.1jaligo , = 2 1000 s , = /2 0.01 , lo = 0 ] . second - order pole : the regime close to the second - order pole one , which provides a maximum of the snr for the gw burst sources given that technical noise is smaller than the sql [ stech = 0.1ssql , j = jaligo , = 1050 s , = /2 0.040 , lo = 0.91 ] . in all cases , d = 0.95 and the losses part of the bandwidth 2 = 1.875 s ( which corresponds to the losses aarm = 10 per bounce in the 4 km long arms ) . examples of the sum noise power ( double - sided ) spectral densities of the detuned interferometer . broadband : double optimization of the advanced ligo interferometer for ns - ns inspiraling and burst sources in presence of the classical noises ( j = jaligo = ( 2 100 ) s , = 3100 s , = 0.80 , lo = /2 0.44 ) . high - frequency : low - power configuration suitable for detection of the gw signals from the millisecond pulsars , similar to one planned for geo hf [ j = 0.1jaligo , = 2 1000 s , = /2 0.01 , lo = 0 ] . second - order pole : the regime close to the second - order pole one , which provides a maximum of the snr for the gw burst sources given that technical noise is smaller than the sql [ stech = 0.1ssql , j = jaligo , = 1050 s , = /2 0.040 , lo = 0.91 ] . in all cases , d = 0.95 and the losses part of the bandwidth 2 = 1.875 s ( which corresponds to the losses aarm = 10 per bounce in the 4 km long arms ) . another example is the configuration suitable for detection of the narrow - band gw radiation from millisecond pulsars . apparently , one of two resonances should coincide with the signal frequency in this case . it is well to bear in mind that in order to create an optical spring with mechanical resonance in a khz region in contemporary and planned gw detectors , an enormous amount of optical power might be required . this is why the optical resonance , whose frequency depends mostly on the detuning , should be used for this purpose . despite one order of magnitude less optical power used ( j = 0.1jaligo ) , several times better sensitivity at frequency 1 khz , than in the broadband regime , can be obtained . note that the mechanical resonance in this case corresponds to 10 hz only and therefore is virtually useless . the second - order pole regime . in order to clarify the main properties of the second - order pole regime , start with the asymptotic case of 0 . in this case , the optical rigidity and the mechanical susceptibility ( 496 ) read 501\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$k(\omega ) = { { mj\delta } \over { { \delta ^2 } - { \omega ^2}}},$$\end{document } 502\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\chi _ { xx}^{{\rm{eff}},\,{\rm{dbl}}}(\omega ) = { 1 \over { k(\omega ) - m{\omega ^2 } } } = { 1 \over m}{{{\delta ^2 } - { \omega ^2 } } \over { j\delta - { \delta ^2}{\omega ^2 } + { \omega ^4}}}.$$\end{document } if condition ( 499 ) is satisfied , then in the close vicinity of the frequency 0 ( see eq . ( 500 ) ) : 503\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\vert\omega - { \omega _ 0}\vert \,\ll { \omega _ 0},$$\end{document } the susceptibility can be approximated as follows : 504\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\chi _ { xx}^{{\rm{eff}},\,{\rm{dbl}}}(\omega ) \approx { { \omega _ 0 ^ 2 } \over { m{{(\omega _ 0 ^ 2 - { \omega ^2})}^2}}}.$$\end{document } note that this susceptibility is proportional to the square of the susceptibility of the ordinary oscillator , 505\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\chi _ { xx}^{{\rm{osc}}}(\omega ) = { 1 \over { m(\omega _ 0 ^ 2 - { \omega ^2})}},$$\end{document } and has the second - order pole at the frequency 0 ( thus the name of this regime ) . this double - resonance feature creates a stronger response to the external forces with spectra concentrated near the frequency 0 , than in the ordinary harmonic oscillator case . the response of the ordinary harmonic oscillator with eigenfrequency 0 on this force increases linearly with time : 506\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${x_{{\rm{osc}}}}(t ) = { { { f_0}t } \over { 2m{\omega _ 0}}}\sin { \omega _ 0}t,$$\end{document } while that of the second - order pole object grows quadratically : 507\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${x_{{\rm{dbl}}}}(t ) = - { { { f_0 } } \over { 8m}}\left({{t^2}\cos { \omega _ 0}t - { { \sin { \omega _ 0}t } \over { { \omega _ 0 } } } } \right).$$\end{document } it follows from eq . ( 151 ) that due to this strong response , the second - order pole test object has a reduced value of the sql around 0 by contrast to the harmonic oscillator . consider the quantum noise of the system , consisting of this test object and the sqm ( that is , the heisenbergs - uncertainty - relation - limited quantum meter with frequency - independent and non - correlated measurement and back - action noises ; see section 4.1.1 ) , which monitors its position . below we show that the real - life long - arm interferometer , under some assumptions , can be approximated by this model . the sum quantum noise power ( double - sided ) spectral density of this system is equal to 508\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s^h}(\omega ) = { 4 \over { { m^2}{l^2}{\omega ^4}}}(\vert\chi _ { xx}^{{\rm{eff}},\,{\rm{dbl}}}(\omega){\vert^2}{s_{{\mathcal x}{\mathcal x } } } + { s_{{\mathcal f}{\mathcal f}}}).$$\end{document } if the frequency is close to 0 : 509\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\omega = { \omega _ 0 } + \nu , \qquad \vert\nu \vert \ll { \omega _ 0},$$\end{document } then 510\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s^h}({\omega _ 0 } + \nu ) \approx { { 2\hbar } \over { m{l^2}\omega _ 0 ^ 4}}\left({{{16{\nu ^4 } } \over { \omega _ q^2 } } + \omega _ q^2 } \right),$$\end{document } and 511\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\xi ^2}({\omega _ 0 } + \nu ) \equiv { { { s^h}({\omega _ 0 } + \nu ) } \over { s_{{\rm{sql}}\,{\rm{f.}}{\rm{m.}}}^h({\omega _ 0 } + \nu ) } } \approx { 1 \over { 2\omega _ 0 ^ 2}}\left({{{16{\nu ^4 } } \over { \omega _ q^2 } } + \omega _ q^2 } \right),$$\end{document } ( compare with eq . ( 165 ) ) , where the frequency q is defined by eq . ( 155 ) . the same minimax optimization as performed in section 4.3.2 for the harmonic oscillator case gives that the optimal value of q is equal to 512\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\omega _ q } = \delta \omega , $ $ \end{document } and in this case , 513\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\xi ^2}({\omega _ 0 } + \nu){\vert_{\vert\nu \vert \leq \delta \omega /2 } } \leq { \xi ^2}({\omega _ 0 } \pm \delta \omega /2 ) = { \left({{{\delta \omega } \over { { \omega _ 0 } } } } \right)^2}.$$\end{document } comparison of eqs . ( 513 ) and ( 171 ) shows that for a given sql - beating bandwidth , the second - order pole system can provide a much stronger sensitivity gain ( i.e. , much smaller value of ) , than the harmonic oscillator , or , alternatively , much broader bandwidth for a given value of . it is noteworthy that the factor ( 513 ) can be made smaller than the normalized oscillator sql 2||0 ( see eq . ( 171 ) ) , which means beating not only the free mass sql , but also the harmonic oscillator one . this consideration is illustrated by the left panel of figure 46 , where the factors for the harmonic oscillator ( 170 ) and of the second - order pole system ( 511 ) are plotted for the same value of the normalized back - action noise spectral density ( q/0 ) = 0.01 , as well as the normalized oscillator sql ( 171 ) . thick solid : the second - order pole system ( 511 ) ; dots : the two - pole system with optimal separation between the poles ( 528 ) , ( 529 ) ; dashes : the harmonic oscillator ( 170 ) ; thin solid sql of the harmonic oscillator ( 171 ) . ( 530 ) ; pluses : numerical optimization of the spectral density ( 514 ) in the lossless case ( = 1 ) ; diamonds : the same for the interferometer with j = ( 2 100 ) s , d = 0.95 and the losses part of the bandwidth 2 = 1.875 s ( which corresponds to the losses aarm = 10 per bounce in the 4 km long arms ) . left panel : the sql beating factors for q/0 = 0.1 . thick solid : the second - order pole system ( 511 ) ; dots : the two - pole system with optimal separation between the poles ( 528 ) , ( 529 ) ; dashes : the harmonic oscillator ( 170 ) ; thin solid sql of the harmonic oscillator ( 171 ) . ( 530 ) ; pluses : numerical optimization of the spectral density ( 514 ) in the lossless case ( = 1 ) ; diamonds : the same for the interferometer with j = ( 2 100 ) s , d = 0.95 and the losses part of the bandwidth 2 = 1.875 s ( which corresponds to the losses aarm = 10 per bounce in the 4 km long arms ) . ( 385 ) for the sum quantum noise power ( double - sided ) spectral density takes the following form : 514\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { s^h}(\omega ) = { \hbar \over { m{l^2}j\gamma}}{1 \over { { \omega ^2}{{\sin}^2}{\phi _ { { \rm{lo } } } } + { { \rm{\gamma}}^2}{{\sin}^2}\varphi}}\left\{{{{\left [ { { \gamma ^2 } - { \delta ^2 } + { \omega ^2 } + { j \over { { \omega ^2}}}(\delta - \gamma \sin 2{\phi _ { { \rm{lo } } } } ) } \right]}^2 } } \right.\quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \,\ , } \\ { \left . { + 4{\gamma ^2}{{\left({\delta - { j \over { { \omega ^2}}}{{\sin}^2}{\phi _ { { \rm{lo } } } } } \right)}^2 } + { \epsilon^2}{{\left\vert\mathcal{d } { ( \omega ) - { { j\delta } \over { { \omega ^2 } } } } \right\vert}^2 } } \right\}. } \\ \end{array}$$\end{document } suppose that the interferometer parameters satisfy approximately the second - order pole conditions . namely , introduce a new parameter defined by the following equation : 515\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$j(\delta - \gamma \sin 2{\phi _ { { \rm{lo } } } } ) = \omega _ 0 ^ 4 - \omega _ 0 ^ 2{\lambda ^2},$$\end{document } where the frequency 0 is defined by eq . ( 500 ) , and assume that 516\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\nu ^2}\sim{\lambda ^2}\sim{\omega _ 0}\gamma \ll \omega _ 0 ^ 2.$$\end{document } keeping only the first non - vanishing terms in , , and in eq . ( 514 ) , we obtain that 517\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s^h}({\omega _ 0 } + \nu ) \approx { { 2\hbar } \over { m{l^2}\omega _ 0 ^ 4}}\left({{1 \over { \omega _ q^2}}\left\{{{{(4{\nu ^2 } - { \lambda ^2})}^2 } + { \epsilon^2}\left [ { { { \left({4{\nu ^2 } - { \lambda ^2 } + { { { \omega _ 0}\gamma } \over { \sqrt 2}}\sin 2{\phi _ { { \rm{lo } } } } } \right)}^2 } + 4{\gamma ^2}\omega _ 0 ^ 2 } \right ] } \right\ } + \omega _ q^2 } \right),$$\end{document } where 518\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\omega _ q^2 = \sqrt 2 \gamma { \omega _ 0}(1 + { \cos ^2}{\phi _ { { \rm{lo}}}}){.}$$\end{document } it follows from eq . ( 517 ) that the parameter is equal to the separation between the two poles of the susceptibility \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal x}_{xx}^{{\rm{eff,}}\,{\rm{dbl}}}$\end{document}. it is evident that the spectral density ( 517 ) represents a direct generalization of eq . ( 510 ) in two aspects . we show below that a small yet non - zero value of a allows one to further increase the sensitivity . the peculiar feature of the second - order pole regime is that , while being , in essence , narrow - band , it can provide an arbitrarily - high snr for the broadband signals , limited only by the level of the additional noise of non - quantum ( technical ) origin . at the same time , in the ordinary harmonic oscillator case , the snr is fundamentally limited . in both the harmonic oscillator and the second - order pole test object cases , the quantum noise spectral density has a deep and narrow minimum , which makes the major part of the snr integral . if the bandwidth of the signal force exceeds the width of this minimum ( which is typically the case in gw experiments , save to the narrow - band signals from pulsars ) , then the snr integral ( 453 ) can be approximated as follows : 519\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\rho ^2 } = { { \vert{h_{\rm{s}}}({\omega _ 0}){\vert^2 } } \over \pi}\int\nolimits_{- \infty}^\infty { { { d\nu } \over { { s^h}({\omega _ 0 } + \nu)}}.}$$\end{document } it is convenient to normalize both the signal force and the noise spectral density by the corresponding sql values , which gives : 520\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\rho ^2 } = { { m{l^2}\omega _ 0 ^ 3 } \over { 4\hbar}}\,\vert{h_{\rm{s}}}({\omega _ 0}){\vert^2}{\sigma ^2},$$\end{document } where 521\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\sigma ^2 } = { 1 \over { \pi { \omega _ 0}}}\int\nolimits_{- \infty}^\infty { { { d\nu } \over { { \xi ^2}({\omega _ 0 } + \nu)}}}$$\end{document } is the dimensionless integral sensitivity measure , which we shall use here , and 522\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\xi ^2}({\omega _ 0 } + \nu ) = { { m{l^2}\omega _ 0 ^ 2 } \over { 4\hbar}}\,{s^h}({\omega _ 0 } + \nu){.}$$\end{document } for a harmonic oscillator , using eq . ( 170 ) , we obtain 523\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\sigma ^2 } = 1.$$\end{document } this result is natural , since the depth of the sum quantum - noise spectral - density minimum ( which makes the dominating part of the integral ( 521 ) ) in the harmonic oscillator case is inversely proportional to its width , see eq . the situation is different for the second - order pole - test object . here , the minimal value of is proportional to ( ) ( see eq . ( 513 ) ) and , therefore , it is possible to expect that the snr will be proportional to 524\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\sigma ^2 } \propto { 1 \over { { { ( \delta \omega)}^2 } } } \times \delta \omega \propto { 1 \over { \delta \omega } } \propto { 1 \over \xi}.$$\end{document } indeed , after substitution of eq . ( 511 ) into ( 521 ) , we obtain : 525\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\sigma ^2 } = { 1 \over { \sqrt 2}}{{{\omega _ 0 } } \over { { \omega _ q } } } = { 1 \over { 2\xi ( { \omega _ 0})}}.$$\end{document } therefore , decreasing the width of the dip in the sum quantum noise spectral density and increasing its depth , it is possible , in principle , to obtain an arbitrarily high value of the snr . of course , it is possible only if there are no other noise sources in the interferometer except for the quantum noise . let there be an additional ( technical ) noise in the system with the spectral density stech( ) . suppose also that this spectral density does not vary much within our frequency band of interest . then the factor can be approximated as follows : 526\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\sigma ^2 } = { 1 \over { \pi { \omega _ 0}}}\int\nolimits_{- \infty}^\infty { { { d\nu } \over { { \xi ^2}({\omega _ 0 } + \nu ) + \xi _ { { \rm{tech}}}^2}},}$$\end{document } where 527\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\xi _ { { \rm{tech}}}^2 = { { { s_{{\rm{tech}}}}({\omega _ 0 } ) } \over { { s_{{\rm{sql}}\,{\rm{f}}{.}{\rm{m}}{.}}}({\omega _ 0})}}.$$\end{document } concerning quantum noise , we consider the regime close ( but not necessarily exactly equal ) to that yielding the second - order pole , that is , we suppose 0 0 . in order to simplify our calculations , we neglect the contribution from optical loss into the sum spectral density ( we show below that it does not affect the final sensitivity much ) . ( 517 ) , one gets 528\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\xi ^2}({\omega _ 0 } + \nu ) = { 1 \over { 2\omega _ 0 ^ 2}}\left[{{{{(4{\nu ^2 } - { \lambda ^2})}^2 } } \over { \omega _ q^2 } } + \omega _ q^2\right].$$\end{document } in the appendix a.3 , we calculate integral eq . ( 526 ) and optimize it over and q . the optimization gives the best sensitivity , for a given value of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\xi _ { { \rm{tech}}}^2$\end{document } , is provided by 529\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\lambda = { \omega _ q } = { \omega _ 0}{\xi _ { { \rm{tech}}}}.$$\end{document } in this case , 530\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\sigma ^2 } = { 1 \over { 2\sqrt 2 { \xi _ { { \rm{tech}}}}}}.$$\end{document } the pure second - order pole regime ( = 0 ) , with the same optimal value of q , provides slightly worse sensitivity : 531\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\sigma ^2 } = { 1 \over { \sqrt { 6\sqrt 3 } { \xi _ { { \rm{tech}}}}}}.$$\end{document } the optimized function ( 528 ) is shown in figure 46 ( left ) for the particular case of = q = 0.10 . in figure 46 ( right ) , the optimal snr ( 530 ) is plotted as a function of the normalized technical noise \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\xi _ { { \rm{tech}}}^2$\end{document}. in order to verify our narrow - band model , we optimized numerically the general normalized snr for the broadband burst - type signals : 532\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\sigma _ { { \rm{burst}}}^2 = { { 2\hbar } \over { \pi m{l^2}\omega _ 0 ^ 2}}\int\nolimits_{- \infty}^\infty { { { d\omega /\omega } \over { { s^h}(\omega ) + s_{{\rm{tech}}}^h}},}$$\end{document } where s is the sum quantum noise of the interferometer defined by eq . ( 514 ) . the only assumption we have made here is that the technical noise power ( double - sided ) spectral density 533\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$s_{{\rm{tech}}}^h = { { 2\hbar } \over { m{l^2}\omega _ 0 ^ 2}}\,\xi _ { { \rm{tech}}}^2$$\end{document } does not depend on frequency , which is reasonable , since only the narrow frequency region around 0 contributes noticeably to the integral ( 532 ) . the result is shown in figure 46 ( right ) for two particular cases : the ideal ( no loss ) case with = 1 , and the realistic case of the interferometer with j = ( 2 100 ) s , d = 0.95 and 2 = 1.875 s ( which corresponds to the loss factor of aarm = 10 per bounce in the 4 km long arms ; see eq . the typical optimized quantum noise spectral density ( for the particular case of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\xi _ { { \rm{tech}}}^2 = 0.1$\end{document } ) is plotted in figure 45 . it is easy to see that the approximations ( 528 ) work very well , even if tech 1 and , therefore , the assumptions ( 516 ) cease to be valid . one can conclude , looking at these plots , that optical losses do not significantly affect the sensitivity of the interferometer , working in the second - order pole regime . the origin of the sensitivity gain is simply the resonance increase of the probe object dynamical response to the signal force , which is , evidently , immune to the optical loss . the only noticeable discrepancy between the analytical model and the numerical calculations for the lossless case , on the one hand , and the numerical calculations for the lossy case , on the other hand , appears only for very small values of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\xi _ { { \rm{tech}}}^2 \sim 0.01$\end{document}. it follows from eqs . ( 518 ) and ( 529 ) that this case corresponds to the proportionally reduced bandwidth of the interferometer , 534\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\gamma \sim { \omega _ 0}\xi _ { { \rm{tech}}}^2 \sim 10\,\,{{\rm{s}}^{- 1}}$$\end{document } ( for a typical value of 0 10 s ) . therefore , the loss - induced part of the total bandwidth 2 , which has no noticeable effect on the unified quantum efficiency [ see eq . normal broadband values of 10 s , degrades it in this narrow - band case . however , it has to be emphasized that the degradation of , for the reasonable values of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\xi _ { { \rm{tech}}}^2$\end{document } , is only about a few percent , and even for the quite unrealistic case of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\xi _ { { \rm{tech}}}^2 = 0.01$\end{document } , does not exceed 25% . in this review , our primary goal was to tell in a clear and understandable way what is meant by quantum measurement in gw detectors . it was conceived as a comprehensive introduction to the quantum noise calculation techniques that are employed currently for the development of advanced interferometric detectors . the target audience are the young researchers , students and postdocs , who have just started their way in this field and need a guide that provides a step - by - step tutorial into the techniques and covers all the current achievements in the field . at the same time , we tried to make this manuscript interesting to all our colleagues from the gw community and , perhaps , from other branches of physics , who might be interested in getting themselves familiar with this area , not necessarily close to their own research field . however , the reality is crude and such a lofty ambition is always a pot of gold at the end of the rainbow . thus , we could not claim this review to be a complete and comprehensive description of the field of quantum measurement . we present here a pretty detailed analysis of the quantum noise features in the first and second generation of gw interferometers , contemplating the techniques considered robust and established . however , many hot topics , related to the planned third generation of gw interferometers [ 44 , 125 , 80 ] remained uncovered . here are only some of them : ( i ) xylophone configurations , ( ii ) multiple - carrier detectors [ 130 , 129 ] , ( iii ) negative optical inertia , ( iv ) intracavity detection schemes [ 18 , 17 , 84 , 86 , 52 ] , etc . it is our determined intention to enjoy the great advantages of the format of living reviews and include those topics in future revisions of this review . we would like to conclude our review by pointing out how the new swiftly - developing areas of modern science and technology , not directly related to gw astronomy and detector science , turn out to be deeply rooted in the quantum measurement theory developed by the gw community . the history of how gw detection and quantum - measurement theory developed and interwove might serve as an example thereof . indeed , from the very first steps towards the experimental observation of gws made by weber in the early 1960s [ 165 , 166 ] , it was realized that the extreme weakness of interaction between the ripples of space - time and matter appeals for unprecedentedly precise measurement . and almost at the same time , braginsky realized that the expected amplitude of the gw - induced oscillations of the bar detector signal mode would be on the order of the zero point oscillations of this mode , as predicted by quantum mechanics ; that is , in order to observe gws , one has to treat a detector quantummechanically and as a consequence there will be a quantum back action , setting a limitation on the achievable sensitivity , the sql . this serendipity had a powerful impact on the quantum measurement theory development , for it set an objective to contrive some ways to overcome this limitation . for decades up to this point , it was a purely theoretical discipline having little in common with experimental science , and , fancy that , become a vital necessity for gw astronomy . and again , for several decades , gw detection has been perhaps the only field where the results of quantum measurement theory were applied , mainly in the struggle with quantum noise , considered as a hindrance towards the noble goal of the detection of gws . and only recently , the same optomechanical interaction , begetting quantum noise and the sql in the interferometric gw detectors , has aroused a keen interest among wide circles of researchers studying the quantum behavior of macroscopic objects and testing the very foundations of quantum mechanics in the macroscopic world [ 91 , 10 ] . all the techniques and concepts developed in the gw community turn out to be highly sought by this new field . such methods , initially developed for future gw detectors , as back - action evasion via properly constructed cross correlation between the measurement and back - action noise sources [ 162 , 159 , 158 , 160 , 161 , 51 , 53 ] , find a use in the optomechanical experiments with micro - and nanoscale mechanical oscillators [ 46 , 114 , 108 , 105 , 106 , 170 , 62 ] . it turns out that gw detectors themselves fit extremely well for testing the fundamental principles of quantum mechanics just for the record low values of the noise , having non - quantum origin , that owes to the ingenuity , patience and dedication by an entire generation of experimental physicists . the very fact that the mechanical differential mode of the km - scale ligo detector has been cooled down to teff = 1.4 k without any special arrangement , just by modifying the standard feedback kernel of the actuators to provide a virtual rigidity , shifting the 10-kg suspended mirrors oscillation frequency from m/2 = 0.74 hz to 150 hz , where the gw detector is most sensitive , tells its own tale . noteworthy also is the experiment on cooling a several - ton auriga bar detector mechanical oscillation mode to teff = 0.17 mk . in principle , some dedicated efforts might yield even cooling to ground state of these definitely macroscopic oscillators [ 54 , 107 ] . one might foresee even more striking , really quantum phenomena , to be demonstrated experimentally by future gw detectors , whose sensitivity will be governed by quantum noise and not limited by the sql . it is possible , e.g. , to prepare the mechanical degree of freedom of the interferometer in a close - to - pure squeezed quantum state , entangle the differential and common motion of the kg - scale mirrors in the epr - like fashion [ 56 , 115 ] , or even prepare it in a highly non - classical schrdinger - cat state [ 135 , 88 ] .

the fast progress in improving the sensitivity of the gravitational - wave detectors , we all have witnessed in the recent years , has propelled the scientific community to the point at which quantum behavior of such immense measurement devices as kilometer - long interferometers starts to matter . the time when their sensitivity will be mainly limited by the quantum noise of light is around the corner , and finding ways to reduce it will become a necessity . therefore , the primary goal we pursued in this review was to familiarize a broad spectrum of readers with the theory of quantum measurements in the very form it finds application in the area of gravitational - wave detection . we focus on how quantum noise arises in gravitational - wave interferometers and what limitations it imposes on the achievable sensitivity . we start from the very basic concepts and gradually advance to the general linear quantum measurement theory and its application to the calculation of quantum noise in the contemporary and planned interferometric detectors of gravitational radiation of the first and second generation . special attention is paid to the concept of the standard quantum limit and the methods of its surmounting .

Introduction Interferometry for GW Detectors: Classical Theory Quantum Nature of Light and Quantum Noise Linear Quantum Measurement Quantum Noise in Conventional GW Interferometers Schemes of GW Interferometers with Sub-SQL Sensitivity Conclusion and Future Directions

the more - than - ten - years - long history of the large - scale laser gravitation - wave ( gw ) detectors ( the first one , tama started to operate in 1999 , and the most powerful pair , the two detectors of the ligo project , in 2001 , not to forget about the two european members of the international interferometric gw detectors network , also having a pretty long history , namely , the german - british interferometer geo 600 located near hannover , germany , and the joint european large - scale detector virgo , operating near pisa , italy ) can be considered both as a great success and a complete failure , depending on the point of view . in the naively designed measurement schemes , built on the basis of a michelson interferometer , kin to the first and the second generation gw detectors , but with sensitivity chiefly limited by quantum noise , the best strategy for reaching a maximal sensitivity at a given spectral frequency would be to make these noise source contributions ( at this frequency ) in the total noise budget equal . at the same time , the gw signal in the considered scheme is confined to the phase quadrature since it comprises the time - dependent part of l and thus the resulting photocurrent will be proportional to : 42\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${i_{{\rm{dc } } } } \propto \overline { { { ( l + s)}^2 } } \simeq { \left({l_{{\rm{dc}}}^{(0 ) } } \right)^2 } + 2l_{{\rm{dc}}}^{(0)}(l_c^{{\rm{out } } } + { n_c})\cos { \phi _ { { \rm{dc } } } } + 2l_{{\rm{dc}}}^{(0)}({g_s } + l_s^{{\rm{out } } } + { n_s})\sin { \phi _ { { \rm{dc}}}},$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$l_{c , s}^{{\rm{out}}}$\end{document } denote the part of the input laser noise that leaked into the output port : 43\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$l_c^{{\rm{out}}}(t ) \simeq l_c^{{\rm{in}}}\delta \epsilon\cos { { { \omega _ 0}\delta l } \over c } - l_s^{{\rm{in}}}\sin { { { \omega _ 0}\delta l } \over c } \simeq l_c^{{\rm{in}}}\delta \epsilon - l_s^{{\rm{in}}}{{{\omega _ 0}\delta l } \over c}\,,$$\end{document } 44\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$l_s^{{\rm{out}}}(t ) \simeq l_c^{{\rm{in}}}\sin { { { \omega _ 0}\delta l } \over c } + l_s^{{\rm{in}}}\delta \epsilon\cos { { { \omega _ 0}\delta l } \over c } \simeq l_c^{{\rm{in}}}{{{\omega _ 0}\delta l } \over c } + l_s^{{\rm{in}}}\delta \epsilon\,,$$\end{document } and nc , s stand for the quantum noise associated with the signal sidebands and entering the interferometer from the signal port . in section 4 , we have talked about the quantum measurement , the general structure of quantum noise implied by the quantum mechanics and the restrictions on the achievable sensitivity it imposes . ( 280 ) , we rewrite the i / o - relations for a fabry - prot cavity in the two - photon quadratures notations : 290\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\hat b_{1,2}}(\omega ) = { { \mathbb r}_{1,2}}(\omega){\hat a_{1,2}}(\omega ) + { \mathbb t } ( \omega){\hat a_{2,1}}(\omega ) + 2\sqrt { { \gamma _ { 1,2}}}{\mathbb l } ( \omega)\hat{\bf{x}}(\omega)\,,$$\end{document } 291\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat e(\omega ) = { 1 \over { \sqrt \tau}}{\mathbb l}(\omega)\left [ { \sqrt { { \gamma _ { 1,2 } } } { { \hat a}_{1,2}}(\omega ) + \sqrt { { \gamma _ { 2,1 } } } { { \hat a}_{2,1}}(\omega ) + \hat{\bf{x}}(\omega ) } \right],$$\end{document } where 292\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat{\bf{x}}(\omega ) = \left [ { \begin{array}{*{20}c } { - { { \rm{e}}_s } } \\ { { { \rm{e}}_c } } \\ \end{array } } \right]\;\;{{{k_p}\hat x(\omega ) } \over { \sqrt \tau}},$$\end{document } 293\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\rm{e}}_c } = \sqrt 2 { \rm re}\;{\rm{e}}\,,\qquad { { \rm{e}}_s } = \sqrt 2{\rm im}\;{\rm{e}}\,,$$\end{document } 294\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathbb l}(\omega ) = { 1 \over { { \mathcal d}(\omega)}}\;\;\left [ { \begin{array}{*{20}c } { \gamma - i\omega } & { - \delta } \\ \delta & { \gamma - i\omega } \\ \end{array } } \right],$$\end{document } 295\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal d}(\omega ) = \ell ( \omega){\ell ^{\ast}}(- \omega ) = { ( \gamma - i\omega)^2 } + { \delta ^2}\,,$$\end{document } 296\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\mathbb r}_{1,2}}(\omega ) = 2{\gamma _ { 1,2}}{\mathbb l}(\omega ) -{\mathbb i } \,,\qquad { \mathbb t}(\omega ) = 2\sqrt { { \gamma _ 1}{\gamma _ 2}}{\mathbb l } ( \omega)\,.$$\end{document } therefore , the i / o - relations in standard form read : 297\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\left [ { \begin{array}{*{20}c } { { { \hat b}_1}(\omega ) } \\ { { { \hat b}_2}(\omega ) } \\ \end{array } } \right ] = { \mathbb m}_{{\rm{fp}}}^{(0)}\cdot\;\left [ { \begin{array}{*{20}c } { { { \hat a}_1}(\omega ) } \\ { { { \hat a}_2}(\omega ) } \\ \end{array } } \right]\ ; + \;\left [ { \begin{array}{*{20}c } { r_1^{{\rm{fp}}}(\omega ) } \\ { r_2^{{\rm{fp}}}(\omega ) } \\ \end{array } } \right]\hat x(\omega)$$\end{document } with optical transfer matrix defined as : 298\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathbb m}_{{\rm{fp}}}^{(0)}(\omega ) = \left [ { \begin{array}{*{20}c } { { { \mathbb r}_1}(\omega ) } & { \mathbb t}{(\omega ) } \\ { { \mathbb t}(\omega ) } & { { { \mathbb r}_2}(\omega ) } \\ \end{array } } \right]$$\end{document } and the response to the cavity elongation \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat x(\omega)$\end{document } defined as : 299\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$r_1^{{\rm{fp}}}(\omega ) = 2{k_p}\sqrt { { { { \gamma _ 1 } } \over \tau } } { \mathbb l}(\omega)\;\cdot\;\left [ { \begin{array}{*{20}c } { - { { \rm{e}}_s } } \\ { { { \rm{e}}_c } } \\ \end{array } } \right]\quad { \rm{and}}\quad r_2^{{\rm{fp}}}(\omega ) = 2{k_p}\sqrt { { { { \gamma _ 2 } } \over \tau}}{\mathbb l } ( \omega)\;\cdot\;\left [ { \begin{array}{*{20}c } { -{{\rm{e}}_s } } \\ { { { \rm{e}}_c } } \\ \end{array } } \right].$$\end{document } note that due to the fact that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${({\mathbb m}_{{\rm{fp}}}^{(0)})^\dagger } = { ( { \mathbb m}_{{\rm{fp}}}^{(0)})^{- 1}}$\end{document } the reflectivity and the transmission matrices 1,2 and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathbb t}$\end{document } satisfy the following unitarity relations : 300\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\mathbb r}_1}{\mathbb r}_1^{\dagger } + { \mathbb t}{\mathbb t}^{\dagger } = { { \mathbb r}_2}{\mathbb r}_2^{\dagger } + { \mathbb t}{\mathbb t}^{\dagger } = { \mathbb i } \,,\qquad { { \mathbb r}_1}{{\mathbb t}^{\dagger } } + { \mathbb t}{\mathbb r}_2^{\dagger } = 0\,.$$\end{document } the mechanical equations of motion of the fabry - prot cavity mirrors , in spectral representation , are the following : 301\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\rm{f}}\chi _ { xx , i}^{- 1}(\omega){\hat x_i}(\omega ) = { \hat f_i}(\omega ) + { g_i}(\omega)\qquad i = 1,2\,,$$\end{document } where xx , i are the mechanical susceptibilities of the mirrors , gi stand for any external classical forces that could act on the mirrors ( for example , a signal force to be detected ) , 302\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\hat f_i } = { { { { \hat { \mathcal i}}_{{\rm{e}}\,i } } + { { \hat { \mathcal i}}_{{\rm{f}}\,i } } - { { \hat { \mathcal i}}_{{\rm{a}}\,i } } - { { \hat { \mathcal i}}_{{\rm{b}}\,i } } } \over c}$$\end{document } are the radiation pressure forces acting on the mirrors , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat { \mathcal i}_{{\rm{e}}i}},\,{\hat { \mathcal i}_{{\rm{f}}i}},{\hat { \mathcal i}_{{\rm{a}}i}},{\hat { \mathcal i}_{{\rm{b}}i}}$\end{document } are the powers of the waves ei , fi , etc . indeed , it can be minimized at any given frequency by setting 396\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal k}(\omega ) = { e^{- r}}\sqrt { { e^{- 2r } } + \epsilon_d^2 } \,,$$\end{document } and in this case , 397\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\xi ^2}(\omega ) \equiv { { { s^h}(\omega ) } \over { s_{{\rm{sql}}\,{\rm{fm}}}^h(\omega ) } } = \sqrt { 1 + \epsilon_d^2{e^{2r } } } \geq 1\,.$$\end{document } the spectral density ( 394 ) was first calculated in the pioneering work of , where the existence of two kinds of quantum noise in optical interferometric devices , namely the measurement ( shot ) noise and the back action ( radiation pressure ) noise , were identified for the first time , and it was shown that the injection of squeezed light with = 0 into the interferometer dark port is equivalent to the increase of the optical pumping power . and only recently , the same optomechanical interaction , begetting quantum noise and the sql in the interferometric gw detectors , has aroused a keen interest among wide circles of researchers studying the quantum behavior of macroscopic objects and testing the very foundations of quantum mechanics in the macroscopic world [ 91 , 10 ] .

the insulin signal transduction pathway in pancreatic -cells is similar to that in most other cell types ( fig . 1 ) . we do not dispute evidence that insulin receptors are expressed in -cells . moreover , it is generally accepted that some elements in the insulin signal transduction pathways play a critical role for -cell survival , growth , and general well - being ( 14 ) . perhaps the best example comes from the global insulin receptor substrate ( irs)-1 and irs-2 knockout mouse models , in which irs-2 was shown to play an especially vital role in the ability of -cells to compensate for insulin resistance ( 57 ) . these landmark studies were a catalyst to change previous thinking in the diabetes research field . before , the predominant thought was that insulin resistance was the main cause of type 2 diabetes , but it was not widely acknowledged until the 1990s that the onset of type 2 diabetes is marked by a failure of the functional -cell mass to meet the metabolic demand ( 810 ) . back then , with the realization that irs-2 signaling in -cells could be important , a plethora of studies blossomed to indicate certain downstream elements in irs-2 signaling pathways also play important roles in -cell function and survival ( fig . its expression is increased by glucose , incretins such as glp-1 , and other factors that increase cytosolic [ ca]i and [ camp]i in -cells ( 1113 ) . by contrast , it can be downregulated by proinflammatory cytokines , physiological stress , and feedback inhibition of normal irs signaling ( 4,1416 ) . it is conceivable that the relatively high expression of irs-2 and its quick turnover in -cells ( 13 ) may offset any need for constitutive activity of the insulin receptor , as it does in the liver ( 17 ) . with a controlled upregulation of irs-2 when -cell compensation is needed to maintain glucose homeostasis and downregulation of irs-2 when -cell compensation is not needed , the responsibility for insulin itself to trigger downstream signaling in -cells could be removed and placed more so on glucose , incretins , neuronal connections , and other more physiologically relevant regulators of -cell function . activation of the irs signaling cascade pathways . a peptide ligand such as insulin or insulin - like growth factor-1 ( igf-1 ) binds to its receptor , activating the intrinsic tyrosine kinase activity of that receptor that then tyrosine phosphorylates ( py ) adaptor molecules such as irs-1 or -2 . other receptor tyrosine kinases , or receptors that activate tyrosine kinases such as janus kinase ( jak ) , can also activate irs signaling . this leads to activation of two major signaling cascades , the ras - raf - mitogen - activated protein kinase ( mapk ) pathway ( orange ) and the phosphatidylinositol-3-kinase ( pi3k)/protein kinase - b ( pkb ; also known as akt ) signaling pathway ( green ) . for the ras - raf - mapk pathway , growth factor receptor bound protein-2 ( grb2)/son of sevenless ( sos ) protein complex binds to specific phosphorylated tyrosines on irs-1/2 , activating the gtp / gdp exchange activity of sos , which loads p21 ( ras ) with gtp to activate ras , leading to phosphorylation of the serine / threonine protein kinase raf-1 , which then phosphorylates the mitogen - activated protein kinase kinase ( mek1 ) , which is then activated to phosphorylate the extracellular signal phospho - activated erk-1/2 can then directly ( or indirectly via phospho - activation of other kinases such as p90 ribosomal serine kinase [ p90 ] ) serine / threonine phosphorylate certain transcription factors , such as cfos and e - twenty - six like transcription factor 1 ( elk-1 ) , to upregulate gene transcription . phospho - activated erk-1/2 can also phosphorylate mapk interacting kinase ( mnk ) 1 and 2 , leading to phosphorylation activation of the eukaryotic initiation factor-4e ( eif4e ) in a complex also containing eif4a and eif4 g to increase general protein synthesis at the level initiation phase of translational control . for the pi3k / pkb signaling pathway , the p85 regulatory subunit of pi3k docks to other specific phosphorylated tyrosine sites on irs-1/2 that then activates its p110 catalytic activity . this catalyzes the phosphorylation of phosphatidylinositol-4 , 5-bisphophaste [ pi(4,5)p2 ] to phosphatidylinositol-3 , 4 , 5-trisphophaste [ pi(3,4,5)p3 ] , which then activates 3-phosphoinositide dependent protein kinase-1 ( pdk1 ) . pdk1 then threonine ( pt ) phosphorylates pkb for pkb activation , which can be amplified by serine phosphorylation ( ps ) of pkb by the target of rapamycin complex-2 ( torc2 ; which includes the protein kinase , mammalian target of rapamycin [ mtor ] and associated proteins rictor and mlst8 ) . pkb - mediated phosphorylation of the tuberous sclerosis protein-1/2 complex ( tsc1/2 ) inhibits its gtpase activating protein activity to then load the ras homolog enriched in brain ( rheb ) protein with gtp ( rheb ) , leading to activation of the torc1 ( which includes mtor and associated proteins raptor and mlst8 ) . this includes the eif4e - binding protein-1 ( 4e - bp1 ) that releases it from eif4e binding , enabling eif4e to associate with eif4a and eif4 g in a complex with mnk , where mnk then phosphorylates eif4e to increase rates of protein synthesis translation . this also shows how the ras / raf / erk and pi3k / pkb signaling pathways can coordinate to give a tight translational control of protein synthesis . torc1 can also phosphorylate and subsequently activate p70 s6-ribosomal kinase ( p70 ) , which can lead to an increase in the elongation phase of protein synthesis translation . torc1 also phosphorylates unc-51like kinases-1/2 ( ulk-1/2 ; also known as autophagy gene-1 ) , which results in inhibition of autophagy . among pkb s other phosphorylation substrates are proteins involved in the apoptotic process such as bcl - antagonist of cell death ( bad ) and x - linked inhibitor of apoptosis protein ( xiap ) , outlining a mechanism whereby pkb is antiapoptotic . pkb phosphorylation of the transcription factors foxo1 and foxo3a causes their removal from the nucleus and promotes their degradation , causing an inhibition of foxo1/3a - mediated transcription . phosphorylation of glycogen synthase kinase-3 ( gsk3 ) by pkb inhibits gsk3 activity , resulting in increased glycogen deposition and cell growth . under certain circumstances , pkb can also influence increases in cell growth by phosphorylating the cell - cycle inhibitor proteins p21 cyclin - dependent kinase inhibitor-1 ( p21 ) and p27 cyclin - dependent - kinase inhibitor ( p27 ) . pkb can also phosphorylate - inhibit phosphodiesterase-3b ( pde3b ) to elevate intracellular camp ( [ camp]i ) levels . many of these irs signaling elements have been shown to be expressed and active and play important roles in pancreatic -cells in terms of certain functions , growth , and survival ( rev . in 24 ) , and these are indicated by a yellow highlighted halo . however , despite this growing body of evidence for the necessity of irs-2regulated signaling pathways in -cells , the identity of any physiologically relevant upstream ligand / receptor interaction that triggers irs signal transduction in -cells in vivo has been rather unclear . of course , the insulin / insulin receptor interaction is an attractive candidate , but there are considerations and circumstances that raise significant doubt of an autocrine effect of insulin on -cells . either the -cells are bathing in a very high concentration of locally secreted insulin , or secreted insulin is rapidly dispensed into the circulation leaving the locality of an islet but then has to complete an entire lap around the body where it is cleared by other tissues before returning to the -cells at a much lower concentration . prolonged exposure to insulin , and/or high concentrations of insulin in all cells that express the insulin receptor effectively desensitizes the irs signaling pathway downstream of the receptor as well as downregulates expression of insulin receptor itself ( 18 ) . internalization of the insulin receptor into an endosomal compartment , once insulin is bound contributes to this desensitization reducing the availability of cell - surface insulin receptors ( 19 ) . some desensitization mechanisms are normal physiological feedback inhibition aimed at preventing potentially harmful effects of prolonged exposure to insulin such as hypoglycemia and/or oncogenesis ( 18 ) ( fig . 2 ) . once the insulin receptor is downregulated and downstream signaling desensitized , some time without insulin is needed to restore normal insulin sensitivity ( 20,21 ) . considering that the insulin receptor is ubiquitously expressed , such feedback inhibition mechanisms provide a dynamic means of locally controlling insulin receptor activity rather than a more complex regulation of controlling insulin receptor gene expression in a tissue - specific manner . indeed , some believe that the chronic hyperinsulinemia found in obesity and obesity - linked type 2 diabetes can make a major contribution to the insulin - resistant state because of these desensitization mechanisms ( 18 ) ( fig . once insulin has activated irs signal transduction pathways in cells , after a period of time there are internal physiological feedback inhibition signals ( indicated by red arrows ) that ensure that the insulin signal is not chronically sustained . under hyperinsulinemic conditions , this feedback results in chronic desensitization of irs signal transduction and contributes to the insulin - resistant state . downstream activation of extracellular signal regulated kinases-1 and -2 ( erk-1/2 ) ( as described in fig . 1 ) can lead to erk-1/2 protein kinase mediated serine phosphorylation ( ps ) of irs-1/2 , which results in dissociation of the insulin receptor and irs-1/2 interaction together with irs-1/2 degradation . 3-phosphoinositide - dependent protein kinase-1 ( pdk1 ) activation can result in downstream activation atypical protein kinase - c ( pkc ) isoforms , such as pkc , which can also serine phosphorylate ( ps ) irs-1/2 to promote their degradation , representing another route of delayed feedback inhibition for insulin signal transduction . in contrast , protein kinase - b ( pkb ; also known as akt ) can serine phosphorylate irs-1/2 at alternative sites to stabilize irs-1/2 tyrosine phosphorylation state and thus enhance downstream signaling . however , pkb - mediated phosphorylation activation of some of its other substrates can have a more dominant - negative feedback effect on irs signaling . both target of rapamycin complex-1 ( torc1 ) and p70 s6-ribosomal kinase ( p70 ) ( the latter amplified by pdk1 phosphorylation ) can serine phosphorylate irs-1/2 to promote their degradation , which then dampens irs signaling . tyrosine phosphatase-2 ( shp2 ) , which upon binding to certain phosphotyrosine residues on irs-1/2 becomes activated and can then remove phosphate from phosphotyrosines on irs-1/2 , thus dampening downstream signaling . foxo1 and -3a have been shown to be critical factors for driving irs-2 expression under basal conditions , especially foxo3a in -cells ( 15 ) . but when irs signaling is triggered by insulin , foxo1/3a transcriptional activity is inactivated , resulting in another route of temporal feedback inhibition by decreasing irs-2 expression . several of these irs signaling feedback mechanisms have indeed been shown to be present in pancreatic -cells ( 24,15,16 ) . another consideration for feedback inhibition of insulin action is that when insulin binds to its receptor , the insulin / insulin receptor complex is internalized into the cell where it dissociates in an endosomal compartment , allowing the free insulin receptor to return to the surface ( 18,19 ) . when insulin levels are high , this cycle is biased toward there being minimal insulin receptors on the surface of the cell with the majority being internalized , and acts as an additional physiological mechanism to prevent prolonged activation of irs signal transduction by insulin . under chronic hyperinsulinemia , insulin receptor internalization makes a contribution to insulin resistance . this long - term hyperinsulinemia also leads to downregulation of insulin receptor gene expression by a mechanism not yet well defined . the concentration of insulin secreted from the pancreas in vivo in response to physiological stimuli has been estimated by measuring insulin concentrations in the portal vein under normal circumstances and can reach high peak concentrations of 5 nmol / l ( 22 ) . as such , it is not too far - fetched to assume that if insulin were not efficiently cleared from the local islet milieu its concentration would be at least equally as high , if not higher . considering that significant insulin receptor downregulation in insulin - targeted primary cells nmol / l ( 23 ) , it is quite possible that the insulin receptor in -cells may be permanently downregulated if insulin is not effectively cleared . in support of this idea , recent in vivo studies in normal mice have indicated that despite a marked effect of insulin in the liver to activate molecular targets , such as sterol regulatory element binding protein-1c , there is a negligible , if any , effect of insulin on islet -cells in the very same animal at the same time ( 15 ) . however , an alternative view is that secreted insulin is rapidly cleared from the islet locale . in favor of this scenario is the fact that pancreatic islets have an extensive microcirculatory network that is required for normal -cell secretory function . in rodents , the islet microcirculation favors blood flow away from -cells passing by the other pancreatic endocrine islet cell types ( including glucagon - producing -cells and somatostatin -cells ) on exiting the islet ( 24 ) . it is thought by some that this unidirectional -cell -cell -cell communication may be involved in coordinate downregulation of glucagon secretion as well as preventing local accumulation of somatostatin to inhibit insulin and glucagon secretion . although human islet cell architecture is different from that in rodents , the same directional plumbing of the islet microcirculatory network is retained in humans ( 25 ) . islet -cells are also polarized , which enables them to effectively secrete insulin into the venous islet microcirculation from where it can be readily cleared from the islet milieu ( 26 ) . if insulin is efficiently dispersed away from the islet then it will be extracted from the circulation by the whole body before returning to the islet via the pancreatic artery at low peripheral picomolar concentrations ( 27 ) . such a depleted concentration of insulin could be ineffective to transduce irs signaling in -cells , which needs to be at 50 nmol / l as indicated from in vitro experiments using primary islet -cells ( 2,3 ) . it has been argued that there is always a degree of constitutive ( unregulated ) insulin secretion from normal islet -cells , and this is continually acting as a local autocrine ligand for the insulin receptor ( 13 ) . but this observation is derived from dedifferentiated tumorigenic -cell lines and transfection of the preproinsulin gene to pituitary cells , and thus it is not necessarily physiologically relevant . in fact , normal primary islet -cells efficiently sort > 99% of the newly synthesized ( pro)insulin to the regulated pathway and there is negligible , if any , constitutive secretion ( 28 ) . notwithstanding this , even if there were constitutive insulin secretion from -cells , the same two scenarios described above would still apply for a negligible autocrine effect of insulin on -cells . both sympathetic and parasympathetic input from the central nervous system ( cns ) can affect -cell function ( 29 ) . positive parasympathetic neurotransmitter signals , such as acetylcholine ( operating through muscarinic [ m3 ] receptors on -cells ) , vasoactive intestinal polypeptide , and pituitary adenylate cyclase - activating polypeptide ( 31 ) , can enter via the vagus to potentiate glucose - induced insulin secretion ( 30,31 ) . conversely , some negative effects to inhibit glucose - induced insulin secretion can be transmitted via sympathetic nerves ( 32 ) . in the pancreas , the vast majority of neurons associate with pancreatic islets ( 33 ) . although it has been suggested that the neuronal connections to pancreatic islets are to endothelial cells ( 34 ) , it is nonetheless clear that the cns can have a major influence on the functional regulation on -cells ( 29,32 ) . indeed , severing the vagus nerve can significantly impair glucose - induced insulin secretion ( 29 ) . in addition , an insulin - induced feedback inhibition of glucose - induced insulin secretion found in vivo is lost when the pancreas is denervated , including in humans ( 3537 ) . it has been suggested that a prior exposure to exogenous insulin can enhance subsequent stimulation of endogenous insulin secretion by glucose in humans in vivo ( 38,39 ) . although this could be consistent with a direct priming effect of insulin on the -cell , an alternative explanation could be that a prior exposure to exogenous insulin downregulates and desensitizes insulin receptor signaling in the brain ( 18 ) . if so , the insulin - induced feedback inhibition of glucose - induced insulin secretion would be less effective . as a consequence , indeed , an inadequate feedback inhibition of insulin on endogenous insulin secretion has been observed in obese / insulin - resistant humans and is thought to play a role in maintaining the persistent hyperinsulinemia under such circumstances ( 36 ) . although the cns influence on pancreatic -cell function is often underestimated , it is attracting renewed interest . some investigators even suggest that endogenously produced incretins may , at least in part , exert their effect on -cells via the cns ( 40,41 ) . intriguingly , compensatory increases in -cell mass and function may be somewhat controlled via the cns ( 42,43 ) . indeed , in vagotomized rats , rates of -cell proliferation decrease by half , which is associated with an 80% reduction in protein kinase - b ( pkb ; also known as akt ) activation , a key element in irs signaling pathways ( fig . 1 ) ( 43 ) . this in turn suggests that the cns may have a degree of control over irs-2regulated signal transduction in -cells . however , it is noted that direct intracerebroventricular administration of insulin into the cns does not affect circulating insulin levels , but does suppress food intake and decrease body weight ( 44 ) . although this is an intriguing observation , it relies on a pharmacological route for insulin delivery and does not reflect the physiological route by which insulin communicates with the cns via the circulation ( 44 ) . despite the evidence that some aspects of -cell function can be neuronally influenced , further research is needed , especially in regard to identifying the specific regions of the cns that directly communicate to the endocrine pancreas . but in terms of understanding the possible influence of insulin itself on the -cell in vivo , it seems that this may not necessarily be a direct autocrine effect but rather a secondary one mediated in the large part via the cns . there are now several transgenic rodent models that imply the need for many of the elements of the irs-2 signaling pathway for -cell function , growth and/or survival ( fig . a critical question is what is the physiologically relevant ligand that activates irs signaling pathways ? is it insulin itself ? perhaps closely related insulin - like growth factor 1 ( igf-1 ) ? or a combination of both ? enticing transgenic mouse models in which the insulin receptor and/or the igf-1 receptor genes have been knocked out in an intended -cell specific manner have been presented ( 4547 ) . intriguingly , deleting the insulin receptor from -cells in this manner ( irko mice ) results in modest glucose intolerance , elevated fasting insulin levels and impaired glucose - induced first - phase insulin secretion ( 45 ) . likewise , deleting the igf-1 receptor by the same technical approach ( igf1r mice ) reveals a similar phenotype ( 46 ) . cross - breeding these two mouse models deletes the receptors for insulin and igf-1 on -cells ( dko mice ) , rendering a more severe diabetic phenotype with apparent loss of -cell secretory capacity and function , together with elevated glucagon levels in the fed state ( 47 ) . such unregulated glucagon release would likely be a major contributor to the hyperglycemic state of these double - knockout ( dko ) animals . these data were very intriguing at the time , but recently the technical approach used to generate these transgenic mouse models has been questioned ( 48,49 ) . the short form of the rat insulin gene promoter ( rip ) used to drive a cre - recombination to generate the -cell specific knockout of the insulin and/or igf-1 receptor genes is unlikely to be exclusively -cell specific ( 4547 ) . indeed , many of the rips and other -cell transcription factor ( e.g. , pdx1 ) gene promoters can not be considered -cell specific because they also drive quite significant cre - expression in several regions of the brain , including the hypothalamus ( 48,49 ) . moreover , the rip - cre mice themselves display mild glucose intolerance , apparently owing to cre - impaired first - phase insulin secretion ( 50 ) . as previously outlined , the cns can have a major influence on metabolic homeostasis , including pancreatic endocrine cell functions involving the -cell . as such , while it is likely that any potential feedback effect of insulin and/or igf-1 on the -cell is blocked in these animal models , it can not be ruled out that the primary defect driving the phenotype of the irko , igf1r , or dko mice originates in the cns and that the -cells respond secondarily to dysfunctional cns control ( 51 ) . in this regard , there are now multiple studies in which knocking out a gene using a rip - cre or pdx1-cre transgenic approach gave an obese , hyperphagic , or metabolically altered phenotype that has been primarily attributed to deleting that gene in the brain / hypothalamus rather than the -cell ( 48,49 ) . in these instances , any apparent -cell defect might be secondary to obesity or changes in metabolic homeostasis controlled by the cns . however , technology has progressed , and there is now a new transgenic mouse model in which the full - length ( 8 kb ) mouse insulin promoter drives cre - expression , apparently in -cells only ( 46 ) . future use of this mip - creer mouse to uniquely delete insulin and/or igf-1 receptors in -cells may move toward resolving some of the controversy behind autocrine action of insulin . in vivo it has been known for more than 70 years that administration of exogenous insulin depletes the pancreas of its insulin stores ( 52 ) . this is often interpreted as a negative effect of insulin on insulin gene expression ( 2 ) . however , this is more likely due to the infused insulin lowering the blood glucose to a hypoglycemic state , which is known to deplete endogenous insulin stores and downregulate insulin gene expression ( 53,54 ) . insulin gene expression is markedly downregulated under starvation conditions , but then rapidly recovers upon refeeding a glucose infusion administered to fasted rats is sufficient to drive increases in insulin gene transcription ( 55 ) . however , if the glucose concentration is clamped in vivo , subsequent insulin infusion to induce hyperinsulinemia can partly reduce insulin gene expression ( 56 ) , but it remains unclear whether this is a direct effect of insulin on the -cell or one acting secondarily via the cns . in hyperglycemic states in vivo , insulin gene expression does not appear to drastically vary at the level of the -cells . although insulin gene expression appears reduced in pancreata from type 2 diabetic rodent models , this can be correlated with loss of -cell mass rather than any relation to hyperinsulinemia ( 57,58 ) . it should be noted that preproinsulin mrna levels are not only transcriptionally regulated , but also at the level of preproinsulin mrna stability . indeed , physiologically relevant increases in glucose stabilize preproinsulin mrna , but this does not appear to be mediated by a local autocrine feedback of secreted insulin ( 59 ) . recently however , there has been a series of in vitro studies using transformed -cell lines or isolated islets to suggest , in contrast to established in vivo studies , that there is a positive effect of insulin to drive insulin gene expression ( 2 ) . these in vitro studies have been complemented by experiments using islets isolated from the aforementioned irko and igf1r transgenic mice ( 4547 ) . however , in these particular mouse models , there could well be a cns influence on the islets of these animals which alters their -cell mass ( 42,43,48,49,51 ) and , as previously observed , preproinsulin mrna levels parallel these changes ( 57 ) . in short , none of these in vitro studies can readily place the potential of insulin itself directly regulating its own gene expression into a proper physiological context . there is some indication that elements in the insulin signaling pathway in -cells , such as foxo1 are involved in influencing insulin gene expression in -cells ( 4 ) . however , whether these elements are directly controlled by an in vivo autocrine feedback action of insulin is not established . the major regulation of insulin production in normal pancreatic -cells occurs at the translational level ( 60 ) . fluctuation in glucose concentrations is the main instigator of this control , but it can be supplemented by other nutrients and incretin peptides ( 60 ) . when primary -cells are exposed to a stimulatory glucose concentration there is a 20- to 30-min lag period ( due to recruitment of preproinsulin mrna containing polyribosome complexes to the rough endoplasmic reticulum , the site of proinsulin biosynthesis ) before increases in proinsulin biosynthesis are observed , that can then reach an impressive 10-fold increase by 60 min . glucose - induced translational control is unique to -cells and specifically directed at proinsulin and the biosynthesis of a subset of 50 secretory granule proteins ( 60 ) . it ensures that insulin stores in -cells are rapidly replenished after a bout of insulin exocytosis . the molecular mechanism for specific glucose - induced translational regulation of proinsulin biosynthesis is quite distinct from that for glucose - stimulated insulin secretion ( 60 ) . for example , unlike ca - dependent regulated insulin secretion , translational control of proinsulin biosynthesis is ca - independent ( 60 ) . many studies have ruled out an autocrine positive feedback of insulin to drive proinsulin biosynthesis ( 5961 ) , but some have been a proponent of this possibility ( 62,63 ) . unfortunately , studies that indicate a positive effect of insulin to stimulate proinsulin biosynthesis are questionable because proinsulin biosynthesis was either not measured directly , or if it was only during the initial 30-min lag period after introduction of the stimulus , and as such these measures are inaccurate ( 62,63 ) . without an extended incubation period up to 60 min , the mechanics for translational control of proinsulin biosynthesis can not be fully appreciated ( 60 ) . thus , the vast majority of evidence indicates that insulin does not have an autocrine effect on its own production in the -cell . as previously noted , there is an in vivo temporal negative feedback of insulin on stimulated insulin secretion that is mediated via the cns in humans ( 3537 ) . however , there is an intriguing suggestion that insulin may also have a positive effect to enhance its own secretion ( 13 ) . but an alternative explanation could be that the effect was mainly mediated via alleviating the negative feedback of insulin through the cns ( 36 ) , as previously discussed . yet , the majority of studies arguing for a positive autocrine effect of insulin to drive insulin secretion have been conducted in vitro where central control no longer operates , or in transgenic mouse models where a primary effect of insulin in the cns to which -cells act secondarily can not be ruled out ( 1,2,51 ) . thus , an in vivo positive autocrine effect of insulin on the -cell remains questionable . but in our minds , a feed - forward positive effect of insulin on insulin secretion from the -cell does not make sense physiologically . if this were the case , the more insulin secreted the bigger the autocrine stimulus would be for further insulin secretion , which would forever be increasing with time and eventually have serious deleterious consequences . in this scenario , it would be difficult to see how insulin secretion would be efficiently shut off to critically avoid hypoglycemia . 2 ) , or natural protective insulin resistance ( 18 ) , would be expected to control this , but such built - in off mechanisms take time ( a few hours ) to be effective ( 20,21 ) . in contrast , it is established that upon removal of a stimulus , such as glucose , insulin secretion returns to basal levels in minutes . as such , in the in vivo physiological context we have no disagreement that multiple downstream elements of the insulin signal transduction pathway are critical for normal -cell function , growth , survival , and general well - being ( fig . however , our thoughts and alternative interpretations have led us to believe that autocrine action of insulin is not established , especially when considering the in vivo physiological context . if insulin is not the appropriate ligand for -cells , then what might be the relevant one ? we mentioned doubts about this previously , but also note that in obesity / insulin resistance , when -cell mass and function may increase in compensation , igf-1 binding proteins also increase , which would actually decrease the an intriguing possibility might be igf-2 , which has been proposed to be cosecreted with insulin in response to incretins , and has an autocrine feedback however , this hypothesis is mostly based on in vitro observations and requires in vivo testing to substantiate . moreover , a possible autocrine feedback of igf-2 on -cells is also subjected to the same arguments of desensitization made here for insulin , so the jury should still be out on igf-2 for the time being . another consideration is that a homologous ligand may not be necessary at all , and it is the tight control of irs-2 expression in -cells through the action of many heterologous factors that acts as a gatekeeper to control downstream -cell homeostasis ( 1117 ) . moreover , the cns may drive compensatory increases in -cell function and mass in response to obesity / insulin resistance ( 42,43 ) . however , there are also a plethora of other untested growth factor ligand candidates to consider that could act on the -cell which , via their specific receptor , could induce tyrosine kinase activity to engage irs adapter molecules to then transduce downstream signaling ( fig . the question that insulin is the physiologically relevant molecule responsible for autocrine regulation of the -cell is still open . doubts and issues remain that are currently difficult to answer convincingly , however as new technologies emerge these could be better addressed experimentally in the near future . but , for the moment , we prefer use of the term irs signal transduction / signaling pathway rather than insulin signal transduction / signaling pathway in reference to elements of these signaling networks in -cells . this will avoid the implication that autocrine action of insulin in vivo is established , when in many quarters it is suggested that the concept remains unproven .

in recent years there has been a growing interest in the possibility of a direct autocrine effect of insulin on the pancreatic -cell . indeed , there have been numerous intriguing articles and several eloquent reviews written on the subject ( 13 ) ; however , the concept is still controversial . although many in vitro experiments , a few transgenic mouse studies , and some human investigations would be supportive of the notion , there exist different insights , other studies , and circumstantial evidence that question the concept . therefore , the idea of autocrine action of insulin remains a conundrum . here we outline a series of thoughts , insights , and alternative interpretations of the available experimental evidence . we ask , how convincing are these , and what are the confusing issues ? we agree that there is a clear contribution of certain downstream elements in the insulin signaling pathway for -cell function and survival , but the question of whether insulin itself is actually the physiologically relevant ligand that triggers this signal transduction remains unsettled .

Elements of insulin signaling pathways in -cells are important What concentrations of insulin are islet -cells exposed to in vivo? Influence of the central nervous system on -cell function in vivo Central issues with -cellspecific transgenic knockout models Does insulin affect insulin gene expression in vivo? Does insulin affect insulin production? Does insulin affect insulin secretion? Final thoughts and conclusions

the insulin signal transduction pathway in pancreatic -cells is similar to that in most other cell types ( fig . we do not dispute evidence that insulin receptors are expressed in -cells . moreover , it is generally accepted that some elements in the insulin signal transduction pathways play a critical role for -cell survival , growth , and general well - being ( 14 ) . these landmark studies were a catalyst to change previous thinking in the diabetes research field . before , the predominant thought was that insulin resistance was the main cause of type 2 diabetes , but it was not widely acknowledged until the 1990s that the onset of type 2 diabetes is marked by a failure of the functional -cell mass to meet the metabolic demand ( 810 ) . back then , with the realization that irs-2 signaling in -cells could be important , a plethora of studies blossomed to indicate certain downstream elements in irs-2 signaling pathways also play important roles in -cell function and survival ( fig . by contrast , it can be downregulated by proinflammatory cytokines , physiological stress , and feedback inhibition of normal irs signaling ( 4,1416 ) . it is conceivable that the relatively high expression of irs-2 and its quick turnover in -cells ( 13 ) may offset any need for constitutive activity of the insulin receptor , as it does in the liver ( 17 ) . with a controlled upregulation of irs-2 when -cell compensation is needed to maintain glucose homeostasis and downregulation of irs-2 when -cell compensation is not needed , the responsibility for insulin itself to trigger downstream signaling in -cells could be removed and placed more so on glucose , incretins , neuronal connections , and other more physiologically relevant regulators of -cell function . this leads to activation of two major signaling cascades , the ras - raf - mitogen - activated protein kinase ( mapk ) pathway ( orange ) and the phosphatidylinositol-3-kinase ( pi3k)/protein kinase - b ( pkb ; also known as akt ) signaling pathway ( green ) . for the pi3k / pkb signaling pathway , the p85 regulatory subunit of pi3k docks to other specific phosphorylated tyrosine sites on irs-1/2 that then activates its p110 catalytic activity . many of these irs signaling elements have been shown to be expressed and active and play important roles in pancreatic -cells in terms of certain functions , growth , and survival ( rev . however , despite this growing body of evidence for the necessity of irs-2regulated signaling pathways in -cells , the identity of any physiologically relevant upstream ligand / receptor interaction that triggers irs signal transduction in -cells in vivo has been rather unclear . of course , the insulin / insulin receptor interaction is an attractive candidate , but there are considerations and circumstances that raise significant doubt of an autocrine effect of insulin on -cells . prolonged exposure to insulin , and/or high concentrations of insulin in all cells that express the insulin receptor effectively desensitizes the irs signaling pathway downstream of the receptor as well as downregulates expression of insulin receptor itself ( 18 ) . internalization of the insulin receptor into an endosomal compartment , once insulin is bound contributes to this desensitization reducing the availability of cell - surface insulin receptors ( 19 ) . considering that the insulin receptor is ubiquitously expressed , such feedback inhibition mechanisms provide a dynamic means of locally controlling insulin receptor activity rather than a more complex regulation of controlling insulin receptor gene expression in a tissue - specific manner . indeed , some believe that the chronic hyperinsulinemia found in obesity and obesity - linked type 2 diabetes can make a major contribution to the insulin - resistant state because of these desensitization mechanisms ( 18 ) ( fig . once insulin has activated irs signal transduction pathways in cells , after a period of time there are internal physiological feedback inhibition signals ( indicated by red arrows ) that ensure that the insulin signal is not chronically sustained . under hyperinsulinemic conditions , this feedback results in chronic desensitization of irs signal transduction and contributes to the insulin - resistant state . 1 ) can lead to erk-1/2 protein kinase mediated serine phosphorylation ( ps ) of irs-1/2 , which results in dissociation of the insulin receptor and irs-1/2 interaction together with irs-1/2 degradation . 3-phosphoinositide - dependent protein kinase-1 ( pdk1 ) activation can result in downstream activation atypical protein kinase - c ( pkc ) isoforms , such as pkc , which can also serine phosphorylate ( ps ) irs-1/2 to promote their degradation , representing another route of delayed feedback inhibition for insulin signal transduction . another consideration for feedback inhibition of insulin action is that when insulin binds to its receptor , the insulin / insulin receptor complex is internalized into the cell where it dissociates in an endosomal compartment , allowing the free insulin receptor to return to the surface ( 18,19 ) . when insulin levels are high , this cycle is biased toward there being minimal insulin receptors on the surface of the cell with the majority being internalized , and acts as an additional physiological mechanism to prevent prolonged activation of irs signal transduction by insulin . the concentration of insulin secreted from the pancreas in vivo in response to physiological stimuli has been estimated by measuring insulin concentrations in the portal vein under normal circumstances and can reach high peak concentrations of 5 nmol / l ( 22 ) . as such , it is not too far - fetched to assume that if insulin were not efficiently cleared from the local islet milieu its concentration would be at least equally as high , if not higher . in support of this idea , recent in vivo studies in normal mice have indicated that despite a marked effect of insulin in the liver to activate molecular targets , such as sterol regulatory element binding protein-1c , there is a negligible , if any , effect of insulin on islet -cells in the very same animal at the same time ( 15 ) . although human islet cell architecture is different from that in rodents , the same directional plumbing of the islet microcirculatory network is retained in humans ( 25 ) . if insulin is efficiently dispersed away from the islet then it will be extracted from the circulation by the whole body before returning to the islet via the pancreatic artery at low peripheral picomolar concentrations ( 27 ) . such a depleted concentration of insulin could be ineffective to transduce irs signaling in -cells , which needs to be at 50 nmol / l as indicated from in vitro experiments using primary islet -cells ( 2,3 ) . it has been argued that there is always a degree of constitutive ( unregulated ) insulin secretion from normal islet -cells , and this is continually acting as a local autocrine ligand for the insulin receptor ( 13 ) . but this observation is derived from dedifferentiated tumorigenic -cell lines and transfection of the preproinsulin gene to pituitary cells , and thus it is not necessarily physiologically relevant . in fact , normal primary islet -cells efficiently sort > 99% of the newly synthesized ( pro)insulin to the regulated pathway and there is negligible , if any , constitutive secretion ( 28 ) . notwithstanding this , even if there were constitutive insulin secretion from -cells , the same two scenarios described above would still apply for a negligible autocrine effect of insulin on -cells . both sympathetic and parasympathetic input from the central nervous system ( cns ) can affect -cell function ( 29 ) . positive parasympathetic neurotransmitter signals , such as acetylcholine ( operating through muscarinic [ m3 ] receptors on -cells ) , vasoactive intestinal polypeptide , and pituitary adenylate cyclase - activating polypeptide ( 31 ) , can enter via the vagus to potentiate glucose - induced insulin secretion ( 30,31 ) . in the pancreas , the vast majority of neurons associate with pancreatic islets ( 33 ) . although it has been suggested that the neuronal connections to pancreatic islets are to endothelial cells ( 34 ) , it is nonetheless clear that the cns can have a major influence on the functional regulation on -cells ( 29,32 ) . indeed , severing the vagus nerve can significantly impair glucose - induced insulin secretion ( 29 ) . although this could be consistent with a direct priming effect of insulin on the -cell , an alternative explanation could be that a prior exposure to exogenous insulin downregulates and desensitizes insulin receptor signaling in the brain ( 18 ) . if so , the insulin - induced feedback inhibition of glucose - induced insulin secretion would be less effective . as a consequence , indeed , an inadequate feedback inhibition of insulin on endogenous insulin secretion has been observed in obese / insulin - resistant humans and is thought to play a role in maintaining the persistent hyperinsulinemia under such circumstances ( 36 ) . although the cns influence on pancreatic -cell function is often underestimated , it is attracting renewed interest . indeed , in vagotomized rats , rates of -cell proliferation decrease by half , which is associated with an 80% reduction in protein kinase - b ( pkb ; also known as akt ) activation , a key element in irs signaling pathways ( fig . this in turn suggests that the cns may have a degree of control over irs-2regulated signal transduction in -cells . however , it is noted that direct intracerebroventricular administration of insulin into the cns does not affect circulating insulin levels , but does suppress food intake and decrease body weight ( 44 ) . despite the evidence that some aspects of -cell function can be neuronally influenced , further research is needed , especially in regard to identifying the specific regions of the cns that directly communicate to the endocrine pancreas . but in terms of understanding the possible influence of insulin itself on the -cell in vivo , it seems that this may not necessarily be a direct autocrine effect but rather a secondary one mediated in the large part via the cns . there are now several transgenic rodent models that imply the need for many of the elements of the irs-2 signaling pathway for -cell function , growth and/or survival ( fig . a critical question is what is the physiologically relevant ligand that activates irs signaling pathways ? is it insulin itself ? enticing transgenic mouse models in which the insulin receptor and/or the igf-1 receptor genes have been knocked out in an intended -cell specific manner have been presented ( 4547 ) . cross - breeding these two mouse models deletes the receptors for insulin and igf-1 on -cells ( dko mice ) , rendering a more severe diabetic phenotype with apparent loss of -cell secretory capacity and function , together with elevated glucagon levels in the fed state ( 47 ) . these data were very intriguing at the time , but recently the technical approach used to generate these transgenic mouse models has been questioned ( 48,49 ) . the short form of the rat insulin gene promoter ( rip ) used to drive a cre - recombination to generate the -cell specific knockout of the insulin and/or igf-1 receptor genes is unlikely to be exclusively -cell specific ( 4547 ) . indeed , many of the rips and other -cell transcription factor ( e.g. moreover , the rip - cre mice themselves display mild glucose intolerance , apparently owing to cre - impaired first - phase insulin secretion ( 50 ) . as previously outlined , the cns can have a major influence on metabolic homeostasis , including pancreatic endocrine cell functions involving the -cell . as such , while it is likely that any potential feedback effect of insulin and/or igf-1 on the -cell is blocked in these animal models , it can not be ruled out that the primary defect driving the phenotype of the irko , igf1r , or dko mice originates in the cns and that the -cells respond secondarily to dysfunctional cns control ( 51 ) . in this regard , there are now multiple studies in which knocking out a gene using a rip - cre or pdx1-cre transgenic approach gave an obese , hyperphagic , or metabolically altered phenotype that has been primarily attributed to deleting that gene in the brain / hypothalamus rather than the -cell ( 48,49 ) . however , technology has progressed , and there is now a new transgenic mouse model in which the full - length ( 8 kb ) mouse insulin promoter drives cre - expression , apparently in -cells only ( 46 ) . future use of this mip - creer mouse to uniquely delete insulin and/or igf-1 receptors in -cells may move toward resolving some of the controversy behind autocrine action of insulin . this is often interpreted as a negative effect of insulin on insulin gene expression ( 2 ) . however , this is more likely due to the infused insulin lowering the blood glucose to a hypoglycemic state , which is known to deplete endogenous insulin stores and downregulate insulin gene expression ( 53,54 ) . however , if the glucose concentration is clamped in vivo , subsequent insulin infusion to induce hyperinsulinemia can partly reduce insulin gene expression ( 56 ) , but it remains unclear whether this is a direct effect of insulin on the -cell or one acting secondarily via the cns . it should be noted that preproinsulin mrna levels are not only transcriptionally regulated , but also at the level of preproinsulin mrna stability . indeed , physiologically relevant increases in glucose stabilize preproinsulin mrna , but this does not appear to be mediated by a local autocrine feedback of secreted insulin ( 59 ) . recently however , there has been a series of in vitro studies using transformed -cell lines or isolated islets to suggest , in contrast to established in vivo studies , that there is a positive effect of insulin to drive insulin gene expression ( 2 ) . these in vitro studies have been complemented by experiments using islets isolated from the aforementioned irko and igf1r transgenic mice ( 4547 ) . however , in these particular mouse models , there could well be a cns influence on the islets of these animals which alters their -cell mass ( 42,43,48,49,51 ) and , as previously observed , preproinsulin mrna levels parallel these changes ( 57 ) . in short , none of these in vitro studies can readily place the potential of insulin itself directly regulating its own gene expression into a proper physiological context . there is some indication that elements in the insulin signaling pathway in -cells , such as foxo1 are involved in influencing insulin gene expression in -cells ( 4 ) . however , whether these elements are directly controlled by an in vivo autocrine feedback action of insulin is not established . the major regulation of insulin production in normal pancreatic -cells occurs at the translational level ( 60 ) . when primary -cells are exposed to a stimulatory glucose concentration there is a 20- to 30-min lag period ( due to recruitment of preproinsulin mrna containing polyribosome complexes to the rough endoplasmic reticulum , the site of proinsulin biosynthesis ) before increases in proinsulin biosynthesis are observed , that can then reach an impressive 10-fold increase by 60 min . it ensures that insulin stores in -cells are rapidly replenished after a bout of insulin exocytosis . many studies have ruled out an autocrine positive feedback of insulin to drive proinsulin biosynthesis ( 5961 ) , but some have been a proponent of this possibility ( 62,63 ) . unfortunately , studies that indicate a positive effect of insulin to stimulate proinsulin biosynthesis are questionable because proinsulin biosynthesis was either not measured directly , or if it was only during the initial 30-min lag period after introduction of the stimulus , and as such these measures are inaccurate ( 62,63 ) . thus , the vast majority of evidence indicates that insulin does not have an autocrine effect on its own production in the -cell . as previously noted , there is an in vivo temporal negative feedback of insulin on stimulated insulin secretion that is mediated via the cns in humans ( 3537 ) . however , there is an intriguing suggestion that insulin may also have a positive effect to enhance its own secretion ( 13 ) . yet , the majority of studies arguing for a positive autocrine effect of insulin to drive insulin secretion have been conducted in vitro where central control no longer operates , or in transgenic mouse models where a primary effect of insulin in the cns to which -cells act secondarily can not be ruled out ( 1,2,51 ) . thus , an in vivo positive autocrine effect of insulin on the -cell remains questionable . but in our minds , a feed - forward positive effect of insulin on insulin secretion from the -cell does not make sense physiologically . if this were the case , the more insulin secreted the bigger the autocrine stimulus would be for further insulin secretion , which would forever be increasing with time and eventually have serious deleterious consequences . in this scenario , it would be difficult to see how insulin secretion would be efficiently shut off to critically avoid hypoglycemia . 2 ) , or natural protective insulin resistance ( 18 ) , would be expected to control this , but such built - in off mechanisms take time ( a few hours ) to be effective ( 20,21 ) . as such , in the in vivo physiological context we have no disagreement that multiple downstream elements of the insulin signal transduction pathway are critical for normal -cell function , growth , survival , and general well - being ( fig . however , our thoughts and alternative interpretations have led us to believe that autocrine action of insulin is not established , especially when considering the in vivo physiological context . we mentioned doubts about this previously , but also note that in obesity / insulin resistance , when -cell mass and function may increase in compensation , igf-1 binding proteins also increase , which would actually decrease the an intriguing possibility might be igf-2 , which has been proposed to be cosecreted with insulin in response to incretins , and has an autocrine feedback however , this hypothesis is mostly based on in vitro observations and requires in vivo testing to substantiate . another consideration is that a homologous ligand may not be necessary at all , and it is the tight control of irs-2 expression in -cells through the action of many heterologous factors that acts as a gatekeeper to control downstream -cell homeostasis ( 1117 ) . moreover , the cns may drive compensatory increases in -cell function and mass in response to obesity / insulin resistance ( 42,43 ) . however , there are also a plethora of other untested growth factor ligand candidates to consider that could act on the -cell which , via their specific receptor , could induce tyrosine kinase activity to engage irs adapter molecules to then transduce downstream signaling ( fig . the question that insulin is the physiologically relevant molecule responsible for autocrine regulation of the -cell is still open . doubts and issues remain that are currently difficult to answer convincingly , however as new technologies emerge these could be better addressed experimentally in the near future . but , for the moment , we prefer use of the term irs signal transduction / signaling pathway rather than insulin signal transduction / signaling pathway in reference to elements of these signaling networks in -cells . this will avoid the implication that autocrine action of insulin in vivo is established , when in many quarters it is suggested that the concept remains unproven .

manipulation of the host immune response is the most precise and effective tool to lower down the disease incidences and to nullify the limitations associated with antibiotic treatment or vaccination . cd molecule ranges from 1 to 166 with differential structure and functions , of these cd14 is the most important molecule known so far , playing a vital role against several enterotoxigenic bacteria . its pattern recognition receptor binds mainly with lps ( lipopolysaccharide ) , lipotechoic acid , arachidonic acid and thus releases various cytokines which act for body 's defence . body 's immunity thus can act against a wide range of pathogens including gram - negative bacteria and gram positive as mycobacterium sp . cd14 functions both as a cell membrane receptor and a soluble receptor for bacterial lps . it has been considered as an important molecule for its role on various diseases , like mastitis , treponemiasis and glomerulonephritis . recombinant protein may be used as therapeutic agent ; cloned gene insertion may be utilized for somatic gene therapy and by the development of transgenic disease resistant animals . detection of snp is useful for analysis of the evolutionary history of species development , assessment of biodiversity , associative studies between polymorphisms , and disease - resistance . scanty reports are available so far for genetic polymorphism of cd14 gene in an animal at coding region ; however , cd14 gene polymorphism study at promoter region is available in human [ 68 ] . in recent days , crossbred ( cb ) cattle are gaining much importance being high milk yielder compared to indigenous cattle in developing countries . cb cattle arise due to crossing of low yielding bos indicus with high yielding bos taurus , preferably holstein friesian or jersey . in india , cb cattle constitute 13.3% of the total cattle population with high growth rate for cb milch cattle ( 34.4% ) , whereas indigenous milch cattle has decreased by 6.1% . there is a tremendous increase in the cb cattle in the country that is , 22.8% but the indigenous cattle declined by 10.2% . however , the major drawback was that cb cattle are poor in adaptability and disease resistance compared to indigenous cattle . scanty reports are available regarding cd14 gene in bos taurus [ 10 , 11 ] . so far no reports are available regarding molecular characterization of cd14 gene in cb cattle or its protein structure . keeping the above facts in mind , the present investigation has been planned to clone and sequence the cd14 gene of crossbred cattle , study of cd14-derived peptide using bioinformatics tools , to compare the sequence homology with other species , and to identify the snp of cd14 gene of cb cattle . blood was collected aseptically by jugular vein puncture over 2.7% ethylenediamine tetraacetic acid from healthy cb cattle . peripheral blood mononuclear cells ( pbm cells ) were isolated from whole blood by density gradient centrifugation using lymphocyte separation media ( lsm ) ( himedia ) . the total rna was isolated from separated cells using trizol ( life technologies , usa ) , following manufacturer 's instructions and was further used for cdna synthesis . the 20 l reaction mixture contained 5 g of total rna , 0.5 g of oligo dt primer ( 1618 mer ) , 40 u of ribonuclease inhibitor , 1000 m of dntp mix , 10 mm of dtt , and 5 u of mumlv reverse transcriptase in reverse transcriptase buffer . the reaction was stopped by heating the mixture at 70c for 10 minutes and chilled on ice . the integrity of the cdna checked by pcr . to amplify full length open reading frame ( orf ) of cd14 gene sequence , a specific primers pair was designed based on the cd14 mrna sequences of cattle ( accession no - acc no . the primers were forward : cd14 - 1-f atggtctgcgtgccctacctg and reverse : cd14 - 70 - 1-r ggagcccgaggcttcgcgtaa . 25 l reaction mixture contained 80100 ng cdna , 3.0 l 10x pcr assay buffer , 0.5 l of 10 mm dntp , 1 u taq dna polymerase , 60 ng of each primer , and 2 mm mgcl2 . pcr - reactions were carried out in a thermocycler ( ptc-200 , mj research , usa ) with cycling conditions as , initial denaturation at 94c for 3 min , denaturation at 94c for 30 sec , annealing at 61c for 35 sec , and extension at 72c for 3 min were carried out for 35 cycles followed by final extension at 72c for 10 min . pcr amplicons verified by 1% agarose gel electrophoresis were purified from gel using gel extraction kit ( qiagen gmbh , hilden , germany ) and ligated into pgem - t easy cloning vector ( promega , madison , wi , usa ) following manufacturers ' instructions . the 10 l of ligated product was directly added to 200 l competent cells , and heat shock was given at 42c for 45 sec . in a water bath , and cells were then immediately transferred on chilled ice for 5 min . , and the bacterial culture was pelleted and plated on lb agar plate containing ampicillin ( 100 mg / ml ) added to agar plate @ 1 : 1000 , iptg ( 200 mg / ml ) and x - gal ( 20 mg / ml ) for blue - white screening . plasmid isolation from overnight - grown culture was done by small - scale alkaline lysis method described by sambrook and russell . recombinant plasmids were characterized by pcr using gene - specific primers and restriction enzyme digestion based on reported nucleotide sequence for cattle . cd14 gene fragment insert in recombinant plasmid was sequenced by automated sequencer ( abi prism ) using dideoxy chain termination method with t7 and sp6 primers ( chromous biotech , bangalore ) . the nucleotide sequence so obtained was analyzed for protein translation , sequence alignments , and contigs comparisons by dnastar version 4.0 , inc . , usa . novel sequence was submitted to the ncbi genbank and accession number ( gu368102 ) was obtained which is available in public domain now . phylogenetic analysis was also performed to determine the evolutionary relationship of cb cattle with other species . nucleotides sequences were then aligned with that of the reported cd14 sequences of different species including bubalus bubalis ( dq457089 ) , bos taurus ( nm_174008 ) , canis familiaris ( xp_848746 ) , capra hircus ( dq457090 ) , homo sapiens ( nm_000591 ) , monkey ( xp_517975 ) , mus musculus ( nm_009841 ) , rattus norvegicus ( np 068512 ) , gallus gallus ( am933591 ) , equus caballus ( af200416 ) , macaca mulatta ( nm_001130433 ) sus scrofa ( ef051626 ) , ovis aries ( ay289201 ) , and oryctolagus cuniculus ( m85233.1 ) using the clusterw method of multiple alignment which is slow and accurate ( megalign programme of lasergene software , dnastar ) . predicted peptide sequence of cd14 gene of cb cattle was derived by edit sequence ( lasergene software , dnastar ) and then aligned with the cd14 peptide of other livestock and avian using megalign sequence programme of lasergene software ( dnastar ) . prediction of signal peptide of cd14 gene was conducted using the software ( signal p 3.0 sewer - prediction results , technical university of denmark ) . di - sulphide bonds were predicted using suitable software ( http://bioinformatics.bc.edu/clotelab/dianna/ ) and by homology search with other species cd14 polypeptide . protein sequence level analysis study was carried out with specific software ( http://www.expasy.org./tools/blast/ ) for determination of leucine rich repeats ( lrr ) , leucine zipper , n - linked glycosylation sites , detection of leucine - rich nuclear export signals ( nes ) , and detection of the position of gpi anchor . detection of leucine - rich nuclear export signals ( nes ) was carried out with netnes 1.1 server , technical university of denmark . analysis of o - linked glycosylation sites was carried out using netoglyc 3.1 server ( http://www.expassy.org/ ) , whereas n - linked glycosylation site were detected by netnglyc 1.0 software ( http://www.expassy.org/ ) . regions for alpha helix and beta sheet were predicted using netsurfp - protein surface accessibility and secondary structure predictions , technical university of denmark . lps - binding site as well as lps - signaling sites were predicted based on homology studies with other species cd14 polypeptide . cd14 gene of cb cattle was observed to be 1.12 kb when checked in 1% agarose gel electrophoresis ( figure 1 ) . the fragment was cloned into pgem - t easy vector . for the confirmation of insert , the transformants were screened using colony pcr followed by plasmid pcr and re digestion of plasmid with ecor - i as restriction enzyme released the insert of approximately 1.12 kb from the plasmid ( figure 2 ) . cd14 gene of cb cattle amplified was observed to be 1122 bp . since this is the first report for study of cd14 gene in cb cattle , comparison was not possible . however , similar nucleotide length was observed in bos taurus [ 11 , 20 , 21 ] , capra hircus , and bubalus bubalis . the dna insert of cd14 gene was found to be exactly 1122 bp with atg as start codon followed by an open reading frame of 1119 nucleotides , after sequencing of a selected representative clone ( figure 3 ) . the comparison of obtained nucleotide sequence and derived amino acid sequence using multiple alignment ( cluster w , slow and accurate ) with available cd14 nucleotide sequence of cattle and buffalo confirmed that the insert was cd14 gene . gc content of cb cattle cd14 gene was found to be as high as 62.57% , similar to bos taurus ( 62.75% ) . this is slightly more than bubalus bubalis ( 62.3% ) and capra hircus ( 62.2% ) , slightly less than homo sapiens ( 62.94% ) . gc content of cb cattle is much higher than rattus norvegicus ( 56.12% ) and lower than canis familiaris ( 64.07% ) . cb cattle cd14 gene is 98.1 , 96.0 , 94.7 , 90.5 , 81.5 , 81.1 , 78.2 , 78.1 , 76.5 , 70.6 , 68.5 , 59.3 , 11.0% identical to bos taurus , bubalus bubalis , ovis aries , capra hircus , sus scrofa , equus caballus , homo sapiens , canis familiaris , oryctolagus cuniculus , mus musculus , rattus norvegicus , macaca mulatta , and gallus gallus cd14 gene ( table 1 ) . phylogenetic analysis of cb cattle with bos taurus and other species indicates that cb cattle and bos taurus are genetically the most similar followed by bubalus bubalis ( figure 4 ) . phylogenetic closeness of buffalo with cattle as observed in the present study has also been reported while studying other genes like growth hormone gene . bos taurus cd14 gene sequence when compared to other species , also revealed that cattle was phylogenetically close to buffalo . comparative sequencing of nucleotide sequences of crossbred cattle ( bos indicus x bos taurus ) with bubalus bubalis revealed similar results [ 23 , 25 ] . phylogenetic analysis of cb cattle with bos taurus and other species indicates that cb cattle and bos taurus are genetically most similar ( 98.1% ) , and thus grouped together . 1.9% dissimilarity between cb cattle and bos taurus may be due to the inheritance of bos indicus in cb cattle . cb cattle were found to be the most distant to mouse and rat among the mammalian species studied . thus it is evident that cb cattle has evolved from an ancestor which is genetically much distant than that of rattus norvegicus or mus musculus . the predicted peptide sequence of cd14 gene of crossbred cattle revealed 373 amino acids precursor corresponding to coding sequence of cd14 gene ( figure 3 ) and a 20 amino acid signal peptide . this is similar to bos taurus and capra hircus , where cd14 peptide also contains 373 amino acids . ( 39939.29 daltons ) than bos taurus ( 39679.96 daltons ) and bubalus bubalis ( 39705.07 daltons ) . cb cattle cd14 peptide sequence was characterized by the presence of two extra strongly basic amino acids and one extra strongly acidic amino acid than bos taurus , whereas less by one hydrophobic and one polar amino acid than bos taurus . protein sequence level analysis study revealed that nine leucine rich repeats ( lrr ) have been identified in predicted peptide sequence of cb cattle cd14 cdna ( table 2 , figure 5 ) . these lrrs are considered to participate in receptor and ligand interactions and have various cellular functions during early phases of the immune response . comparison of cb cattle cd14 gene with other spp . revealed that homo sapiens , mus musculus , bos taurus , capra hircus , and bubalus bubalis was reported to have 10 , 10 , 10 , 7 , and 6 lrr , respectively [ 23 , 27 ] . it has already been reported that lrr in extracellular domain is responsible for the recognition of pathogens . crystal structure of mus musculus cd14 gene has been depicted with the grooves responsible for receptor recognition and binding . since cd14 molecules can bind to a wide range of substances including lipopolysaccharides from gram - negative bacteria , lipoarabinomannan of mycobacteria , mannuronic acid polymers of pseudomonas sp . , and to peptidoglycans of staphylococcus aureus , it is expected that there should be sufficient variability in the pathogen recognition and receptor binding site , which is primarily comprised of lrr region of the cd14 molecule . thus , the presence of lrr coding region may be the region for maximum variability to enable the cd14 molecule to bind with a wide range of substances . from the sequence alignment studies for the derived amino acid sequences for different species , it was observed that the particular amino acid leucine was almost unaltered within the leucine rich repeats , and the variations were observed for other amino acids . although there are no reports regarding the number of lrr and its relation to disease susceptibility , a trend has been observed with more the species is resistant , less the no . of lrr . as reported from a separate study , buffalo is most resistant among common farm animals , namely , cattle , goat , sheep , and buffalo . so , it may be possible that the lesser number of lrr , the more it is resistant to diseases . the cd14 derived - peptide sequence of crossbred cattle contained 4 putative n - linked glycosylation sites ( figure 5 , table 3 ) whereas , there is report of three , five , four , five , and four glycosylation sites for n - linked glycosylation in bos taurus , mus musculus , homo sapiens , rattus norvegicus , bubalus bubalis , respectively . n - linked glycosylation is vital for the molecule as it supports the molecule to be present either in membranous or soluble form . cb cattle contain 5 sites for o - linked glycosylation ( figure 5 ) at amino acid positions 128 , 131 , 134 , 145 , 147 , which differs from bubalus bubalis containing 3 sites . however , o - linked glycosylation was reported to be absent in homo sapiens purified native cd14 molecule . glycosylation is needed when hydrophilic clusters of carbohydrates alter the polarity and solubility of protein or protein folding . protein sequence level analysis study revealed that in cb cattle , there was a glycosyl phosphatidyl inositol ( gpi ) anchor located at c - terminus near 345th position of the cd14 molecule ( figure 5 ) . it differs from bubalus bubalis containing glycosyl phosphatidyl inositol ( gpi ) anchor located at c - terminus near 353rd position of the cd14 molecule . similar findings were reported by wright in other species like and mus musculus . gpi anchor is vital for the molecule as the basic function of gpi in mammalian cell system is to make a bridge between cd14 and cell surface . however , in case of avains , cd14 is trans - membrane rather than gpi anchored . alignment of derived peptide sequence of cb cattle with other species ( figure 5 ) revealed that leucine residues are conserved across the species ( figure 5 ) . cystine residue has also been observed to be conserved across species ( figure 5 ) . five disulphide bridges have been predicted between cysteine residues in cd14 polypeptide of cb cattle of which only four were visualized in figure 5 . since cys287 is a tyrosine in the mouse cd14 sequence , in the homology study of cd14 sequences for different species ( figure 5 ) , the fifth disulphide bond is not visualized . the crystal structure of mouse cd14 also sheds light on the sites of cotranslational modifications of human cd14 . similar five disulfide bonds are implicated from the biochemical studies in human . in human cd14 , a differential impact is observed for the five disulfide bonds on cd14 folding , with the first two being indispensable , the third and fourth being important , and the last ( cys287cys333 ) being dispensable . when mapped to the crystal structure of mouse cd14 , the first two disulfide bonds are found in the sheets in the inner concave surface ( the core structure ) , whereas the third and fourth disulfide bonds are in the loops and helices on the outer surface ( the peripheral structure ) . the last disulfide bond ( cys287cys333 ) is not seen in the structure , because the construct used for crystallization has the truncation of c - terminal 33 amino acids , and cys287 is a tyrosine in the mouse cd14 sequence . the first disulfide bond is therefore essential for the structural and functional integrity of cd14 . the third and fourth disulfide bonds contribute to but do not determine cd14 folding , because the substitutions of these cysteines with alanines decrease but do not abrogate cd14 secretion . the last disulfide bond ( cys287cys333 ) has no effect on cd14 folding . in cb cattle , four lps - binding sites were depicted from 29th32nd , 44th47th , 55th59th , and 75th80th amino acid positions ( figure 5 ) . the structural characteristics of the binding site may explain the broad ligand specificity of cd14 . although the hydrophobic bottom and walls of the pocket are rigid , the generous size of the pocket may allow structural variation in the hydrophobic portion of the ligand . structural diversity in the hydrophilic part of the ligands could be explained by the considerable flexibility of the hydrophilic rim combined with the multiplicity of grooves available for ligand binding . this is also the case of mus musculus cd14 molecule reported by sambrook and russell . lps - binding site is in agreement with the present study that n - terminal region of homo sapiens cd14 molecule is responsible for lps binding . three lps signaling sites were predicted for amino acid positions as 27th33th , 109th119th , and 169th171th amino acid ( figure 5 ) . similar observations were reported by sambrook and russell in mus musculus with four lps - binding sites and three lps signaling sites . this is similar to mus musculus cd14 molecule , as reported by sambrook and russell . in terms of secondary structure prediction , five regions were detected for helix conformation as 412 , 6773 , 241244 , 343346 , and 360368 , and eleven regions were identified for strand conformation as 60 - 61 , 118122 , 145147 , 173176 , 181 - 182 , 197199 , 225227 , 252254 , 278280 , 299301 , 321324 amino acid positions ( figure 5 ) . however , in mus musculus , 7 alpha helix , and 13 beta sheets were identified . the monomeric subunit of cd14 contains eleven strands , and 9 of them , from 2 , 4 to 11 , overlap with conserved leucine - rich repeats ( lrrs ) ( figure 5 ) . leucine - rich nuclear export signals were detected for 7 sites at amino acid positions 12 , 15 , 16 , 17 , 117 , 122 , 127 ( figure 5 ) . a nuclear export signal ( nes ) is a short amino acid sequence of 4 hydrophobic residues in a protein that targets it for export from the cell nucleus to the cytoplasm through the nuclear pore complex . however , it is the first report of prediction of the site of leucine - rich nuclear export signal in cd14 gene in animal . dna - binding motif as leucine zipper pattern was detected from amino acid position 279 of cb cd14 peptide ( figure 5 ) , which is reported here for the first time in cd14 molecule . these motifs are usually found as part of a dna - binding domain in various transcription factors and are therefore involved in regulating gene expression . the leucine zipper is a supersecondary structure that functions as a dimerization domain , and its presence generates adhesion forces in parallel alpha helices . however , the site of leucine zipper in cd14 gene is predicted here for the first time in any animal . domain linker site was predicted for amino acid positions 121146 and 283334 ( figure 5 ) . linker are likely to act as a scaffold to prevent unfavourable interactions between folding domains . recent advances in protein engineering have come from creating multifunctional chimeric proteins containing modules from various proteins . these modules are typically joined via an oligopeptide linker , the correct design of which is crucial for the desired function of the chimeric protein . analysis of the properties of naturally occurring interdomain linkers is useful with the aim to design linkers for domain fusion leading to chimeric protein formation . cb cattle cd14 molecule is predicted to be mostly expressed on cell membrane or cell surface . since cd14 is a receptor molecule , it is obvious to be expressed on cell surface . moreover , in the present study , cd14 cdna was prepared from mrna expressed on cells of liver tissue . cd14 exists in two forms as membranous and soluble form [ 42 , 43 ] . in the present study , cd14 cdna cloned is the membranous form as it contains gpi anchor for the attachment with cell membrane as well as glycosylated form [ 42 , 43 ] . 3d model of cd14 molecule of cb cattle was predicted from amino acid position 24 to 331 ( figure 6 ) , with horseshoe - shaped structure , with alternating alpha helix and beta chains . cd14 exists as dimmer with the help of leucine zipper when acting as a receptor molecule . the finding is similar to study conducted by sambrook and russell , where they studied crystal structure of cd14 molecule of mus musculus . based on homology study for cd14 3d structure with mus musculus and homo sapiens , lps - binding pocket was predicted at the amino terminal end of cb cattle cd14 molecule , helix were located at the convex surface , and strand were located at the concave surface of horse - shoe shaped cd14 structure , represented in figure 6 . as expected , 27 nucleotide changes were observed , when compared with the published sequences of cd14 gene of bos taurus , out of which 25 were newly reported . 6 synonymous changes and 18 amino acid changes were reported out of which 17 were new changes ( table 4 ) . similar higher degrees of mutations have been observed in some other genes as 25 snps in leptin gene in korean cattle , 96 snps from tlrs , and their associated intracellular signaling molecules in human . nonsynonymous substitutions exceeding synonymous substitutions indicate the evolution of this protein through positive selection among domestic animals . snp for the cd14 gene of crossbred cattle was reported here for the first time . an interesting observation is that the cd14 nucleotide sequence from 1 to 258 has no major snp detected , except one a230c . this may be the reason that 1 to 60 nucleotide codes for signal peptide , when no variation is expected . the present finding is similar to bubalus bubalis , when monomorphism was reported for this nucleotide region except for one nucleotide change that is too within lps - binding site . this allele is most probably contributed by bos indicus , since none of the bos taurus contains this allele . another interesting observation is that about 50% of the nucleotide changes , including 3 synonymous changes have been reported from 602nd to 800th nucleotide region of cd14 gene of cb cattle , that is , within 199 nucleotide . so , this region may be the hyper - variable region containing mutational hot spot . similar findings were also reported in bubalus bubalis , where 42 snp were identified with 39 nonsynonymous changes , leading to 23 amino acid changes . cd14 gene of crossbred cattle ( bos indicus crossed with bos taurus ) has been characterized for the first time in the present study . 25 snps with 17 amino acid changes were newly reported , and the site for mutational hot - spot , detected in cb cattle cd14 gene . hyper - variable regions containing mutational hot spot have been reported from 602nd to 800th nucleotide region of cd14 gene of cb cattle , that is , within 199 nucleotide . further research need to be directed to find out the association of allelic variants with traits related to disease incidences , to establish it as marker . gene insert containing the resistant variety of cd14 gene of cb cattle can be used for somatic gene therapy , particularly against mastitis . transgenic animal production with cb cattle cd14 gene insert may provide the scope for future research to develop disease - resistant stock .

cd14 is an important molecule for innate immunity that can act against a wide range of pathogens . the present paper has characterized cd14 gene of crossbred ( cb ) cattle ( bos indicusbos taurus ) . cloning and sequence analysis of cd14 cdna revealed 1119 nucleotide long open reading frame encoding 373 amino acids protein and 20 amino acids signal peptide . cb cattle cd14 gene exhibited a high percentage of nucleotide identity ( 59.398.1% ) with the corresponding mammalian homologs . cattle and buffalo appear to have diverged from a common ancestor in phylogenetic analysis . 25 snps with 17 amino acid changes were newly reported and the site for mutational hot - spot was detected in cb cattle cd14 gene . non - synonymous substitutions exceeding synonymous substitutions indicate the evolution of this protein through positive selection among domestic animals . predicted protein structures obtained from deduced amino acid sequence indicated cb cattle cd14 molecule to be a receptor with horse shoe - shaped structure . the sites for lps binding , lps signalling , leucine - rich repeats , putative n - linked glycosylation , o - linked glycosylation , glycosyl phosphatidyl inositol anchor , disulphide bridges , alpha helix , beta strand , leucine rich nuclear export signal , leucine zipper and domain linker were predicted . most of leucine and cysteine residues remain conserved across the species .

1. Introduction 2. Materials and Methods 3. Results and Discussion 4. Conclusion

cd molecule ranges from 1 to 166 with differential structure and functions , of these cd14 is the most important molecule known so far , playing a vital role against several enterotoxigenic bacteria . body 's immunity thus can act against a wide range of pathogens including gram - negative bacteria and gram positive as mycobacterium sp . it has been considered as an important molecule for its role on various diseases , like mastitis , treponemiasis and glomerulonephritis . detection of snp is useful for analysis of the evolutionary history of species development , assessment of biodiversity , associative studies between polymorphisms , and disease - resistance . scanty reports are available so far for genetic polymorphism of cd14 gene in an animal at coding region ; however , cd14 gene polymorphism study at promoter region is available in human [ 68 ] . in recent days , crossbred ( cb ) cattle are gaining much importance being high milk yielder compared to indigenous cattle in developing countries . cb cattle arise due to crossing of low yielding bos indicus with high yielding bos taurus , preferably holstein friesian or jersey . in india , cb cattle constitute 13.3% of the total cattle population with high growth rate for cb milch cattle ( 34.4% ) , whereas indigenous milch cattle has decreased by 6.1% . there is a tremendous increase in the cb cattle in the country that is , 22.8% but the indigenous cattle declined by 10.2% . however , the major drawback was that cb cattle are poor in adaptability and disease resistance compared to indigenous cattle . scanty reports are available regarding cd14 gene in bos taurus [ 10 , 11 ] . so far no reports are available regarding molecular characterization of cd14 gene in cb cattle or its protein structure . keeping the above facts in mind , the present investigation has been planned to clone and sequence the cd14 gene of crossbred cattle , study of cd14-derived peptide using bioinformatics tools , to compare the sequence homology with other species , and to identify the snp of cd14 gene of cb cattle . blood was collected aseptically by jugular vein puncture over 2.7% ethylenediamine tetraacetic acid from healthy cb cattle . to amplify full length open reading frame ( orf ) of cd14 gene sequence , a specific primers pair was designed based on the cd14 mrna sequences of cattle ( accession no - acc no . pcr - reactions were carried out in a thermocycler ( ptc-200 , mj research , usa ) with cycling conditions as , initial denaturation at 94c for 3 min , denaturation at 94c for 30 sec , annealing at 61c for 35 sec , and extension at 72c for 3 min were carried out for 35 cycles followed by final extension at 72c for 10 min . , and the bacterial culture was pelleted and plated on lb agar plate containing ampicillin ( 100 mg / ml ) added to agar plate @ 1 : 1000 , iptg ( 200 mg / ml ) and x - gal ( 20 mg / ml ) for blue - white screening . cd14 gene fragment insert in recombinant plasmid was sequenced by automated sequencer ( abi prism ) using dideoxy chain termination method with t7 and sp6 primers ( chromous biotech , bangalore ) . phylogenetic analysis was also performed to determine the evolutionary relationship of cb cattle with other species . predicted peptide sequence of cd14 gene of cb cattle was derived by edit sequence ( lasergene software , dnastar ) and then aligned with the cd14 peptide of other livestock and avian using megalign sequence programme of lasergene software ( dnastar ) . prediction of signal peptide of cd14 gene was conducted using the software ( signal p 3.0 sewer - prediction results , technical university of denmark ) . di - sulphide bonds were predicted using suitable software ( http://bioinformatics.bc.edu/clotelab/dianna/ ) and by homology search with other species cd14 polypeptide . protein sequence level analysis study was carried out with specific software ( http://www.expasy.org./tools/blast/ ) for determination of leucine rich repeats ( lrr ) , leucine zipper , n - linked glycosylation sites , detection of leucine - rich nuclear export signals ( nes ) , and detection of the position of gpi anchor . detection of leucine - rich nuclear export signals ( nes ) was carried out with netnes 1.1 server , technical university of denmark . analysis of o - linked glycosylation sites was carried out using netoglyc 3.1 server ( http://www.expassy.org/ ) , whereas n - linked glycosylation site were detected by netnglyc 1.0 software ( http://www.expassy.org/ ) . regions for alpha helix and beta sheet were predicted using netsurfp - protein surface accessibility and secondary structure predictions , technical university of denmark . lps - binding site as well as lps - signaling sites were predicted based on homology studies with other species cd14 polypeptide . cd14 gene of cb cattle was observed to be 1.12 kb when checked in 1% agarose gel electrophoresis ( figure 1 ) . cd14 gene of cb cattle amplified was observed to be 1122 bp . since this is the first report for study of cd14 gene in cb cattle , comparison was not possible . the dna insert of cd14 gene was found to be exactly 1122 bp with atg as start codon followed by an open reading frame of 1119 nucleotides , after sequencing of a selected representative clone ( figure 3 ) . the comparison of obtained nucleotide sequence and derived amino acid sequence using multiple alignment ( cluster w , slow and accurate ) with available cd14 nucleotide sequence of cattle and buffalo confirmed that the insert was cd14 gene . gc content of cb cattle cd14 gene was found to be as high as 62.57% , similar to bos taurus ( 62.75% ) . gc content of cb cattle is much higher than rattus norvegicus ( 56.12% ) and lower than canis familiaris ( 64.07% ) . cb cattle cd14 gene is 98.1 , 96.0 , 94.7 , 90.5 , 81.5 , 81.1 , 78.2 , 78.1 , 76.5 , 70.6 , 68.5 , 59.3 , 11.0% identical to bos taurus , bubalus bubalis , ovis aries , capra hircus , sus scrofa , equus caballus , homo sapiens , canis familiaris , oryctolagus cuniculus , mus musculus , rattus norvegicus , macaca mulatta , and gallus gallus cd14 gene ( table 1 ) . phylogenetic analysis of cb cattle with bos taurus and other species indicates that cb cattle and bos taurus are genetically the most similar followed by bubalus bubalis ( figure 4 ) . phylogenetic closeness of buffalo with cattle as observed in the present study has also been reported while studying other genes like growth hormone gene . bos taurus cd14 gene sequence when compared to other species , also revealed that cattle was phylogenetically close to buffalo . comparative sequencing of nucleotide sequences of crossbred cattle ( bos indicus x bos taurus ) with bubalus bubalis revealed similar results [ 23 , 25 ] . phylogenetic analysis of cb cattle with bos taurus and other species indicates that cb cattle and bos taurus are genetically most similar ( 98.1% ) , and thus grouped together . 1.9% dissimilarity between cb cattle and bos taurus may be due to the inheritance of bos indicus in cb cattle . cb cattle were found to be the most distant to mouse and rat among the mammalian species studied . thus it is evident that cb cattle has evolved from an ancestor which is genetically much distant than that of rattus norvegicus or mus musculus . the predicted peptide sequence of cd14 gene of crossbred cattle revealed 373 amino acids precursor corresponding to coding sequence of cd14 gene ( figure 3 ) and a 20 amino acid signal peptide . this is similar to bos taurus and capra hircus , where cd14 peptide also contains 373 amino acids . cb cattle cd14 peptide sequence was characterized by the presence of two extra strongly basic amino acids and one extra strongly acidic amino acid than bos taurus , whereas less by one hydrophobic and one polar amino acid than bos taurus . protein sequence level analysis study revealed that nine leucine rich repeats ( lrr ) have been identified in predicted peptide sequence of cb cattle cd14 cdna ( table 2 , figure 5 ) . comparison of cb cattle cd14 gene with other spp . revealed that homo sapiens , mus musculus , bos taurus , capra hircus , and bubalus bubalis was reported to have 10 , 10 , 10 , 7 , and 6 lrr , respectively [ 23 , 27 ] . it has already been reported that lrr in extracellular domain is responsible for the recognition of pathogens . crystal structure of mus musculus cd14 gene has been depicted with the grooves responsible for receptor recognition and binding . since cd14 molecules can bind to a wide range of substances including lipopolysaccharides from gram - negative bacteria , lipoarabinomannan of mycobacteria , mannuronic acid polymers of pseudomonas sp . , and to peptidoglycans of staphylococcus aureus , it is expected that there should be sufficient variability in the pathogen recognition and receptor binding site , which is primarily comprised of lrr region of the cd14 molecule . thus , the presence of lrr coding region may be the region for maximum variability to enable the cd14 molecule to bind with a wide range of substances . from the sequence alignment studies for the derived amino acid sequences for different species , it was observed that the particular amino acid leucine was almost unaltered within the leucine rich repeats , and the variations were observed for other amino acids . although there are no reports regarding the number of lrr and its relation to disease susceptibility , a trend has been observed with more the species is resistant , less the no . as reported from a separate study , buffalo is most resistant among common farm animals , namely , cattle , goat , sheep , and buffalo . the cd14 derived - peptide sequence of crossbred cattle contained 4 putative n - linked glycosylation sites ( figure 5 , table 3 ) whereas , there is report of three , five , four , five , and four glycosylation sites for n - linked glycosylation in bos taurus , mus musculus , homo sapiens , rattus norvegicus , bubalus bubalis , respectively . n - linked glycosylation is vital for the molecule as it supports the molecule to be present either in membranous or soluble form . cb cattle contain 5 sites for o - linked glycosylation ( figure 5 ) at amino acid positions 128 , 131 , 134 , 145 , 147 , which differs from bubalus bubalis containing 3 sites . however , o - linked glycosylation was reported to be absent in homo sapiens purified native cd14 molecule . protein sequence level analysis study revealed that in cb cattle , there was a glycosyl phosphatidyl inositol ( gpi ) anchor located at c - terminus near 345th position of the cd14 molecule ( figure 5 ) . it differs from bubalus bubalis containing glycosyl phosphatidyl inositol ( gpi ) anchor located at c - terminus near 353rd position of the cd14 molecule . however , in case of avains , cd14 is trans - membrane rather than gpi anchored . alignment of derived peptide sequence of cb cattle with other species ( figure 5 ) revealed that leucine residues are conserved across the species ( figure 5 ) . cystine residue has also been observed to be conserved across species ( figure 5 ) . five disulphide bridges have been predicted between cysteine residues in cd14 polypeptide of cb cattle of which only four were visualized in figure 5 . the crystal structure of mouse cd14 also sheds light on the sites of cotranslational modifications of human cd14 . in human cd14 , a differential impact is observed for the five disulfide bonds on cd14 folding , with the first two being indispensable , the third and fourth being important , and the last ( cys287cys333 ) being dispensable . the last disulfide bond ( cys287cys333 ) is not seen in the structure , because the construct used for crystallization has the truncation of c - terminal 33 amino acids , and cys287 is a tyrosine in the mouse cd14 sequence . the first disulfide bond is therefore essential for the structural and functional integrity of cd14 . in cb cattle , four lps - binding sites were depicted from 29th32nd , 44th47th , 55th59th , and 75th80th amino acid positions ( figure 5 ) . the structural characteristics of the binding site may explain the broad ligand specificity of cd14 . structural diversity in the hydrophilic part of the ligands could be explained by the considerable flexibility of the hydrophilic rim combined with the multiplicity of grooves available for ligand binding . this is also the case of mus musculus cd14 molecule reported by sambrook and russell . lps - binding site is in agreement with the present study that n - terminal region of homo sapiens cd14 molecule is responsible for lps binding . three lps signaling sites were predicted for amino acid positions as 27th33th , 109th119th , and 169th171th amino acid ( figure 5 ) . in terms of secondary structure prediction , five regions were detected for helix conformation as 412 , 6773 , 241244 , 343346 , and 360368 , and eleven regions were identified for strand conformation as 60 - 61 , 118122 , 145147 , 173176 , 181 - 182 , 197199 , 225227 , 252254 , 278280 , 299301 , 321324 amino acid positions ( figure 5 ) . however , in mus musculus , 7 alpha helix , and 13 beta sheets were identified . the monomeric subunit of cd14 contains eleven strands , and 9 of them , from 2 , 4 to 11 , overlap with conserved leucine - rich repeats ( lrrs ) ( figure 5 ) . leucine - rich nuclear export signals were detected for 7 sites at amino acid positions 12 , 15 , 16 , 17 , 117 , 122 , 127 ( figure 5 ) . a nuclear export signal ( nes ) is a short amino acid sequence of 4 hydrophobic residues in a protein that targets it for export from the cell nucleus to the cytoplasm through the nuclear pore complex . however , it is the first report of prediction of the site of leucine - rich nuclear export signal in cd14 gene in animal . dna - binding motif as leucine zipper pattern was detected from amino acid position 279 of cb cd14 peptide ( figure 5 ) , which is reported here for the first time in cd14 molecule . the leucine zipper is a supersecondary structure that functions as a dimerization domain , and its presence generates adhesion forces in parallel alpha helices . however , the site of leucine zipper in cd14 gene is predicted here for the first time in any animal . domain linker site was predicted for amino acid positions 121146 and 283334 ( figure 5 ) . analysis of the properties of naturally occurring interdomain linkers is useful with the aim to design linkers for domain fusion leading to chimeric protein formation . cb cattle cd14 molecule is predicted to be mostly expressed on cell membrane or cell surface . since cd14 is a receptor molecule , it is obvious to be expressed on cell surface . moreover , in the present study , cd14 cdna was prepared from mrna expressed on cells of liver tissue . in the present study , cd14 cdna cloned is the membranous form as it contains gpi anchor for the attachment with cell membrane as well as glycosylated form [ 42 , 43 ] . 3d model of cd14 molecule of cb cattle was predicted from amino acid position 24 to 331 ( figure 6 ) , with horseshoe - shaped structure , with alternating alpha helix and beta chains . cd14 exists as dimmer with the help of leucine zipper when acting as a receptor molecule . the finding is similar to study conducted by sambrook and russell , where they studied crystal structure of cd14 molecule of mus musculus . based on homology study for cd14 3d structure with mus musculus and homo sapiens , lps - binding pocket was predicted at the amino terminal end of cb cattle cd14 molecule , helix were located at the convex surface , and strand were located at the concave surface of horse - shoe shaped cd14 structure , represented in figure 6 . as expected , 27 nucleotide changes were observed , when compared with the published sequences of cd14 gene of bos taurus , out of which 25 were newly reported . 6 synonymous changes and 18 amino acid changes were reported out of which 17 were new changes ( table 4 ) . nonsynonymous substitutions exceeding synonymous substitutions indicate the evolution of this protein through positive selection among domestic animals . snp for the cd14 gene of crossbred cattle was reported here for the first time . the present finding is similar to bubalus bubalis , when monomorphism was reported for this nucleotide region except for one nucleotide change that is too within lps - binding site . another interesting observation is that about 50% of the nucleotide changes , including 3 synonymous changes have been reported from 602nd to 800th nucleotide region of cd14 gene of cb cattle , that is , within 199 nucleotide . similar findings were also reported in bubalus bubalis , where 42 snp were identified with 39 nonsynonymous changes , leading to 23 amino acid changes . cd14 gene of crossbred cattle ( bos indicus crossed with bos taurus ) has been characterized for the first time in the present study . 25 snps with 17 amino acid changes were newly reported , and the site for mutational hot - spot , detected in cb cattle cd14 gene . hyper - variable regions containing mutational hot spot have been reported from 602nd to 800th nucleotide region of cd14 gene of cb cattle , that is , within 199 nucleotide . further research need to be directed to find out the association of allelic variants with traits related to disease incidences , to establish it as marker . gene insert containing the resistant variety of cd14 gene of cb cattle can be used for somatic gene therapy , particularly against mastitis . transgenic animal production with cb cattle cd14 gene insert may provide the scope for future research to develop disease - resistant stock .

nowadays , these lesions in children are being routinely evaluated by mri . in our experience , mri helps to characterize these lesions and narrow the differential diagnosis . in some cases , in combination with clinical features there are a few reviews on imaging of multifocal liver lesions and liver lesions in general in children . however , to the best of our knowledge , there are no original studies published on imaging of multifocal liver lesions in children presenting the actual experience in daily practice . in this manuscript , multiple liver lesions of the same pathologic entity as well as multiple lesions of different pathologic entity are included and all are referred as multifocal liver lesions . the purpose of this study is to present our experience with mri evaluation of multifocal liver lesions in children at our institution . this study also aims at describing the mr imaging features of common and uncommon multifocal liver lesions . we retrospectively reviewed consecutive liver mri in children ( < 18 years ) with multifocal liver lesions performed between january 2007 and december 2012 . a radiology fellow compiled the list of liver and abdominal mri from picture archiving and communication system ( pacs ) using the filters mri abdomen and mri liver in the examination column . he then prepared the final list of mr examinations by looking at the reports mentioning more than two liver lesions . non - diagnostic studies and those with a single liver lesion were excluded . mr imaging was performed using either a 1.5 t siemens ( avanto ; siemens medical system , erlangen , germany ) or 1.5 t and 3 t philips ( achieva ; philips medical system , best , the netherlands ) mri scanners . acquired sequences included a combination of coronal single - shot t2w , axial t1w gradient - echo sequence with in- and out - phase , axial t2w fast spin - echo with respiratory triggering , balanced ssfp ( steady state free precession ) ( true fisp / btfe ) , diffusion - weighted images , pre - contrast axial and coronal t1w 3d gradient - echo sequence [ volume interpolated body examination ( vibe ) ( siemens)/t1w high - resolution isotropic volume examination ( thrive ) ( philips ) ] . post - contrast images were obtained including axial t1w 3d gradient - echo sequence in arterial , portal venous , and equilibrium phases during dynamic injection of gadolinium - based contrast media and axial t1 fast spin - echo with fat saturation at 5 min after contrast injection . the typical scan time for t1w 3d gradient - echo sequence ranged between 15 and 20 s. the dynamic imaging was performed using manual timing with arterial phase starting at 16 s , portal venous phase at 55 s , and equilibrium phase at 2 min . all mris were evaluated by two pediatric radiologists ( gbc with 7 and ko with 27 years of experience in reading pediatric body mri , respectively ) independently . the number ( number of the lesions was noted up to 10 ; more than 10 lesions were rounded to 10 ) , size , and signal characteristics of lesions were noted on t1w , t2w , in- and out - phase , arterial phase , portal venous phase , and equilibrium phase images . patient charts were reviewed by a radiology fellow after the completion of the imaging review for specific clinical details including known primary tumor , follow - up , and histology if biopsy was available . the final lesion diagnosis was based on histology if available . in cases without biopsy , the diagnosis was based on characteristic imaging features , mainly on mri , clinical features , and any available corroborative evidence such as lesion stability on follow - up imaging . the period between the first imaging on which the lesion was detected and the latest available imaging was considered follow - up period for lesion size change . stability in size over a period and/or gradual marginal growth supports benign nature of the lesion . mr imaging was performed using either a 1.5 t siemens ( avanto ; siemens medical system , erlangen , germany ) or 1.5 t and 3 t philips ( achieva ; philips medical system , best , the netherlands ) mri scanners . children below 6 years of age were scanned under general anesthesia . acquired sequences included a combination of coronal single - shot t2w , axial t1w gradient - echo sequence with in- and out - phase , axial t2w fast spin - echo with respiratory triggering , balanced ssfp ( steady state free precession ) ( true fisp / btfe ) , diffusion - weighted images , pre - contrast axial and coronal t1w 3d gradient - echo sequence [ volume interpolated body examination ( vibe ) ( siemens)/t1w high - resolution isotropic volume examination ( thrive ) ( philips ) ] . post - contrast images were obtained including axial t1w 3d gradient - echo sequence in arterial , portal venous , and equilibrium phases during dynamic injection of gadolinium - based contrast media and axial t1 fast spin - echo with fat saturation at 5 min after contrast injection . the typical scan time for t1w 3d gradient - echo sequence ranged between 15 and 20 s. the dynamic imaging was performed using manual timing with arterial phase starting at 16 s , portal venous phase at 55 s , and equilibrium phase at 2 min . all mris were evaluated by two pediatric radiologists ( gbc with 7 and ko with 27 years of experience in reading pediatric body mri , respectively ) independently . the number ( number of the lesions was noted up to 10 ; more than 10 lesions were rounded to 10 ) , size , and signal characteristics of lesions were noted on t1w , t2w , in- and out - phase , arterial phase , portal venous phase , and equilibrium phase images . patient charts were reviewed by a radiology fellow after the completion of the imaging review for specific clinical details including known primary tumor , follow - up , and histology if biopsy was available . the final lesion diagnosis was based on histology if available . in cases without biopsy , the diagnosis was based on characteristic imaging features , mainly on mri , clinical features , and any available corroborative evidence such as lesion stability on follow - up imaging . the period between the first imaging on which the lesion was detected and the latest available imaging was considered follow - up period for lesion size change . stability in size over a period and/or gradual marginal growth supports benign nature of the lesion . a total of 48 children ( 22 boys , 26 girls ; age between 3 months and 18 years with average age 10.58 years ) had mri exams for the evaluation of multiple liver lesions during the study period . only 7/48 children were otherwise healthy at presentation without significant associated or previous disease conditions . background / associated diseases in children ( n=48 ) overall , 19/48 children had > 10 lesions , 12/48 had 5 - 9 lesions , and 17/48 had < 4 lesions . the appearance of the background liver parenchyma on mri was normal in 33/48 children , cirrhotic in 10/48 , with fatty infiltration in 3/48 , and siderotic in 2/48 children . it was based on a combination of imaging , clinical features , and follow - up ( follow - up period from 6 months to 5 years ; average 26 months ) . the lesion stability or only marginal growth over the follow - up period was considered as one of the corroborative evidences for the benign nature of the lesion . in the remaining 7/48 children , three of 48 children had two different types of hepatic lesions [ adenoma and focal nodular hyperplasia ( fnh ) ; regenerative nodule ( rn ) and fnh ; and fnh and hemangioma ] , giving 51 lesion diagnoses in 48 children . final diagnoses of multifocal liver lesions in 48 children ( n=51 ) * seventeen of 48 children ( 7 boys , 10 girls ; age 2 - 17 years with average age 10.41 years ) had multiple fnh . the appearance of background liver parenchyma on mri was normal in 12/17 children , with fatty infiltration in 1/17 , and cirrhotic in 4/17 . four of 17 children had > 10 lesions , 6/17 had 5 - 9 lesions , and remaining 7/17 had < 4 lesions . most lesions were well - defined with the largest measuring 6.4 4 cm in transverse dimensions . most fnh were iso - to - hypointense on t1w , slightly hyperintense on t2w , showed prompt arterial enhancement , and became iso - to - slightly hyperintense on portal venous phase and equilibrium phase images [ figure 1 ] . one child with li fraumeni syndrome and previous history of astrocytoma had multiple biopsy - proven fnh that contained fat . the lesions are slightly hyperintense on t2w fat suppressed ( a ) isointense on pre - contrast t1w 3d gradient - echo image ( b ) show avid enhancement on arterial phase ( c ) remain slightly hyperintense to parenchyma on portal venous phase ( d ) and become isointense to parenchyma on equilibrium phase image ( e ) eight of 48 children ( 2 boys , 6 girls ; age from 3 months to 17 years with average age 8.66 years ) had multiple hemangiomas . three of these children were otherwise healthy and remaining one each had history of lymphoma , ovarian rhabdomyosarcoma , neurofibromatosis type 1 , sensory neural hearing loss , and meningitis . four of eight children showed > 10 lesions , 3/8 had two lesions each , and one child had seven lesions . all lesions were hypo- or iso - intense on t1w , hyperintense on t2w , and showed prompt arterial enhancement and remained hyperintense to parenchyma on equilibrium phase images [ figure 2 ] . multiple small lesions are hyperintense on coronal stir image ( a ) hypointense on coronal t1w image ( b ) and show homogeneous enhancement on post - contrast t1w fat sat image ( c ) the lesions are hypoechoic on ultrasound color image ( d ) and show peripheral vascularity seven of 48 children ( 5 boys , 2 girls ; age from 7 months to 14 years with average age 8 years ) had biopsy - proven metastases . primary tumors included neuroblastoma ( three instances ) and papillary renal cell carcinoma , undifferentiated sarcoma of the kidney , adrenocortical carcinoma , and desmoplastic small round cell tumor of the abdomen ( one case each ) . background liver parenchyma had normal appearance in six children and was siderotic in one child . four of seven children showed > 10 lesions and remaining three showed < 4 lesions with the largest measuring 14.3 8.9 cm . enhancement pattern was variable in all phases with some showing hyper - enhancement and the others remaining hypointense to parenchyma on equilibrium . a few metastases in two children showed central scar , but none showed fat content [ figure 3 ] . axial t2w fat saturated ( a ) and t1w ( b ) images show multiple lobular masses in the liver ( asterisk ) . the primary tumor in the left kidney ( arrowheads ) is also seen rns were seen in 6/48 children ( 3 boys , 3 girls ; age from 14 months to 17 years with average age 12.11 years ) , one of which had pathological confirmation from explanted liver . associated conditions in these children included chronic myeloid leukemia , congenital hepatic fibrosis , hypoplastic left heart syndrome with fontan procedure , langerhans cell histiocytosis , biliary atresia , and previously resected hepatoblastoma with cirrhosis in the residual liver . the hepatic parenchyma showed features of cirrhosis in four children , iron deposition in one child , and normal appearance in another one child . three children showed > 10 lesions , and remaining three children had five , -four , and three lesions , respectively . the largest nodule measured 2.6 3.5 cm , but most nodules were less than 2 cm in diameter [ figure 4 ] . enhancement pattern was variable with nodules showing hyper - enhancement on arterial phase in two children . otherwise , the enhancement was predominantly isointense to the parenchyma on all phases . axial t2w fat saturated ( a ) axial t1w out - phase ( b ) images , pre - contrast t1w thrive image ( c ) and post - contrast t1w fat saturated images in arterial ( d ) portal venous ( e ) and delayed ( f ) phases show regenerative nodules ( arrows ) . the nodule shows hypointense signal on t2w , hyperintense signal on pre - contrast t1w images , hyper - enhancement on arterial and portal venous phase images , and becomes almost isointense to parenchyma on equilibrium phase images . more nodules are seen on arterial and portal venous phase images ; additional nodule is seen on post - contrast images as compared to pre - contrast images in this case . explanted liver in this child confirmed presence of multiple regenerative nodules three children had adenomas , two of which had pathological confirmation . one child proved to have adenomatosis and had normal appearance of background liver parenchyma on mri ; the second one had glycogen storage disorder ( gsd ) with fatty infiltration of parenchyma ; and the third child had polyarteritis nodosa with budd - chiari syndrome and cirrhotic liver . the child with adenomatosis had > 10 lesions and remaining two had six lesions each . fat content was seen in all lesions except one of the lesions in the child with gsd [ figure 5 ] . hemorrhage was seen in the largest lesion in the child with adenomatosis that was resected . signal characteristics on t1w , t2w , and equilibrium phase images were variable with most lesions showing slight hyper - enhancement on arterial phase images . axial t2w fat saturated ( a ) axial t1w in - phase ( b ) axial t1w out - phase ( c ) images , and post - contrast t1w fat saturated images in arterial ( d ) portal venous ( e ) and delayed ( f ) phases show one of the adenomas ( arrows ) . the lesion shows hyperintense signal on t2w , isointense signal on in- and out - phase images without any fat content , hyper - enhancement on arterial and portal venous phase images , and remains hyperintense to parenchyma on equilibrium phase images . other five adenomas in this child showed fat content three children , one each with hyper ige syndrome , gsd , and primary sclerosing cholangitis , had abscesses . intravenous contrast was injected in one child during mri scan that showed ring enhancement of the lesions [ figure 6 ] . another child had contrast - enhanced ct scan a day earlier that showed peripheral enhancement . axial t2w fat saturated ( a ) axial t1w ( b ) and post - contrast t1w fat saturated ( c ) images show two conglomerate abscesses in the right lobe ( arrows ) . the lesions show well - circumscribed enhancing wall that is dark on t2w image other multifocal liver lesions included one case each of angiomyolipoma in a child with tuberous sclerosis , epithelioid hemangioendothelioma , focal fatty infiltration , hepatocellular carcinoma ( hcc ) , infarcts , nodular regenerative hyperplasia ( nrh ) , and hepatic cysts . epithelioid hemangioendotheliomas were seen in a 15-year - old girl with multiple lesions in multiple organs including lungs , liver , and bones . the hepatic lesions were small , peripheral , and showed target appearance on t2w images with central hyperintensity [ figure 7 ] . there was also hepatic capsular retraction with some of the lesions [ figure 7 ] . axial t2w fat saturated ( a ) axial t1w in - phase ( b ) images , and post - contrast t1w fat saturated images in arterial ( c ) portal venous ( d ) and delayed ( e ) phases show multiple lesions ( arrows ) . some lesions have target appearance with bright center and relatively less bright periphery on t2w image . the lesions do not show significant enhancement on arterial phase ( c ) show only minimal enhancement on portal venous phase ( d ) and fill completely with homogeneous enhancement on delayed phase image ( e ) the findings are characteristic for epithelioid hemangioendothelioma ( ehe ) in this pathologically proved case two foci of hcc were seen in the cirrhotic liver in a child with tyrosinemia . these nodules were hyperintense on t1w images and hypointense on t2w images relative to hepatic parenchyma . multiple infarcts were seen within a cirrhotic liver with portal hypertension in a child with cystic fibrosis [ figure 8 ] . right , left , and main portal veins as well as hepatic artery were patent on mri . these lobulated conglomerate areas were periportal in distribution and showed only minimal smooth peripheral enhancement . they were confused with abscesses on initial imaging review , but clinical features were not fitting with abscesses . axial t2w fat saturated ( a ) axial t1w ( b ) and post - contrast t1w fat saturated ( c ) images show multiple lobulated lesions that are predominantly central in location adjacent to portal tracts ( arrows ) . these areas show high signal on t2w ( a ) low signal on t1w ( b ) and show smooth peripheral enhancement ( c ) . gray scale ( d ) and color ( e ) ultrasound images show these areas as hypoechoic areas with increased vascularity surrounding them . it was based on a combination of imaging , clinical features , and follow - up ( follow - up period from 6 months to 5 years ; average 26 months ) . the lesion stability or only marginal growth over the follow - up period was considered as one of the corroborative evidences for the benign nature of the lesion . in the remaining 7/48 children , three of 48 children had two different types of hepatic lesions [ adenoma and focal nodular hyperplasia ( fnh ) ; regenerative nodule ( rn ) and fnh ; and fnh and hemangioma ] , giving 51 lesion diagnoses in 48 children . seventeen of 48 children ( 7 boys , 10 girls ; age 2 - 17 years with average age 10.41 years ) had multiple fnh . the appearance of background liver parenchyma on mri was normal in 12/17 children , with fatty infiltration in 1/17 , and cirrhotic in 4/17 . four of 17 children had > 10 lesions , 6/17 had 5 - 9 lesions , and remaining 7/17 had < 4 lesions . most lesions were well - defined with the largest measuring 6.4 4 cm in transverse dimensions . most fnh were iso - to - hypointense on t1w , slightly hyperintense on t2w , showed prompt arterial enhancement , and became iso - to - slightly hyperintense on portal venous phase and equilibrium phase images [ figure 1 ] . one child with li fraumeni syndrome and previous history of astrocytoma had multiple biopsy - proven fnh that contained fat . the lesions are slightly hyperintense on t2w fat suppressed ( a ) isointense on pre - contrast t1w 3d gradient - echo image ( b ) show avid enhancement on arterial phase ( c ) remain slightly hyperintense to parenchyma on portal venous phase ( d ) and become isointense to parenchyma on equilibrium phase image ( e ) eight of 48 children ( 2 boys , 6 girls ; age from 3 months to 17 years with average age 8.66 years ) had multiple hemangiomas . three of these children were otherwise healthy and remaining one each had history of lymphoma , ovarian rhabdomyosarcoma , neurofibromatosis type 1 , sensory neural hearing loss , and meningitis . four of eight children showed > 10 lesions , 3/8 had two lesions each , and one child had seven lesions . all lesions were hypo- or iso - intense on t1w , hyperintense on t2w , and showed prompt arterial enhancement and remained hyperintense to parenchyma on equilibrium phase images [ figure 2 ] . hemangiomas ( arrowheads ) . multiple small lesions are hyperintense on coronal stir image ( a ) hypointense on coronal t1w image ( b ) and show homogeneous enhancement on post - contrast t1w fat sat image ( c ) the lesions are hypoechoic on ultrasound color image ( d ) and show peripheral vascularity seven of 48 children ( 5 boys , 2 girls ; age from 7 months to 14 years with average age 8 years ) had biopsy - proven metastases . primary tumors included neuroblastoma ( three instances ) and papillary renal cell carcinoma , undifferentiated sarcoma of the kidney , adrenocortical carcinoma , and desmoplastic small round cell tumor of the abdomen ( one case each ) . background liver parenchyma had normal appearance in six children and was siderotic in one child . four of seven children showed > 10 lesions and remaining three showed < 4 lesions with the largest measuring 14.3 8.9 cm . enhancement pattern was variable in all phases with some showing hyper - enhancement and the others remaining hypointense to parenchyma on equilibrium . a few metastases in two children showed central scar , but none showed fat content [ figure 3 ] . axial t2w fat saturated ( a ) and t1w ( b ) images show multiple lobular masses in the liver ( asterisk ) . rns were seen in 6/48 children ( 3 boys , 3 girls ; age from 14 months to 17 years with average age 12.11 years ) , one of which had pathological confirmation from explanted liver . associated conditions in these children included chronic myeloid leukemia , congenital hepatic fibrosis , hypoplastic left heart syndrome with fontan procedure , langerhans cell histiocytosis , biliary atresia , and previously resected hepatoblastoma with cirrhosis in the residual liver . the hepatic parenchyma showed features of cirrhosis in four children , iron deposition in one child , and normal appearance in another one child . three children showed > 10 lesions , and remaining three children had five , -four , and three lesions , respectively . the largest nodule measured 2.6 3.5 cm , but most nodules were less than 2 cm in diameter [ figure 4 ] . all nodules were either iso- or hyperintense on t1w images . on t2w images , four children had hyperintense and two children had hypointense nodules . enhancement pattern was variable with nodules showing hyper - enhancement on arterial phase in two children . otherwise , the enhancement was predominantly isointense to the parenchyma on all phases . axial t2w fat saturated ( a ) axial t1w out - phase ( b ) images , pre - contrast t1w thrive image ( c ) and post - contrast t1w fat saturated images in arterial ( d ) portal venous ( e ) and delayed ( f ) phases show regenerative nodules ( arrows ) . the nodule shows hypointense signal on t2w , hyperintense signal on pre - contrast t1w images , hyper - enhancement on arterial and portal venous phase images , and becomes almost isointense to parenchyma on equilibrium phase images . more nodules are seen on arterial and portal venous phase images ; additional nodule is seen on post - contrast images as compared to pre - contrast images in this case . one child proved to have adenomatosis and had normal appearance of background liver parenchyma on mri ; the second one had glycogen storage disorder ( gsd ) with fatty infiltration of parenchyma ; and the third child had polyarteritis nodosa with budd - chiari syndrome and cirrhotic liver . the child with adenomatosis had > 10 lesions and remaining two had six lesions each . fat content was seen in all lesions except one of the lesions in the child with gsd [ figure 5 ] . hemorrhage was seen in the largest lesion in the child with adenomatosis that was resected . signal characteristics on t1w , t2w , and equilibrium phase images were variable with most lesions showing slight hyper - enhancement on arterial phase images . axial t2w fat saturated ( a ) axial t1w in - phase ( b ) axial t1w out - phase ( c ) images , and post - contrast t1w fat saturated images in arterial ( d ) portal venous ( e ) and delayed ( f ) phases show one of the adenomas ( arrows ) . the lesion shows hyperintense signal on t2w , isointense signal on in- and out - phase images without any fat content , hyper - enhancement on arterial and portal venous phase images , and remains hyperintense to parenchyma on equilibrium phase images . three children , one each with hyper ige syndrome , gsd , and primary sclerosing cholangitis , had abscesses . intravenous contrast was injected in one child during mri scan that showed ring enhancement of the lesions [ figure 6 ] . another child had contrast - enhanced ct scan a day earlier that showed peripheral enhancement . axial t2w fat saturated ( a ) axial t1w ( b ) and post - contrast t1w fat saturated ( c ) images show two conglomerate abscesses in the right lobe ( arrows ) . other multifocal liver lesions included one case each of angiomyolipoma in a child with tuberous sclerosis , epithelioid hemangioendothelioma , focal fatty infiltration , hepatocellular carcinoma ( hcc ) , infarcts , nodular regenerative hyperplasia ( nrh ) , and hepatic cysts . epithelioid hemangioendotheliomas were seen in a 15-year - old girl with multiple lesions in multiple organs including lungs , liver , and bones . the hepatic lesions were small , peripheral , and showed target appearance on t2w images with central hyperintensity [ figure 7 ] . there was also hepatic capsular retraction with some of the lesions [ figure 7 ] . axial t2w fat saturated ( a ) axial t1w in - phase ( b ) images , and post - contrast t1w fat saturated images in arterial ( c ) portal venous ( d ) and delayed ( e ) phases show multiple lesions ( arrows ) . some lesions have target appearance with bright center and relatively less bright periphery on t2w image . these peripheral lesions also show capsular retraction ( arrowheads ) . the lesions do not show significant enhancement on arterial phase ( c ) show only minimal enhancement on portal venous phase ( d ) and fill completely with homogeneous enhancement on delayed phase image ( e ) the findings are characteristic for epithelioid hemangioendothelioma ( ehe ) in this pathologically proved case two foci of hcc were seen in the cirrhotic liver in a child with tyrosinemia . these nodules were hyperintense on t1w images and hypointense on t2w images relative to hepatic parenchyma . multiple infarcts were seen within a cirrhotic liver with portal hypertension in a child with cystic fibrosis [ figure 8 ] . right , left , and main portal veins as well as hepatic artery were patent on mri . these lobulated conglomerate areas were periportal in distribution and showed only minimal smooth peripheral enhancement . they were confused with abscesses on initial imaging review , but clinical features were not fitting with abscesses . axial t2w fat saturated ( a ) axial t1w ( b ) and post - contrast t1w fat saturated ( c ) images show multiple lobulated lesions that are predominantly central in location adjacent to portal tracts ( arrows ) . these areas show high signal on t2w ( a ) low signal on t1w ( b ) and show smooth peripheral enhancement ( c ) . gray scale ( d ) and color ( e ) ultrasound images show these areas as hypoechoic areas with increased vascularity surrounding them . multifocal liver lesions in children include hemangioma , fnh , metastases , hepatoblastoma , mesenchymal hamartoma , adenoma , hcc , infective process , and rarely nrh , epithelioid hemangioendothelioma , and lymphoma . the frequency of these lesions in our study does not necessarily represent the true picture because it evaluates only lesions assessed by mri and not all the lesions seen in the clinic or a hospital . not all multifocal lesions are evaluated by mri at our institution , for example , initial evaluation of suspected hepatoblastoma or stage 4s neuroblastoma is still performed by ct scan that allows staging ct chest and neck to be done in the same short anesthesia time . this is possibly one of the reasons we did not see any cases of multifocal hepatoblastoma and found only one case of multifocal hcc . nonetheless , barring these exceptions , most liver lesions in children , solitary or multifocal , especially benign lesions , are now increasingly evaluated by mri at many institutions . hence , our study represents day - to - day experience in terms of types of lesions seen and their approximate frequency . fnh was the most common multifocal liver lesion that was evaluated by mri in our study . fnh is being seen with increasing frequency in children , especially in those who have been previously treated for malignancy . fnh tends to be multiple , smaller in size ( typically around 2 cm ) , and most of them lack central scar in these cases . similar to previous reports in the literature , most children with multifocal fnh in our study had history of previous cancer . most fnh were iso- to hypointense on t1w , slightly hyperintense on t2w , showed prompt arterial enhancement , and became isointense to slightly hyperintense on portal venous phase and equilibrium phase images . hemangiomas are the most common benign tumor of infancy and about half of them are multifocal . as reported in the literature , all hemangiomas in our study were hypo- or iso - intense on t1w and hyperintense on t2w images . peripheral enhancement followed by progressive centripetal enhancement is a typical feature of the hemangioma that can be seen in infantile hepatic hemangioma . however , smaller lesions may not show this feature and may show complete homogenous enhancement of the lesion in the arterial phase itself that is retained on the equilibrium and delayed phase images . this feature was seen in most of our hemangiomas that were small in size with the largest measuring 1.3 2.2 cm . these may be difficult to differentiate from vascular metastases in children with known primary malignancy . multiple liver lesions in children , even in those with previous history of cancer , are more likely to represent fnh than metastases as shown in the literature and is reflected in our study . imaging features of metastases are variable and nonspecific on all sequences as shown in this study . hepatocyte - specific contrast media can help to differentiate them from common benign liver lesion such as fnh . rns seen in cirrhosis are formed by localized proliferation of hepatocytes and their supporting stroma , and are surrounded by fibrosis . four of six children with rn in our study had frank changes of cirrhosis on mri . rn can be micronodular ( <3 mm ) or macronodular ( > 3 mm ) , and are usually < 2 cm in size . rns are typically hypointense on t2w , variable in signal on t1w images , and show enhancement similar to liver parenchyma on all phases including arterial phase . in our study , most nodules were either iso- or hyperintense on t1w images and showed enhancement similar to liver parenchyma . however , on t2w images , four children had hyperintense and two children had hypointense nodules . nrh is a distinct entity from rn of cirrhosis that also shows rns of hepatocytes seen in non - cirrhotic liver and can be associated with portal hypertension in many cases . nrh is seen variety of conditions including budd - chiari syndrome , congenital anomalies of portal vein , congenital hepatic fibrosis , myeloproliferative and autoimmune disorders , lupus , and chemotherapy among others . hepatocellular adenoma in children is seen with predispositions like use of oral contraceptives in girls , gsd , and anomalies of hepatic vasculature . the inflammatory subtype is predisposed for hemorrhage , while inflammatory and beta - catenin - mutated subtypes are at risk for developing into hcc in up to 5 - 10% cases . fat content , suggested by signal drop on out - of - phase image , is the characteristic feature of adenomas . most adenomas are hyperintense on t1w and t2w images and show hyper - enhancement in the arterial phase . hepatic abscesses are typically seen in immunocompromised children , especially those with immunodeficiencies , those on various cancer therapies , or recipients of transplantation . peripheral enhancement and perilesional edema along with clinical features help to differentiate abscesses from other lesions . one of the common causes of hepatic metastases in children is neuroblastoma in which the liver is markedly enlarged . other rare cases of multifocal liver lesions were seen in our series including epithelioid hemangioendothelioma that showed characteristic imaging features described in the literature . limitations of this study include retrospective nature and lack of pathologic confirmation in many cases . most cases without pathologic confirmation were of fnh and hemangioma . however , because of common occurrence and typical mri features in most cases , fnh are mainly diagnosed by mri and are rarely biopsied . one of the limitations of this study was lack of cases with imaging using hepatocyte - specific contrast media that have improved sensitivity and specificity of non - invasive diagnosis of hepatic lesions in children and adults . it helps achieve reasonable differential diagnoses and guide further investigation and management . in some cases

objective : the purpose of this study is to present our experience with mri evaluation of multifocal liver lesions in children and describe the mri characteristics of these lesions.patients and methods : a retrospective review of consecutive mri exams performed for the evaluation of multiple liver lesions between 1 january 2007 and 31 december 2012 was done to note the number of lesions , the size of the largest lesion , mr signal characteristics , and background liver . final diagnosis was assigned to each case based on pathology in the available cases and a combination of clinical features , imaging features , and follow - up in the remaining cases.results:a total of 48 children ( 22 boys , 26 girls ; age between 3 months and 18 years with average age 10.58 years and median age 11 years ) were included in the study . totally 51 lesion diagnoses were seen in 48 children that included 17 focal nodular hyperplasia ( fnh ) , 8 hemangiomas , 7 metastases , 6 regenerative nodules , 3 adenomas , 3 abscesses , and one each of angiomyolipoma , epithelioid hemangioendothelioma , focal fatty infiltration , hepatocellular carcinoma , hepatic infarction , nodular regenerative hyperplasia , and hepatic cyst . background liver was normal in 33 , cirrhotic in 10 , fatty in 3 , and siderotic in 2 children . most fnh , hemangiomas , and regenerative nodules showed characteristic mri features , while metastases were variable in signal pattern.conclusion:many commonly seen multifocal liver lesions in children have characteristic mri features . mri can help to arrive at reasonable differential diagnoses for multifocal liver lesions in children and guide further investigation and management .

Introduction Patients and Methods MRI technique Imaging analysis Chart review and final diagnosis Results Standard of reference Focal nodular hyperplasia Hemangiomas Metastases Regenerative nodules Adenomas Abscesses Other lesions Discussion Conclusion

nowadays , these lesions in children are being routinely evaluated by mri . in our experience , mri helps to characterize these lesions and narrow the differential diagnosis . in some cases , in combination with clinical features there are a few reviews on imaging of multifocal liver lesions and liver lesions in general in children . however , to the best of our knowledge , there are no original studies published on imaging of multifocal liver lesions in children presenting the actual experience in daily practice . in this manuscript , multiple liver lesions of the same pathologic entity as well as multiple lesions of different pathologic entity are included and all are referred as multifocal liver lesions . the purpose of this study is to present our experience with mri evaluation of multifocal liver lesions in children at our institution . this study also aims at describing the mr imaging features of common and uncommon multifocal liver lesions . we retrospectively reviewed consecutive liver mri in children ( < 18 years ) with multifocal liver lesions performed between january 2007 and december 2012 . acquired sequences included a combination of coronal single - shot t2w , axial t1w gradient - echo sequence with in- and out - phase , axial t2w fast spin - echo with respiratory triggering , balanced ssfp ( steady state free precession ) ( true fisp / btfe ) , diffusion - weighted images , pre - contrast axial and coronal t1w 3d gradient - echo sequence [ volume interpolated body examination ( vibe ) ( siemens)/t1w high - resolution isotropic volume examination ( thrive ) ( philips ) ] . the number ( number of the lesions was noted up to 10 ; more than 10 lesions were rounded to 10 ) , size , and signal characteristics of lesions were noted on t1w , t2w , in- and out - phase , arterial phase , portal venous phase , and equilibrium phase images . patient charts were reviewed by a radiology fellow after the completion of the imaging review for specific clinical details including known primary tumor , follow - up , and histology if biopsy was available . the final lesion diagnosis was based on histology if available . in cases without biopsy , the diagnosis was based on characteristic imaging features , mainly on mri , clinical features , and any available corroborative evidence such as lesion stability on follow - up imaging . the period between the first imaging on which the lesion was detected and the latest available imaging was considered follow - up period for lesion size change . acquired sequences included a combination of coronal single - shot t2w , axial t1w gradient - echo sequence with in- and out - phase , axial t2w fast spin - echo with respiratory triggering , balanced ssfp ( steady state free precession ) ( true fisp / btfe ) , diffusion - weighted images , pre - contrast axial and coronal t1w 3d gradient - echo sequence [ volume interpolated body examination ( vibe ) ( siemens)/t1w high - resolution isotropic volume examination ( thrive ) ( philips ) ] . the number ( number of the lesions was noted up to 10 ; more than 10 lesions were rounded to 10 ) , size , and signal characteristics of lesions were noted on t1w , t2w , in- and out - phase , arterial phase , portal venous phase , and equilibrium phase images . patient charts were reviewed by a radiology fellow after the completion of the imaging review for specific clinical details including known primary tumor , follow - up , and histology if biopsy was available . the final lesion diagnosis was based on histology if available . in cases without biopsy , the diagnosis was based on characteristic imaging features , mainly on mri , clinical features , and any available corroborative evidence such as lesion stability on follow - up imaging . the period between the first imaging on which the lesion was detected and the latest available imaging was considered follow - up period for lesion size change . a total of 48 children ( 22 boys , 26 girls ; age between 3 months and 18 years with average age 10.58 years ) had mri exams for the evaluation of multiple liver lesions during the study period . background / associated diseases in children ( n=48 ) overall , 19/48 children had > 10 lesions , 12/48 had 5 - 9 lesions , and 17/48 had < 4 lesions . the appearance of the background liver parenchyma on mri was normal in 33/48 children , cirrhotic in 10/48 , with fatty infiltration in 3/48 , and siderotic in 2/48 children . it was based on a combination of imaging , clinical features , and follow - up ( follow - up period from 6 months to 5 years ; average 26 months ) . the lesion stability or only marginal growth over the follow - up period was considered as one of the corroborative evidences for the benign nature of the lesion . in the remaining 7/48 children , three of 48 children had two different types of hepatic lesions [ adenoma and focal nodular hyperplasia ( fnh ) ; regenerative nodule ( rn ) and fnh ; and fnh and hemangioma ] , giving 51 lesion diagnoses in 48 children . final diagnoses of multifocal liver lesions in 48 children ( n=51 ) * seventeen of 48 children ( 7 boys , 10 girls ; age 2 - 17 years with average age 10.41 years ) had multiple fnh . the appearance of background liver parenchyma on mri was normal in 12/17 children , with fatty infiltration in 1/17 , and cirrhotic in 4/17 . four of 17 children had > 10 lesions , 6/17 had 5 - 9 lesions , and remaining 7/17 had < 4 lesions . the lesions are slightly hyperintense on t2w fat suppressed ( a ) isointense on pre - contrast t1w 3d gradient - echo image ( b ) show avid enhancement on arterial phase ( c ) remain slightly hyperintense to parenchyma on portal venous phase ( d ) and become isointense to parenchyma on equilibrium phase image ( e ) eight of 48 children ( 2 boys , 6 girls ; age from 3 months to 17 years with average age 8.66 years ) had multiple hemangiomas . three of these children were otherwise healthy and remaining one each had history of lymphoma , ovarian rhabdomyosarcoma , neurofibromatosis type 1 , sensory neural hearing loss , and meningitis . four of eight children showed > 10 lesions , 3/8 had two lesions each , and one child had seven lesions . multiple small lesions are hyperintense on coronal stir image ( a ) hypointense on coronal t1w image ( b ) and show homogeneous enhancement on post - contrast t1w fat sat image ( c ) the lesions are hypoechoic on ultrasound color image ( d ) and show peripheral vascularity seven of 48 children ( 5 boys , 2 girls ; age from 7 months to 14 years with average age 8 years ) had biopsy - proven metastases . primary tumors included neuroblastoma ( three instances ) and papillary renal cell carcinoma , undifferentiated sarcoma of the kidney , adrenocortical carcinoma , and desmoplastic small round cell tumor of the abdomen ( one case each ) . background liver parenchyma had normal appearance in six children and was siderotic in one child . the primary tumor in the left kidney ( arrowheads ) is also seen rns were seen in 6/48 children ( 3 boys , 3 girls ; age from 14 months to 17 years with average age 12.11 years ) , one of which had pathological confirmation from explanted liver . associated conditions in these children included chronic myeloid leukemia , congenital hepatic fibrosis , hypoplastic left heart syndrome with fontan procedure , langerhans cell histiocytosis , biliary atresia , and previously resected hepatoblastoma with cirrhosis in the residual liver . explanted liver in this child confirmed presence of multiple regenerative nodules three children had adenomas , two of which had pathological confirmation . one child proved to have adenomatosis and had normal appearance of background liver parenchyma on mri ; the second one had glycogen storage disorder ( gsd ) with fatty infiltration of parenchyma ; and the third child had polyarteritis nodosa with budd - chiari syndrome and cirrhotic liver . fat content was seen in all lesions except one of the lesions in the child with gsd [ figure 5 ] . hemorrhage was seen in the largest lesion in the child with adenomatosis that was resected . signal characteristics on t1w , t2w , and equilibrium phase images were variable with most lesions showing slight hyper - enhancement on arterial phase images . axial t2w fat saturated ( a ) axial t1w in - phase ( b ) axial t1w out - phase ( c ) images , and post - contrast t1w fat saturated images in arterial ( d ) portal venous ( e ) and delayed ( f ) phases show one of the adenomas ( arrows ) . other five adenomas in this child showed fat content three children , one each with hyper ige syndrome , gsd , and primary sclerosing cholangitis , had abscesses . the lesions show well - circumscribed enhancing wall that is dark on t2w image other multifocal liver lesions included one case each of angiomyolipoma in a child with tuberous sclerosis , epithelioid hemangioendothelioma , focal fatty infiltration , hepatocellular carcinoma ( hcc ) , infarcts , nodular regenerative hyperplasia ( nrh ) , and hepatic cysts . epithelioid hemangioendotheliomas were seen in a 15-year - old girl with multiple lesions in multiple organs including lungs , liver , and bones . the lesions do not show significant enhancement on arterial phase ( c ) show only minimal enhancement on portal venous phase ( d ) and fill completely with homogeneous enhancement on delayed phase image ( e ) the findings are characteristic for epithelioid hemangioendothelioma ( ehe ) in this pathologically proved case two foci of hcc were seen in the cirrhotic liver in a child with tyrosinemia . it was based on a combination of imaging , clinical features , and follow - up ( follow - up period from 6 months to 5 years ; average 26 months ) . the lesion stability or only marginal growth over the follow - up period was considered as one of the corroborative evidences for the benign nature of the lesion . in the remaining 7/48 children , three of 48 children had two different types of hepatic lesions [ adenoma and focal nodular hyperplasia ( fnh ) ; regenerative nodule ( rn ) and fnh ; and fnh and hemangioma ] , giving 51 lesion diagnoses in 48 children . seventeen of 48 children ( 7 boys , 10 girls ; age 2 - 17 years with average age 10.41 years ) had multiple fnh . the appearance of background liver parenchyma on mri was normal in 12/17 children , with fatty infiltration in 1/17 , and cirrhotic in 4/17 . four of 17 children had > 10 lesions , 6/17 had 5 - 9 lesions , and remaining 7/17 had < 4 lesions . most fnh were iso - to - hypointense on t1w , slightly hyperintense on t2w , showed prompt arterial enhancement , and became iso - to - slightly hyperintense on portal venous phase and equilibrium phase images [ figure 1 ] . the lesions are slightly hyperintense on t2w fat suppressed ( a ) isointense on pre - contrast t1w 3d gradient - echo image ( b ) show avid enhancement on arterial phase ( c ) remain slightly hyperintense to parenchyma on portal venous phase ( d ) and become isointense to parenchyma on equilibrium phase image ( e ) eight of 48 children ( 2 boys , 6 girls ; age from 3 months to 17 years with average age 8.66 years ) had multiple hemangiomas . three of these children were otherwise healthy and remaining one each had history of lymphoma , ovarian rhabdomyosarcoma , neurofibromatosis type 1 , sensory neural hearing loss , and meningitis . four of eight children showed > 10 lesions , 3/8 had two lesions each , and one child had seven lesions . multiple small lesions are hyperintense on coronal stir image ( a ) hypointense on coronal t1w image ( b ) and show homogeneous enhancement on post - contrast t1w fat sat image ( c ) the lesions are hypoechoic on ultrasound color image ( d ) and show peripheral vascularity seven of 48 children ( 5 boys , 2 girls ; age from 7 months to 14 years with average age 8 years ) had biopsy - proven metastases . primary tumors included neuroblastoma ( three instances ) and papillary renal cell carcinoma , undifferentiated sarcoma of the kidney , adrenocortical carcinoma , and desmoplastic small round cell tumor of the abdomen ( one case each ) . background liver parenchyma had normal appearance in six children and was siderotic in one child . rns were seen in 6/48 children ( 3 boys , 3 girls ; age from 14 months to 17 years with average age 12.11 years ) , one of which had pathological confirmation from explanted liver . associated conditions in these children included chronic myeloid leukemia , congenital hepatic fibrosis , hypoplastic left heart syndrome with fontan procedure , langerhans cell histiocytosis , biliary atresia , and previously resected hepatoblastoma with cirrhosis in the residual liver . three children showed > 10 lesions , and remaining three children had five , -four , and three lesions , respectively . one child proved to have adenomatosis and had normal appearance of background liver parenchyma on mri ; the second one had glycogen storage disorder ( gsd ) with fatty infiltration of parenchyma ; and the third child had polyarteritis nodosa with budd - chiari syndrome and cirrhotic liver . fat content was seen in all lesions except one of the lesions in the child with gsd [ figure 5 ] . hemorrhage was seen in the largest lesion in the child with adenomatosis that was resected . signal characteristics on t1w , t2w , and equilibrium phase images were variable with most lesions showing slight hyper - enhancement on arterial phase images . axial t2w fat saturated ( a ) axial t1w in - phase ( b ) axial t1w out - phase ( c ) images , and post - contrast t1w fat saturated images in arterial ( d ) portal venous ( e ) and delayed ( f ) phases show one of the adenomas ( arrows ) . three children , one each with hyper ige syndrome , gsd , and primary sclerosing cholangitis , had abscesses . other multifocal liver lesions included one case each of angiomyolipoma in a child with tuberous sclerosis , epithelioid hemangioendothelioma , focal fatty infiltration , hepatocellular carcinoma ( hcc ) , infarcts , nodular regenerative hyperplasia ( nrh ) , and hepatic cysts . epithelioid hemangioendotheliomas were seen in a 15-year - old girl with multiple lesions in multiple organs including lungs , liver , and bones . the lesions do not show significant enhancement on arterial phase ( c ) show only minimal enhancement on portal venous phase ( d ) and fill completely with homogeneous enhancement on delayed phase image ( e ) the findings are characteristic for epithelioid hemangioendothelioma ( ehe ) in this pathologically proved case two foci of hcc were seen in the cirrhotic liver in a child with tyrosinemia . multifocal liver lesions in children include hemangioma , fnh , metastases , hepatoblastoma , mesenchymal hamartoma , adenoma , hcc , infective process , and rarely nrh , epithelioid hemangioendothelioma , and lymphoma . the frequency of these lesions in our study does not necessarily represent the true picture because it evaluates only lesions assessed by mri and not all the lesions seen in the clinic or a hospital . not all multifocal lesions are evaluated by mri at our institution , for example , initial evaluation of suspected hepatoblastoma or stage 4s neuroblastoma is still performed by ct scan that allows staging ct chest and neck to be done in the same short anesthesia time . this is possibly one of the reasons we did not see any cases of multifocal hepatoblastoma and found only one case of multifocal hcc . nonetheless , barring these exceptions , most liver lesions in children , solitary or multifocal , especially benign lesions , are now increasingly evaluated by mri at many institutions . fnh tends to be multiple , smaller in size ( typically around 2 cm ) , and most of them lack central scar in these cases . peripheral enhancement followed by progressive centripetal enhancement is a typical feature of the hemangioma that can be seen in infantile hepatic hemangioma . however , smaller lesions may not show this feature and may show complete homogenous enhancement of the lesion in the arterial phase itself that is retained on the equilibrium and delayed phase images . this feature was seen in most of our hemangiomas that were small in size with the largest measuring 1.3 2.2 cm . multiple liver lesions in children , even in those with previous history of cancer , are more likely to represent fnh than metastases as shown in the literature and is reflected in our study . imaging features of metastases are variable and nonspecific on all sequences as shown in this study . hepatocyte - specific contrast media can help to differentiate them from common benign liver lesion such as fnh . rn can be micronodular ( <3 mm ) or macronodular ( > 3 mm ) , and are usually < 2 cm in size . rns are typically hypointense on t2w , variable in signal on t1w images , and show enhancement similar to liver parenchyma on all phases including arterial phase . hepatocellular adenoma in children is seen with predispositions like use of oral contraceptives in girls , gsd , and anomalies of hepatic vasculature . peripheral enhancement and perilesional edema along with clinical features help to differentiate abscesses from other lesions . one of the common causes of hepatic metastases in children is neuroblastoma in which the liver is markedly enlarged . other rare cases of multifocal liver lesions were seen in our series including epithelioid hemangioendothelioma that showed characteristic imaging features described in the literature . limitations of this study include retrospective nature and lack of pathologic confirmation in many cases . one of the limitations of this study was lack of cases with imaging using hepatocyte - specific contrast media that have improved sensitivity and specificity of non - invasive diagnosis of hepatic lesions in children and adults . it helps achieve reasonable differential diagnoses and guide further investigation and management .

branchial fistulas and cysts , involving soft tissues of neck , are uncommon anomalies of embryonic development that are commonly encountered by otolaryngologists . in fact , approximately 17% of all pediatric cervical masses are due to branchial anomalies . although branchial cleft cysts are benign , superinfection , mass effect , and surgical complications account for its morbidity . branchial apparatus , seen in the early embryonic life , has a vital role to play in the development of head and neck structures . branchia is the greek word for gill , and the same word represents these anomalies owing to their resemblance to gills of certain species as fish . six paired branchial arches , which appear in the fourth week of embryonic life , give rise to many structures of the head and neck . each branchial arch consists of core of mesenchyme covered externally by ectoderm and internally by endoderm . many anomalies of the head and neck region have been attributed to the aberrant development of these structures . depending on the anatomic location , branchial anomalies are classified into first , second , third , and fourth anomalies . the course of a particular branchial anomaly is caudal to the structures derived from the corresponding arch and dorsal to the structures that develop from the following arch . cysts are considered to be entrapped remnants of branchial cleft or sinuses ; sinuses are remnants of cleft or pouches ; and fistulae result from persistence of both pouch and cleft . different anomalies of the head and neck area have been attributed to the maldevelopment of branchial apparatus . the importance of knowing the development of branchial apparatus and their anomalies is in applying the knowledge during surgery , as vital structures like facial nerve and parotid are in intimate relation with many of these anomalies . we performed a ten - year retrospective study to analyze the pathophysiology , clinical features , and management of branchial anomalies . ours is a retrospective study of 30 cases of branchial anomalies , which presented to the department of otolaryngology , head and neck surgery , kasturba medical college , over a period of 10 years from 2000 to 2010 . family history and previous history of infection and/or surgery were noted . the side and site of the lesion and the site of opening of sinuses and fistula were noted . patients with sinus and fistulas underwent sino-/fistulogram , by injecting contrast material urografin into the tract ( figure 1 ) . all patients were operated upon . in cases of acute infection , patients were put on intravenous antibiotics and in cases of abscess incision and drainage were done . such patients were taken up for surgical excision of tract four weeks later . during surgery , a conscious attempt was made to remove some fascia and tissues adjacent to the branchial tracts along their path to avoid leaving behind ramifications that might lead to recurrences . this fistulous communication between the external auditory canal and the neck in the upper part of anterior border of sternocleidomastoid ( scm ) muscle was identified by injecting methylene blue dye into the neck opening which was seen coming of an opening in the external auditory canal . the tract was then dissected from the surrounding tissue and followed till its opening in the external auditory canal ( figure 2 ) . the tract was excised off its attachment to the external auditory canal and wound closed in layers . scm muscle was retracted away from the field taking care not to injure the greater auricular nerve . elliptical skin incision was made over the skin opening and the dissection proceeded in the direction of the tract . this second incision was given at the level of the hyoid and the whole tract was brought out through this incision . it was then followed to its opening into the pharynx . during their course towards the oropharynx , the second branchial fistulae , were seen passing between the carotid bifurcations , where they were in close relation to the hypoglossal nerve . the third branchial fistulae were seen piercing the thyrohyoid membrane to open into the pyriform fossa ( figure 3 ) . the tracts were followed to the pharynx and were excised ; the pharyngeal defects were sutured . the suture lines were reinforced by a second layer of suture in the pharyngeal musculature . the fourth branchial fistulae were seen opening into the lower part of neck near the scm muscle . the tract then passed inferiorly into the mediastinum , looping around the arch of aorta in the left and subclavian artery in the right and back in to the neck , ascending posterior to the carotid . the tract was followed from its neck opening into the mediastinum using blunt finger dissection . the intramediastinal part of the fistulae was left behind with their ends ligated and the rest of the tract was dissected out from the superior mediastinum up to the pyriform fossa ( figure 4 ) . thirty patients with 34 branchial anomalies were studied retrospectively over a period of 10 years from 2000 to 2010 in the department of otolaryngology , head and neck surgery , kasturba medical college , mangalore . there was maximum incidence of second branchial anomalies with 17(50% ) cases . among the first branchial anomalies , seven ( 20.59% ) cases belonged to work ii , while six ( 17.65% ) cases belonged to work i ( according to the work classification ) . of these , branchial cyst constituted eight ( 26.53% ) cases while branchial fistula constituted nine ( 26.47% ) cases . fourth branchial arch anomaly was seen in two ( 5.88% ) patients ( figure 5 ) . among the anatomical types of the lesion , we had a maximum incidence of fistula seen in 20 ( 58.82% ) cases , followed by cyst in 14 ( 41.12% ) cases . the youngest patient in our study was one and a half years and the oldest one 48 years . the mean age of presentation was 18.67 years with a standard deviation of 11.06 . only 1 ( 3.3% ) the mean age of onset among this late onset group was 15.97 years . among the first branchial anomalies , the maximum incidence of the lesion was seen in the 1120 age group with 5 ( 14.70% ) cases followed by 4 ( 11.76% ) cases in the 610 and 2140 age groups . in the second arch anomalies , we had four ( 11.76% ) cases in the 610 age group and seven ( 20.59% ) cases in the 1120 and five ( 14.70% ) in 2040 age groups . considering all the anomalies together , 55.88% were males and 44.12% were females with a male to female ratio of 1.27 : 1 . in the first branchial anomalies , the incidence in males was 17.65% and 20.59% in females . among the second arch anomalies the overall incidence of the anomalies was more on the right side ( 57.08% ) while 42.92% of the lesions occurred on the left side . in the first arch anomalies , 8 ( 23.53% ) cases were present on the right and 5 ( 14.71% ) cases on the left . in the second arch anomalies 11 ( 32.35% ) cases occurred on the right side and six ( 17.65% ) cases on the left including one patient with bilateral branchial cysts ( figure 6 ) . the third anomalies occurred on the right side and the fourth anomalies on the left side . in all anomalies put together , the most common clinical feature was a swelling seen in 21 ( 61.76 ) and fistula opening in 18 ( 52.94% ) cases . 26.47% of the patients had pain at the site of the lesion . among the first arch anomaly patients , swelling in the neck and postauricular region was the most common presenting feature ( 29.41% ) . the most common presenting feature of second branchial arch anomaly was neck swelling , seen in 26.47% , while 23.53% presented with opening in the neck . the third arch anomaly patient had swelling and opening in the neck along with discharge . the fourth arch anomaly patient had swelling and opening in the neck along with discharge and pain . ct scan and ultrasound were done in all cases of third and fourth arch anomalies and nine cases of second arch anomaly . acute infection was treated by a course of antibiotics in 18 ( 60% ) cases and incision and drainage in one case ( before proceeding to the excision of the lesion ) . 73.33% of the cases were managed by single incision , while 23.33% required stepladder incision . though described first in the early nineteenth century , the origin and classification of different branchial anomalies are highly controversial even today . second branchial anomalies are considered to be the commonest with figures up to 95% being reported . the remainder of branchial anomalies is derived from first branchial remnants ( 18% ) with third and fourth branchial anomalies being quite rare . several theories proposed for the development of branchial anomalies include branchial apparatus theory , cervical sinus theory , thymopharyngeal theory , and inclusion theory . of these , the widely accepted theory is that branchial anomalies result from incomplete involution of the branchial apparatus . according to ford et al . , most of the branchial anomalies arise from the second branchial cleft ( 92.45% ) . remaining is derived from first arch remnants ( 4.72% ) and third ( 1.87% ) and fourth arch anomalies ( 0.94% ) are quite rare . bajaj et al . also reported higher incidence of second branchial anomalies ( 78% ) in their series of 80 patients . choi and zalzal who reported a higher incidence of first branchial arch anomalies ( 25% ) in their series still had the maximum incidence of second branchial arch anomalies ( 40% ) . in our series , we had the maximum incidence of second arch anomalies ( 50% ) followed by first arch anomalies ( 38.24% ) . choi and zalzal reported a maximum incidence of sinuses , followed by fistula . in our series cysts were the most common lesion followed by sinuses . the age of onset of these anomalies has been seen to vary according to the type of the lesion . choi and zalzal have noted that mean age of presentation of cyst ( 18.35 years ) was late compared to that of fistulae ( 6.28 years ) and sinuses ( 7.82 years ) . this finding was confirmed in our study . in our group , it was found that fistulas ( 1.14 years ) had an early age of onset followed by that of sinuses ( 4.21 years ) . cysts ( 7.51 years ) were found to have a late onset compared to the other two lesions . ford et al . have pointed out that the branchial anomalies occur more on the right side . they had 60% incidence on the right side and 40% on the left side . in the present study , we have a similar picture with right side incidence of 55.38% and left side incidence of 44.62% . in the study by choi and zalzal , the most common presenting features were discharge from the openings , cervical mass , and repeated infection . in our study , the most common clinical feature was a swelling seen in 21 ( 61.76 ) . an initial correct diagnosis is crucial because experience shows that recurrence rates after surgical excision of branchial anomalies are 14% and 22% with previous infection and surgery , respectively , whereas the recurrence rate for primary lesion is 3% . although physical examination and history are the most important elements in the diagnosis , radio - diagnostic studies can add valuable information to the evaluation of a congenital neck mass . a ct scan is an accurate and noninvasive diagnostic tool , which can confirm the diagnosis or suggest an alternative diagnosis , define both the location and extent of a neck lesion , and delineate infectious process or possible malignant degeneration . ct scan is useful in evaluating first branchial anomaly and the position of facial nerve . ct scan is reported to be more useful than mri in evaluating branchial anomalies . in case of sinus or fistula , sinogram or conray contrast study can delineate the course of branchial anomaly . in the series by choi and zalzal , ct scan was performed on 15.38% of patients , sonogram / contrast study was performed on 7.69% of patients , and ultrasound / mri was done on 1.92% of patients . in our series , intraoperative methylene blue injection was performed in all cases ; ct scan was done in 10.77% of cases . surgery is definitive mode of treatment because there is lack of spontaneous regression , a high rate of recurrent infection , the possibility of other diagnoses , and rare malignant degeneration . acute inflammation is treated medically unless incision and drainage or aspiration of an abscess is required . three to four weeks should pass after an acute infection before a definitive surgical exploration is undertaken . in our series 60% of patients took medical treatment and 3.33% underwent drainage of abscess before definitive surgical excision of the lesion . surgical excision of lesion was done in all patients . in the series by ford et al . it is our observation that while methylene blue dye enhances visualization of the larger and more proximal ( in relation to the punctum ) part of the tracts and ramifications , it does not demarcate the most peripheral ramifications and hence a conscious attempt must be made to follow the tracts till the end . using magnification loops or the microscope at the time of dissection may enhance prospects of complete removal . while it may be impossible to guarantee a complete removal , we believe that this along with the practice of excision of the tract along with surrounding tissue has led to a low recurrence rate seen in our series . in our series , this relatively high incidence can be attributed to the fact that a high percentage of the patients in this series were from a low socioeconomic stratum . though facial nerve paralysis / weakness has been reported in patients undergoing superficial parotidectomy for first branchial cleft anomalies , none of our patients had involvement of facial nerve . first branchial cleft anomalies are thought to develop as a result of incomplete obliteration of the cleft between the mandibular process of the first arch and the second arch . a sinus will have an opening in the upper neck or in the floor of the external auditory canal , and a fistula will have an opening in both of these sites . the first branchial cleft anomalies have been classified as type i or type ii by work . a cystic mass in the postauricular area extends medially and anteriorly along the external auditory canal . type ii is considered to be a duplication of the cartilaginous external auditory canal and pinna . a sinus passes from an external opening high in the neck along the anterior border of scm muscle , superficial or deep to the facial nerve in close relation to the parotid gland . it can either end blindly at the floor of the cartilaginous external auditory canal or open in to the external auditory canal , which is called the collaural fistula . in both types entrapment of desquamating squamous epithelium will result in the production of a cholesteatoma process resulting in erosion of bony external meatus , tympanic annulus , and hypotympanum . in the series by belenky and medina 66.66% of patients belonged to work i and 33.33% to work ii . in the study by nofsinger et al . in our study , work i constituted 17.65% and work ii constituted 20.59% . in the study by triglia et al . on the first branchial cleft anomalies , 30.77% were male and 69.23% female . in the study by belenky and medina the incidence in male was 22.22% and in female was 77.77% . in our study , 17.65% were male and 20.59% were female . the symptoms and signs related to these anomalies in this series are similar to those described by various authors . in general , both types of anomalies may present as a progressively enlarging or recurrent mass or as a draining sinus . incision and drainage of an abscess are frequently needed before definitive surgical treatment can be performed . histopathology of the work i lesions in our study showed that 100% of cases lined by squamous epithelium and 20% had cartilage components in the subepithelial layer . in the study by belenky and medina 16.7% of work i had cartilage component in the subepithelium . intraoperatively the lesion was found superficial to the facial nerve in 100% of our cases . belenky and medina reported that the lesion was superficial to facial nerve in 88.88% of cases . in the study by triglia et al . lesion was deep to facial nerve in 39% cases . in the series by nofsinger et al . , the lesion was deep to the facial nerve in 55% of cases . [ 4 , 11 ] that it is advisable to perform a superficial parotidectomy in cases of first cleft anomalies while identifying the tract in relation to the facial nerve . during embryonic development , the second arch grows caudally , enveloping the third , fourth , and sixth arches and fusing with skin caudal to these arches , forming a deep groove ( cervical sinus ) . a persistent fistula of the second branchial cleft and pouch usually has its external opening in the neck near mid or lower part of scm muscle . as it ascends it pierces platysma . at the level of hyoid it curves medially and passes between the external and internal carotids in relation to the hypoglossal and glossopharyngeal nerves . it opens in to the oropharynx usually in the intratonsillar cleft of palatine tonsil . in series of 98 cases by ford 78% presented by the age of five years and in vast majority there was history of intermittent discharge and infection of neck sinus since birth . in seven percent there was history of incision and drainage of an associated neck abscess . in his series 60% sinus opening was on the right side and 40% on left . in our series of 17 cases 70% presented at age above 11 years . . there may be only a simple sinus opening that extends up the neck for a variable distance . branchial fistulas commonly present with persistent mucoid discharge from an opening in the skin of the neck . but they have been documented as to present as parapharyngeal mass located in the supratonsillar fossa and extending to the lateral nasopharynx . exceedingly rarely , a branchial cleft anomaly may be found to be malignant on presentation . the completeness of a fistula is diagnosed by a dye test in which methylene blue is injected through the outer opening and appears in the throat . a negative preoperative outcome on the test might become positive under general anaesthesia because of muscle relaxation . occasionally , the fistula tract may be blocked by secretion or granulation giving negative fistula test . in many a case , saliva is seen dribbling from the neck opening , which itself proves the completeness of the tract ( figure 7 ) . in the 62 pediatric second branchial cleft anomalies , bajaj et al . reported 50 of them to be unilateral and 12 to be bilateral . the fistulous tract can be approached through a series of stepladder incision first encompassing the sinus opening and second overlying the carotid bifurcation . subsequently the parapharyngeal portion of the fistula can be approached perorally after tonsillectomy . a wide cervicotomy incision ( hockey stick ) can also be used which allows for adequate exposure of neck structure for accurate dissection . in all our cases , we traced the fistula up to the tonsillar area and excised the tract . third branchial anomalies are rare and constitute less than 1% of all such cases . here the fistula opening is seen in the lower neck and it passes along the carotid sheath and then passes between the glossopharyngeal and hypoglossal nerve , piercing the thyrohyoid membrane to enter pharynx in the region of pyriform fossa . third pouch remnants are described as passing superior to superior laryngeal nerve and posterior to the common carotid artery . a persistent fistula of the fourth branchial cleft and pouch is theoretically possible but is very rare . here the fistula opens in to the lower part of neck near the scm muscle . the tract then passes inferiorly between superior and recurrent laryngeal nerve into the mediastinum , looping around the arch of aorta in the left and subclavian artery in the right and passes back in to the neck , ascending posterior to the carotid . then it passes between the thyroid and cricoid cartilages and opens in to the pyriform fossa . , almost all the fourth arch anomalies reported occurred in the left side . in our series also , the fourth arch fistula occurred in the left side . , these anomalies can be dangerous because of rapid enlargement leading to tracheal compression and respiratory distress . noncommunicating or noninfected communicating cysts may present as cold thyroid nodules . when infected , diagnosis and successful excision of a pyriform fossa sinus are very challenging and require meticulous approach . a history of recurrent upper respiratory tract infection , neck or thyroid pain and tenderness , and neck mass is common . other presentations include cellulites , hoarseness , odynophagia , thyroiditis , abscess , and stridor . a combination of ultrasound and ct with or without oral contrast will assist in the diagnosis . all these patients came to us with after recurrences following surgeries done elsewhere . in cases of fourth cleft anomalies , the third branchial anomalies showed a fistulous opening in the lower neck , which , on dissection , passed between the glossopharyngeal and hypoglossal nerve as classically described , piercing the thyrohyoid membrane to enter pharynx in the region of pyriform fossa . in the cases of fourth branchial anomalies , the intramediastinal part of the fistula was left behind with their ends ligated and the rest of the tract was dissected out from the superior mediastinum up to the pyriform fossa . our series confirms a higher incidence of first branchial cleft anomaly among females , the cause of which needs to be investigated.none of our patients had involvement of facial nerve . all patients had tracts going superficial to the facial nerve.all the fourth arch anomalies in our series occurred on the left side which is consistent with the literature.while it may be impossible to guarantee a complete removal , injection of dye and microscopic removal may be vital in preventing recurrences . we believe that this along with the practice of excision of the tract along with surrounding tissue has led to a low recurrence rate seen in our series . our series confirms a higher incidence of first branchial cleft anomaly among females , the cause of which needs to be investigated . all the fourth arch anomalies in our series occurred on the left side which is consistent with the literature . while it may be impossible to guarantee a complete removal , injection of dye and microscopic removal may be vital in preventing recurrences . we believe that this along with the practice of excision of the tract along with surrounding tissue has led to a low recurrence rate seen in our series . diagnosis is rather easy with a proper knowledge of the anatomy of the branchial anomalies . confirming the extent of the tract is mandatory before any surgery as these lesions pass in relation to some of the most vital structures of the neck . surgery must always be the treatment option for these lesions due to the fact that these lesions do not regress spontaneously and they have a high incidence of recurrent infection . surgery also gives a chance to diagnose by means of histopathology , the rare occurence of branchogenic carcinoma .

objective . to find out the incidence of involvement of individual arches , anatomical types of lesions , the age and sex incidence , the site and side of predilection , the common clinical features , the common investigations , treatment , and complications of the different anomalies . setting . academic department of otolaryngology , head and neck surgery . design . a 10 year retrospective study . participants . 30 patients with clinically proven branchial anomalies including patients with bilateral disease totaling 34 lesions . main outcome measures . the demographical data , clinical features , type of branchial anomalies , and the management details were recorded and analyzed . results and observations . the mean age of presentation was 18.67 years . male to female sex ratio was 1.27 : 1 with a male preponderance . of the 34 lesions , maximum incidence was of second arch anomalies ( 50% ) followed by first arch . we had two cases each of third and fourth arch anomalies . only 1 ( 3.3% ) patients of the 30 presented with lesion at birth . the most common pathological type of lesions was fistula ( 58.82% ) followed by cyst . 41.18% of the lesions occurred on the right side . all the patients underwent surgical excision . none of our patients had involvement of facial nerve in first branchial anomaly . all patients had tracts going superficial to the facial nerve . conclusion . confirming the extent of the tract is mandatory before any surgery as these lesions pass in relation to some of the most vital structures of the neck . surgery should always be the treatment option . injection of dye , microscopic removal and inclusion of surrounding tissue while excising the tract leads to a decreased incidence of recurrence .

1. Introduction 2. Materials and Methods 3. Results and Observations 4. Discussion 5. Individual Branchial Anomalies 6. Highlights 7. Conclusion

in fact , approximately 17% of all pediatric cervical masses are due to branchial anomalies . although branchial cleft cysts are benign , superinfection , mass effect , and surgical complications account for its morbidity . branchial apparatus , seen in the early embryonic life , has a vital role to play in the development of head and neck structures . branchia is the greek word for gill , and the same word represents these anomalies owing to their resemblance to gills of certain species as fish . six paired branchial arches , which appear in the fourth week of embryonic life , give rise to many structures of the head and neck . many anomalies of the head and neck region have been attributed to the aberrant development of these structures . depending on the anatomic location , branchial anomalies are classified into first , second , third , and fourth anomalies . the course of a particular branchial anomaly is caudal to the structures derived from the corresponding arch and dorsal to the structures that develop from the following arch . cysts are considered to be entrapped remnants of branchial cleft or sinuses ; sinuses are remnants of cleft or pouches ; and fistulae result from persistence of both pouch and cleft . different anomalies of the head and neck area have been attributed to the maldevelopment of branchial apparatus . the importance of knowing the development of branchial apparatus and their anomalies is in applying the knowledge during surgery , as vital structures like facial nerve and parotid are in intimate relation with many of these anomalies . we performed a ten - year retrospective study to analyze the pathophysiology , clinical features , and management of branchial anomalies . ours is a retrospective study of 30 cases of branchial anomalies , which presented to the department of otolaryngology , head and neck surgery , kasturba medical college , over a period of 10 years from 2000 to 2010 . the side and site of the lesion and the site of opening of sinuses and fistula were noted . patients with sinus and fistulas underwent sino-/fistulogram , by injecting contrast material urografin into the tract ( figure 1 ) . such patients were taken up for surgical excision of tract four weeks later . during surgery , a conscious attempt was made to remove some fascia and tissues adjacent to the branchial tracts along their path to avoid leaving behind ramifications that might lead to recurrences . this fistulous communication between the external auditory canal and the neck in the upper part of anterior border of sternocleidomastoid ( scm ) muscle was identified by injecting methylene blue dye into the neck opening which was seen coming of an opening in the external auditory canal . the tract was then dissected from the surrounding tissue and followed till its opening in the external auditory canal ( figure 2 ) . the tract was excised off its attachment to the external auditory canal and wound closed in layers . elliptical skin incision was made over the skin opening and the dissection proceeded in the direction of the tract . this second incision was given at the level of the hyoid and the whole tract was brought out through this incision . during their course towards the oropharynx , the second branchial fistulae , were seen passing between the carotid bifurcations , where they were in close relation to the hypoglossal nerve . the tracts were followed to the pharynx and were excised ; the pharyngeal defects were sutured . the tract then passed inferiorly into the mediastinum , looping around the arch of aorta in the left and subclavian artery in the right and back in to the neck , ascending posterior to the carotid . the intramediastinal part of the fistulae was left behind with their ends ligated and the rest of the tract was dissected out from the superior mediastinum up to the pyriform fossa ( figure 4 ) . thirty patients with 34 branchial anomalies were studied retrospectively over a period of 10 years from 2000 to 2010 in the department of otolaryngology , head and neck surgery , kasturba medical college , mangalore . there was maximum incidence of second branchial anomalies with 17(50% ) cases . among the first branchial anomalies , seven ( 20.59% ) cases belonged to work ii , while six ( 17.65% ) cases belonged to work i ( according to the work classification ) . among the anatomical types of the lesion , we had a maximum incidence of fistula seen in 20 ( 58.82% ) cases , followed by cyst in 14 ( 41.12% ) cases . the youngest patient in our study was one and a half years and the oldest one 48 years . the mean age of presentation was 18.67 years with a standard deviation of 11.06 . only 1 ( 3.3% ) the mean age of onset among this late onset group was 15.97 years . among the first branchial anomalies , the maximum incidence of the lesion was seen in the 1120 age group with 5 ( 14.70% ) cases followed by 4 ( 11.76% ) cases in the 610 and 2140 age groups . in the second arch anomalies , we had four ( 11.76% ) cases in the 610 age group and seven ( 20.59% ) cases in the 1120 and five ( 14.70% ) in 2040 age groups . considering all the anomalies together , 55.88% were males and 44.12% were females with a male to female ratio of 1.27 : 1 . in the first branchial anomalies , the incidence in males was 17.65% and 20.59% in females . among the second arch anomalies the overall incidence of the anomalies was more on the right side ( 57.08% ) while 42.92% of the lesions occurred on the left side . in the first arch anomalies , 8 ( 23.53% ) cases were present on the right and 5 ( 14.71% ) cases on the left . in the second arch anomalies 11 ( 32.35% ) cases occurred on the right side and six ( 17.65% ) cases on the left including one patient with bilateral branchial cysts ( figure 6 ) . the third anomalies occurred on the right side and the fourth anomalies on the left side . in all anomalies put together , the most common clinical feature was a swelling seen in 21 ( 61.76 ) and fistula opening in 18 ( 52.94% ) cases . 26.47% of the patients had pain at the site of the lesion . among the first arch anomaly patients , swelling in the neck and postauricular region was the most common presenting feature ( 29.41% ) . the most common presenting feature of second branchial arch anomaly was neck swelling , seen in 26.47% , while 23.53% presented with opening in the neck . the third arch anomaly patient had swelling and opening in the neck along with discharge . the fourth arch anomaly patient had swelling and opening in the neck along with discharge and pain . ct scan and ultrasound were done in all cases of third and fourth arch anomalies and nine cases of second arch anomaly . acute infection was treated by a course of antibiotics in 18 ( 60% ) cases and incision and drainage in one case ( before proceeding to the excision of the lesion ) . 73.33% of the cases were managed by single incision , while 23.33% required stepladder incision . though described first in the early nineteenth century , the origin and classification of different branchial anomalies are highly controversial even today . second branchial anomalies are considered to be the commonest with figures up to 95% being reported . the remainder of branchial anomalies is derived from first branchial remnants ( 18% ) with third and fourth branchial anomalies being quite rare . several theories proposed for the development of branchial anomalies include branchial apparatus theory , cervical sinus theory , thymopharyngeal theory , and inclusion theory . of these , the widely accepted theory is that branchial anomalies result from incomplete involution of the branchial apparatus . , most of the branchial anomalies arise from the second branchial cleft ( 92.45% ) . remaining is derived from first arch remnants ( 4.72% ) and third ( 1.87% ) and fourth arch anomalies ( 0.94% ) are quite rare . also reported higher incidence of second branchial anomalies ( 78% ) in their series of 80 patients . choi and zalzal who reported a higher incidence of first branchial arch anomalies ( 25% ) in their series still had the maximum incidence of second branchial arch anomalies ( 40% ) . in our series , we had the maximum incidence of second arch anomalies ( 50% ) followed by first arch anomalies ( 38.24% ) . choi and zalzal reported a maximum incidence of sinuses , followed by fistula . in our series cysts were the most common lesion followed by sinuses . the age of onset of these anomalies has been seen to vary according to the type of the lesion . choi and zalzal have noted that mean age of presentation of cyst ( 18.35 years ) was late compared to that of fistulae ( 6.28 years ) and sinuses ( 7.82 years ) . in our group , it was found that fistulas ( 1.14 years ) had an early age of onset followed by that of sinuses ( 4.21 years ) . cysts ( 7.51 years ) were found to have a late onset compared to the other two lesions . have pointed out that the branchial anomalies occur more on the right side . they had 60% incidence on the right side and 40% on the left side . in the present study , we have a similar picture with right side incidence of 55.38% and left side incidence of 44.62% . in the study by choi and zalzal , the most common presenting features were discharge from the openings , cervical mass , and repeated infection . in our study , the most common clinical feature was a swelling seen in 21 ( 61.76 ) . an initial correct diagnosis is crucial because experience shows that recurrence rates after surgical excision of branchial anomalies are 14% and 22% with previous infection and surgery , respectively , whereas the recurrence rate for primary lesion is 3% . although physical examination and history are the most important elements in the diagnosis , radio - diagnostic studies can add valuable information to the evaluation of a congenital neck mass . a ct scan is an accurate and noninvasive diagnostic tool , which can confirm the diagnosis or suggest an alternative diagnosis , define both the location and extent of a neck lesion , and delineate infectious process or possible malignant degeneration . ct scan is useful in evaluating first branchial anomaly and the position of facial nerve . ct scan is reported to be more useful than mri in evaluating branchial anomalies . in case of sinus or fistula , sinogram or conray contrast study can delineate the course of branchial anomaly . in the series by choi and zalzal , ct scan was performed on 15.38% of patients , sonogram / contrast study was performed on 7.69% of patients , and ultrasound / mri was done on 1.92% of patients . surgery is definitive mode of treatment because there is lack of spontaneous regression , a high rate of recurrent infection , the possibility of other diagnoses , and rare malignant degeneration . in our series 60% of patients took medical treatment and 3.33% underwent drainage of abscess before definitive surgical excision of the lesion . surgical excision of lesion was done in all patients . it is our observation that while methylene blue dye enhances visualization of the larger and more proximal ( in relation to the punctum ) part of the tracts and ramifications , it does not demarcate the most peripheral ramifications and hence a conscious attempt must be made to follow the tracts till the end . while it may be impossible to guarantee a complete removal , we believe that this along with the practice of excision of the tract along with surrounding tissue has led to a low recurrence rate seen in our series . in our series , this relatively high incidence can be attributed to the fact that a high percentage of the patients in this series were from a low socioeconomic stratum . though facial nerve paralysis / weakness has been reported in patients undergoing superficial parotidectomy for first branchial cleft anomalies , none of our patients had involvement of facial nerve . first branchial cleft anomalies are thought to develop as a result of incomplete obliteration of the cleft between the mandibular process of the first arch and the second arch . a sinus will have an opening in the upper neck or in the floor of the external auditory canal , and a fistula will have an opening in both of these sites . the first branchial cleft anomalies have been classified as type i or type ii by work . type ii is considered to be a duplication of the cartilaginous external auditory canal and pinna . a sinus passes from an external opening high in the neck along the anterior border of scm muscle , superficial or deep to the facial nerve in close relation to the parotid gland . it can either end blindly at the floor of the cartilaginous external auditory canal or open in to the external auditory canal , which is called the collaural fistula . in both types entrapment of desquamating squamous epithelium will result in the production of a cholesteatoma process resulting in erosion of bony external meatus , tympanic annulus , and hypotympanum . on the first branchial cleft anomalies , 30.77% were male and 69.23% female . in the study by belenky and medina the incidence in male was 22.22% and in female was 77.77% . in general , both types of anomalies may present as a progressively enlarging or recurrent mass or as a draining sinus . histopathology of the work i lesions in our study showed that 100% of cases lined by squamous epithelium and 20% had cartilage components in the subepithelial layer . intraoperatively the lesion was found superficial to the facial nerve in 100% of our cases . belenky and medina reported that the lesion was superficial to facial nerve in 88.88% of cases . lesion was deep to facial nerve in 39% cases . , the lesion was deep to the facial nerve in 55% of cases . [ 4 , 11 ] that it is advisable to perform a superficial parotidectomy in cases of first cleft anomalies while identifying the tract in relation to the facial nerve . during embryonic development , the second arch grows caudally , enveloping the third , fourth , and sixth arches and fusing with skin caudal to these arches , forming a deep groove ( cervical sinus ) . a persistent fistula of the second branchial cleft and pouch usually has its external opening in the neck near mid or lower part of scm muscle . at the level of hyoid it curves medially and passes between the external and internal carotids in relation to the hypoglossal and glossopharyngeal nerves . it opens in to the oropharynx usually in the intratonsillar cleft of palatine tonsil . in series of 98 cases by ford 78% presented by the age of five years and in vast majority there was history of intermittent discharge and infection of neck sinus since birth . in his series 60% sinus opening was on the right side and 40% on left . branchial fistulas commonly present with persistent mucoid discharge from an opening in the skin of the neck . but they have been documented as to present as parapharyngeal mass located in the supratonsillar fossa and extending to the lateral nasopharynx . in many a case , saliva is seen dribbling from the neck opening , which itself proves the completeness of the tract ( figure 7 ) . in the 62 pediatric second branchial cleft anomalies , bajaj et al . subsequently the parapharyngeal portion of the fistula can be approached perorally after tonsillectomy . in all our cases , we traced the fistula up to the tonsillar area and excised the tract . third branchial anomalies are rare and constitute less than 1% of all such cases . third pouch remnants are described as passing superior to superior laryngeal nerve and posterior to the common carotid artery . a persistent fistula of the fourth branchial cleft and pouch is theoretically possible but is very rare . here the fistula opens in to the lower part of neck near the scm muscle . the tract then passes inferiorly between superior and recurrent laryngeal nerve into the mediastinum , looping around the arch of aorta in the left and subclavian artery in the right and passes back in to the neck , ascending posterior to the carotid . then it passes between the thyroid and cricoid cartilages and opens in to the pyriform fossa . , almost all the fourth arch anomalies reported occurred in the left side . in our series also , the fourth arch fistula occurred in the left side . a history of recurrent upper respiratory tract infection , neck or thyroid pain and tenderness , and neck mass is common . other presentations include cellulites , hoarseness , odynophagia , thyroiditis , abscess , and stridor . in cases of fourth cleft anomalies , the third branchial anomalies showed a fistulous opening in the lower neck , which , on dissection , passed between the glossopharyngeal and hypoglossal nerve as classically described , piercing the thyrohyoid membrane to enter pharynx in the region of pyriform fossa . in the cases of fourth branchial anomalies , the intramediastinal part of the fistula was left behind with their ends ligated and the rest of the tract was dissected out from the superior mediastinum up to the pyriform fossa . our series confirms a higher incidence of first branchial cleft anomaly among females , the cause of which needs to be investigated.none of our patients had involvement of facial nerve . all patients had tracts going superficial to the facial nerve.all the fourth arch anomalies in our series occurred on the left side which is consistent with the literature.while it may be impossible to guarantee a complete removal , injection of dye and microscopic removal may be vital in preventing recurrences . we believe that this along with the practice of excision of the tract along with surrounding tissue has led to a low recurrence rate seen in our series . our series confirms a higher incidence of first branchial cleft anomaly among females , the cause of which needs to be investigated . all the fourth arch anomalies in our series occurred on the left side which is consistent with the literature . while it may be impossible to guarantee a complete removal , injection of dye and microscopic removal may be vital in preventing recurrences . we believe that this along with the practice of excision of the tract along with surrounding tissue has led to a low recurrence rate seen in our series . diagnosis is rather easy with a proper knowledge of the anatomy of the branchial anomalies . confirming the extent of the tract is mandatory before any surgery as these lesions pass in relation to some of the most vital structures of the neck . surgery must always be the treatment option for these lesions due to the fact that these lesions do not regress spontaneously and they have a high incidence of recurrent infection .

eugen bleuler ( 1857 - 1939 ) was born and raised in the village of zollikon near zurich in switzerland . after graduating in medicine study trips took him to paris to work with jean - martin charcot , to munich where he trained under bernhard von gudden , and to london . he completed his residential training at the university hospital of psychiatry in zurich , known as burghlzli , and was appointed director of the mental asylum of rheinau in 1886 . after living with and caring for long - term psychiatric patients in rheinau for more than 12 years , he returned to zurich as professor of psychiatry at burghlzli in 1898 , and held this position until his retirement in 1927 . whilst today bleuler is perhaps best known for the introduction of the term and concept schizophrenia , or more precisely the group of schizophrenias , this paper considers his work on schizophrenia principally in terms of its relationship to long - standing and complex theoretical debates in psychiatric nosology . these concern the very nature of mental illness , in particular the relationships between nature and mind and the individual and society . debates about how nature , ie , the brain and body , is related to the mind were highly topical with regard to mental illness in bleuler 's time and remain so today , as nicely captured in lipowski 's interrogative : psychiatry : mindless or brainless , both or neither ? bleuler would have sided neither with a mindless nor with a brainless psychiatry , but would have acknowledged both brain and mind , as well as social factors , as equally important elements in mental health and illness . he can thus be considered an early proponent of a bio - psychosocial model in psychiatry . reviewing bleuler 's conception of schizophrenia against the background of his overall clinical and theoretical work and contrasting it with kraepelin 's earlier concept dementia praecox , this paper provides a critical overview of bleuler 's key nosological principles and links his work with present day debates about naturalism , essentialism , and stigma . bleuler 's work stands in the conceptually rich tradition of turn - of - the - century psychiatry and psychology and can be considered a synthesis of various contemporary concepts and theories ( see ref 5 for an overview ) . amongst these are the theories of emil kraepelin ( 1856 - 1926 ) and sigmund freud ( 1856 - 1939 ) , who , through their different theoretical emphases , introduced him to two in one view opposed , in another complementary traditions in psychiatry . emil kraepelin promoted the biological tradition , viewing mental illnesses as natural disease entities ( natrliche krankheitseinheiten ) , ie , entities given by nature and existing independently from the psychiatric practitioner and researcher . acknowledging that science at the time did not yet allow identification of these posited disease entities , he based his preliminary nosology on an alternative principle , namely following karl ludwig kahibaum ( 1828 - 1899)on the course and prognosis of a given illness . this led to a dichotomous nosology based on the distinction between the concept of dementia praecox , characterized by a fatal course , and the concept of manic - depressive insanity , characterized by a positive course . this nosological system had gained wide acceptance amongst psychiatrists by the turn of the century . at the same time , sigmund freud , founder of psychoanalysis , struggled to find recognition for his new concepts in academic psychiatry . also rooted in a biological tradition , yet offering a new psychological explanation of mental illness as the unfolding of unconscious intrapsychic conflicts , freud 's theory was met with skepticism , if not outright rejection , as a dogmatic world - view . bleuler was the only influential contemporary academic psychiatrist who not only joined in the debate about psychoanalysis , but also , while always remaining circumspect , implemented psychoanalytic treatment and research at burghlzli . bleuler 's thinking was further shaped by the german philosopher and psychologist johann friedrich herbart ( 1776 - 1841 ) . championing a scientific approach to psychology , herbart was one of the major proponents of association theory . this saw human mental life as consisting in a multitude of basic , individual mental acts that are combined ( associated ) into more complex cognitive functions . as will be shown in the following section , bleuler , like the viennese psychiatrist erwin stransky ( 1877 - 1962 ) , integrated aspects of this atomistic approach to psycho(patho)logy in his work on dementia praecox and its reconceptualization as schizophrenia . in 1908 , bleuler publicly introduced the term and concept schizophrenia in a lecture given at the meeting of the deutscher verein fur psychatrie ( german psychiatric association ) in berlin . in the opening paragraph , he summarized his reasons for abandoning kraepelin 's earlier concept dementia praecox : i wish to emphasize that in kraepelin 's , dementia praecox it is neither a question of an essential dementia nor of a necessary precociousness . for this reason , and because from the expression dementia praecox one can not form further adjectives nor substantives , i am taking the liberty of employing the word schizophrenia for revising the kraepelinian concept . in my opinion the breaking up or splitting of psychic functioning is an excellent symptom of the whole group [ ... ] ( translation from ref 20 ) whilst this passage also underscores the importance of linguistic labels in psychiatry , the wish to rename kraepelin 's dementia praecox is only a secondary motive in bleuler 's introduction of schizophrenia . having gathered epidemiological data on the prognosis and end states of patients admitted with a diagnosis of dementia praecox , he came to the conclusion that this group of patients could not be coherently defined by a specific prognosis , ie , that this kraepelinian nosological principle had to be rejected . importantly , however , bleuler wanted to maintain the nosological unity of the group of patients that kraepelin identified by dementia praecox and believed that there was something specifically schizophrenic behind the general manifestations of the disease . he shared kraepelin 's assumption of an underlying physical disease process that the sciences of his day could not yet identify , and having rejected kraepelin 's principle of prognosis he set about searching for alternative criteria to define the essence of schizophrenia . to this purpose , bleuler turned to psychology , where , influenced by herbart 's atomistic view of the mind , he identified the alteration of associations , ie , of the way in which basic mental acts meaningfully combine into more complex units , as schizophrenia 's most fundamental feature . this idea is more fully developed in bleuler 's 1911 volume dementia praecox or the group of schizophrenias : the connections between associations are lost . the disease interrupts the threads that give direction to our thoughts in an irregular fashion , sometimes affecting only a few , sometimes a large proportion of them . thus , the result of the thought process is rendered unusual , and often logically incorrect , bleuler develops a symptomatology organized around two dichotomous distinctions : that between basic and accessory and that between primary and secondary symptoms . basic symptoms are those which are necessarily present in any case of schizophrenia ; accessory symptoms may or may not occur . the distinction of primary and secondary refers to both etiology and pathogenesis , with primary symptoms being caused directly by the assumed neurobiological disease process , whilst secondary symptoms are seen as the potentially understandable reactions of the psyche to the disturbing primary symptoms . the alteration of associations is the only symptom that bleuler regarded as both basic and primary , and can thus be described as the core disturbance in the bleulerian conception of schizophrenia . importantly , the alteration of associations is not to be equated with formal thought disorder but to be understood as a disturbance affecting all aspects , both cognitive and affective , of mental life . also , bleuler was keen to stress that the alteration of associations had been identified by empirical observation , not by theoretical speculation , and that it was accessible to experimental testing . two other phenomena that bleuler characterized as basic ( but not primary ) symptoms were ambivalence and autism . by ambivalence , he understood the simultaneous presence of contradictory ideas and emotions : autism described the phenomenon of a patient 's getting lost in personal ideas , emotions , and intentions without being able to adapt to the external reality , resulting in a reduction of communication . linking bleuler 's implementation of psychoanalytic , or more generally psychodynamic , ideas , with his distinction between primary and secondary symptoms and thus to the interaction between brain / body and mind , a further distinction has to be introduced , namely that between form and content . bleuler himself did not systematically introduce nor use this distinction , but it is implicit in his statement that what is psychologically understandable is the content of schizophrenic symptoms , ie , why a specific hallucination or delusion occurs . as the following quotation makes clear , he thus assumed a neurobiological disease process giving rise to a primary symptom , the alteration of associations , to which understandably the psyche reacts giving rise to secondary symptoms , with individually meaningful content : it goes without saying that the disease process can not give rise to the complex psychological symptoms which we are accustomed to consider first and foremost . this process can not account for the fact that it is a specific delusional idea or a specific hallucination that occurs . the process can only lead to certain fundamental disturbances of the psyche on the basis of which , in conjunction with precipitating and determining factors , hallucinations and delusional ideas emerge . the characterization of schizophrenic symptoms as bearing individually meaningful content is one of the important novelties in bleuler 's understanding of schizophrenia . in the words of his son manfred bleuler : one of bleuler 's main aims in choosing and following his career was to arrive at an understanding of schizophrenic symptoms as expressions of an inner psychodynamic life . [ ... ] he studied the schizophrenic 's life essentially in the same way as we study the inner life of neurotics , of healthy men , and of ourselves . in his summary of the development of the schizophrenia concept at the burghlzli hospital over the course of nearly 70 years ( 1902 - 1971 ) , manfred bleuler expanded on this attitude and orientation so central to his father 's work : he stressed that the view of schizophrenic symptoms as secondary phenomena , ie , understandable intellectually and emotionally , also made them in principle accessible to therapy . such therapy consisted of two pillars , firstly personal communication ( today analytically oriented psychotherapy ) in order to help patients by understanding their intentions and skills in adapting to reality ; and secondly multiple joint activities , such as work and leisure activities in groups of patients . during his time at the rheinau psychiatric hospital , eugen bleuler devoted as much time as possible to such personal contacts with his patients ; later , during his years at burghlzli , he suffered severely from a lack of time to do this . unlike kraepelin , then , bleuler saw no contradiction between the assumption of an underlying neurobiological disease process and the assumption of psychological understandability , but integrated both in his conception of schizophrenia . the nosological divide between psychotic and neurotic disorders , commonly drawn along the line of biologically determined versus psychologically understandable disease , is thus blurred . in the light of his keen awareness of social factors in the course of the disease and his efforts to address those therapeutically , bleuler can thus be seen as an early proponent of a bio - psychosocial understanding of mental illness . whilst the belief in a specifically schizophrenic feature spurred bleuler 's reconceptualization , he was anxious to leave his concept open for scientific revision and to accommodate the possibility that such a unifying feature might indeed not exist . he therefore spoke cautiously of the group of schizophrenias and intended his account to be a preliminary one . bleuler 's nosological changes were met with enthusiasm by some , with criticism by others , and the reception of his work varied between countries ie , psychiatric cultures . whilst in switzerland , for example , the concept of schizophrenia was quickly adopted , it was criticized in germany for carrying too much psychoanalytical baggage and for relying on the poorly defined concept of association . in britain , where kraepelin 's prognosis - based nosology had been criticized early on for promoting therapeutic nihilism and unjustified counsels of despair , the reception of bleuler 's schizophrenia was impeded by its perceived close connection with kraepelin 's dementia praecox . once the break with kraepelinian nosology implicit in the new concept was recognized , this latter aspect was especially welcomed . later the concept was developed further , giving rise to derivative concepts , such as schizoidism . later the concept was developed further , giving rise to derivative concepts such as schizoidism , which , whilst present in bleuler 's work on schizophrenia from the outset , was only explicitly introduced under that label by kurt binswanger ( 1887 - 1981 ) in 1920 . bleuler 's later writings on general psychology and philosophy received little attention from either his contemporaries or later workers . this may be due to the rather speculative , and in parts obscure , nature of works like psychoids : organizing principle of organic development or in these works , bleuler proposes a comprehensive life science in which physical , mental , and social phenomena are not seen as separate or even opposed , but as equal aspects of a single integrative life principle . in the light of the portrayal of bleuler as a proponent of a bio - psychosocial understanding of mental illness given above , these works might be interpreted as his attempt to theorize the relation and interaction between brain / body , mind and the social sphere . bleuler 's work stands in the conceptually rich tradition of turn - of - the - century psychiatry and psychology and can be considered a synthesis of various contemporary concepts and theories ( see ref 5 for an overview ) . amongst these are the theories of emil kraepelin ( 1856 - 1926 ) and sigmund freud ( 1856 - 1939 ) , who , through their different theoretical emphases , introduced him to two in one view opposed , in another complementary traditions in psychiatry . emil kraepelin promoted the biological tradition , viewing mental illnesses as natural disease entities ( natrliche krankheitseinheiten ) , ie , entities given by nature and existing independently from the psychiatric practitioner and researcher . acknowledging that science at the time did not yet allow identification of these posited disease entities , he based his preliminary nosology on an alternative principle , namely following karl ludwig kahibaum ( 1828 - 1899)on the course and prognosis of a given illness . this led to a dichotomous nosology based on the distinction between the concept of dementia praecox , characterized by a fatal course , and the concept of manic - depressive insanity , characterized by a positive course . this nosological system had gained wide acceptance amongst psychiatrists by the turn of the century . at the same time , sigmund freud , founder of psychoanalysis , struggled to find recognition for his new concepts in academic psychiatry . also rooted in a biological tradition , yet offering a new psychological explanation of mental illness as the unfolding of unconscious intrapsychic conflicts , freud 's theory was met with skepticism , if not outright rejection , as a dogmatic world - view . bleuler was the only influential contemporary academic psychiatrist who not only joined in the debate about psychoanalysis , but also , while always remaining circumspect , implemented psychoanalytic treatment and research at burghlzli . bleuler 's thinking was further shaped by the german philosopher and psychologist johann friedrich herbart ( 1776 - 1841 ) . championing a scientific approach to psychology , herbart was one of the major proponents of association theory . this saw human mental life as consisting in a multitude of basic , individual mental acts that are combined ( associated ) into more complex cognitive functions . as will be shown in the following section , bleuler , like the viennese psychiatrist erwin stransky ( 1877 - 1962 ) , integrated aspects of this atomistic approach to psycho(patho)logy in his work on dementia praecox and its reconceptualization as schizophrenia . in 1908 , bleuler publicly introduced the term and concept schizophrenia in a lecture given at the meeting of the deutscher verein fur psychatrie ( german psychiatric association ) in berlin . in the opening paragraph , he summarized his reasons for abandoning kraepelin 's earlier concept dementia praecox : i wish to emphasize that in kraepelin 's , dementia praecox it is neither a question of an essential dementia nor of a necessary precociousness . for this reason , and because from the expression dementia praecox one can not form further adjectives nor substantives , i am taking the liberty of employing the word schizophrenia for revising the kraepelinian concept . in my opinion the breaking up or splitting of psychic functioning is an excellent symptom of the whole group [ ... ] ( translation from ref 20 ) whilst this passage also underscores the importance of linguistic labels in psychiatry , the wish to rename kraepelin 's dementia praecox is only a secondary motive in bleuler 's introduction of schizophrenia . having gathered epidemiological data on the prognosis and end states of patients admitted with a diagnosis of dementia praecox , he came to the conclusion that this group of patients could not be coherently defined by a specific prognosis , ie , that this kraepelinian nosological principle had to be rejected . importantly , however , bleuler wanted to maintain the nosological unity of the group of patients that kraepelin identified by dementia praecox and believed that there was something specifically schizophrenic behind the general manifestations of the disease . he shared kraepelin 's assumption of an underlying physical disease process that the sciences of his day could not yet identify , and having rejected kraepelin 's principle of prognosis he set about searching for alternative criteria to define the essence of schizophrenia . to this purpose , bleuler turned to psychology , where , influenced by herbart 's atomistic view of the mind , he identified the alteration of associations , ie , of the way in which basic mental acts meaningfully combine into more complex units , as schizophrenia 's most fundamental feature . this idea is more fully developed in bleuler 's 1911 volume dementia praecox or the group of schizophrenias : the connections between associations are lost . the disease interrupts the threads that give direction to our thoughts in an irregular fashion , sometimes affecting only a few , sometimes a large proportion of them . thus , the result of the thought process is rendered unusual , and often logically incorrect , bleuler develops a symptomatology organized around two dichotomous distinctions : that between basic and accessory and that between primary and secondary symptoms . basic symptoms are those which are necessarily present in any case of schizophrenia ; accessory symptoms may or may not occur . the distinction of primary and secondary refers to both etiology and pathogenesis , with primary symptoms being caused directly by the assumed neurobiological disease process , whilst secondary symptoms are seen as the potentially understandable reactions of the psyche to the disturbing primary symptoms . the alteration of associations is the only symptom that bleuler regarded as both basic and primary , and can thus be described as the core disturbance in the bleulerian conception of schizophrenia . importantly , the alteration of associations is not to be equated with formal thought disorder but to be understood as a disturbance affecting all aspects , both cognitive and affective , of mental life . also , bleuler was keen to stress that the alteration of associations had been identified by empirical observation , not by theoretical speculation , and that it was accessible to experimental testing . two other phenomena that bleuler characterized as basic ( but not primary ) symptoms were ambivalence and autism . by ambivalence , he understood the simultaneous presence of contradictory ideas and emotions : autism described the phenomenon of a patient 's getting lost in personal ideas , emotions , and intentions without being able to adapt to the external reality , resulting in a reduction of communication . linking bleuler 's implementation of psychoanalytic , or more generally psychodynamic , ideas , with his distinction between primary and secondary symptoms and thus to the interaction between brain / body and mind , a further distinction has to be introduced , namely that between form and content . bleuler himself did not systematically introduce nor use this distinction , but it is implicit in his statement that what is psychologically understandable is the content of schizophrenic symptoms , ie , why a specific hallucination or delusion occurs . as the following quotation makes clear , he thus assumed a neurobiological disease process giving rise to a primary symptom , the alteration of associations , to which understandably the psyche reacts giving rise to secondary symptoms , with individually meaningful content : it goes without saying that the disease process can not give rise to the complex psychological symptoms which we are accustomed to consider first and foremost . this process can not account for the fact that it is a specific delusional idea or a specific hallucination that occurs . the process can only lead to certain fundamental disturbances of the psyche on the basis of which , in conjunction with precipitating and determining factors , hallucinations and delusional ideas emerge . the characterization of schizophrenic symptoms as bearing individually meaningful content is one of the important novelties in bleuler 's understanding of schizophrenia . in the words of his son manfred bleuler : one of bleuler 's main aims in choosing and following his career was to arrive at an understanding of schizophrenic symptoms as expressions of an inner psychodynamic life . [ ... ] he studied the schizophrenic 's life essentially in the same way as we study the inner life of neurotics , of healthy men , and of ourselves . in his summary of the development of the schizophrenia concept at the burghlzli hospital over the course of nearly 70 years ( 1902 - 1971 ) , manfred bleuler expanded on this attitude and orientation so central to his father 's work : he stressed that the view of schizophrenic symptoms as secondary phenomena , ie , understandable intellectually and emotionally , also made them in principle accessible to therapy . such therapy consisted of two pillars , firstly personal communication ( today analytically oriented psychotherapy ) in order to help patients by understanding their intentions and skills in adapting to reality ; and secondly multiple joint activities , such as work and leisure activities in groups of patients . during his time at the rheinau psychiatric hospital , eugen bleuler devoted as much time as possible to such personal contacts with his patients ; later , during his years at burghlzli , he suffered severely from a lack of time to do this . unlike kraepelin , then , bleuler saw no contradiction between the assumption of an underlying neurobiological disease process and the assumption of psychological understandability , but integrated both in his conception of schizophrenia . the nosological divide between psychotic and neurotic disorders , commonly drawn along the line of biologically determined versus psychologically understandable disease , is thus blurred . in the light of his keen awareness of social factors in the course of the disease and his efforts to address those therapeutically , bleuler can thus be seen as an early proponent of a bio - psychosocial understanding of mental illness . whilst the belief in a specifically schizophrenic feature spurred bleuler 's reconceptualization , he was anxious to leave his concept open for scientific revision and to accommodate the possibility that such a unifying feature might indeed not exist . he therefore spoke cautiously of the group of schizophrenias and intended his account to be a preliminary one . bleuler 's nosological changes were met with enthusiasm by some , with criticism by others , and the reception of his work varied between countries ie , psychiatric cultures . whilst in switzerland , for example , the concept of schizophrenia was quickly adopted , it was criticized in germany for carrying too much psychoanalytical baggage and for relying on the poorly defined concept of association . in britain , where kraepelin 's prognosis - based nosology had been criticized early on for promoting therapeutic nihilism and unjustified counsels of despair , the reception of bleuler 's schizophrenia was impeded by its perceived close connection with kraepelin 's dementia praecox . once the break with kraepelinian nosology implicit in the new concept was recognized , this latter aspect was especially welcomed . later the concept was developed further , giving rise to derivative concepts , such as schizoidism . later the concept was developed further , giving rise to derivative concepts such as schizoidism , which , whilst present in bleuler 's work on schizophrenia from the outset , was only explicitly introduced under that label by kurt binswanger ( 1887 - 1981 ) in 1920 . bleuler 's later writings on general psychology and philosophy received little attention from either his contemporaries or later workers . this may be due to the rather speculative , and in parts obscure , nature of works like psychoids : organizing principle of organic development or in these works , bleuler proposes a comprehensive life science in which physical , mental , and social phenomena are not seen as separate or even opposed , but as equal aspects of a single integrative life principle . in the light of the portrayal of bleuler as a proponent of a bio - psychosocial understanding of mental illness given above , these works might be interpreted as his attempt to theorize the relation and interaction between brain / body , mind and the social sphere . when we examine bleuler 's intellectual background and the major theoretical debates in psychiatry around the turn of the century , it is hard not to notice important similarities with ongoing debates today . born of the rejection of kraepelin 's principle of prognosis , on which his dementia praecox was based , the view of a variety of possible courses of schizophrenia , often summarized in a simplified manner as a rule of thirds , belongs to the stock - in - trade of present - day psychiatry . yet current debates about renaming schizophrenia would seem to suggest that the kraepelinian understanding still lingers amongst professionals and lay persons alike : one of the declared aims of renaming schizophrenia in japan , for instance , was to replace the view of an incurable condition by one associated with therapeutic optimism , in other words , to replace a kraepelinian by a bleulerian understanding . this , it was hoped , would enable better access to psychiatric care and reduce stigma . why , one might ask , was the bleulerian understanding not taken up from the outset ? we shall return to this question later after first reconsidering other features of bleuler 's concept . stemming from the search for something specifically schizophrenic , bleuler 's approach stands in the tradition of essentialist views of mental illness , to which it further contributes through the identification of the alteration of associations as schizophrenia 's clinical core . whilst not explicitly addressing its phenomenological status , ie , the question of whether this core disturbance has itself phenomenal quality , bleuler was adamant that it was not a theoretically inferred construct , but a phenomenon open to empirical observation and testing . he thus challenges the view recently put forward by mishara and schwartz , that only nonessentialist , phenomenological accounts of mental illness can provide hypotheses that can be tested by experimental neuroscience . with regard to the relationship between neurobiological and psychological understandings and approaches in psychiatry , it seems that whilst psychological theories and therapeutic approaches have gained recognition in research and treatment , there is currently a strong tendency to naturalize the mind , ie , to hold the mind to be exhausted by nature as understood by the natural sciences . efforts to naturalize mental illness come in many forms , and are observable not only in psychiatry but in all the mind sciences , including psychology and philosophy ( of mind ) . despite these efforts , the authors of the latest edition of the diagnostic and statistical manual of mental disorders ( dsm-5 ) have not included biomarkers , regarding them as still incapable of carving nature at its joints , ie , correctly and reliably identifying natural disease entities . this leaves current psychiatry in a position very much akin to bleuler 's : whilst a neurobiological basis of mental illness is generally taken for granted , it is not considered to provide a sufficiently firm foundation for a psychiatric nosology . bleuler 's theoretical position regarding this question is not altogether clear . in the work on schizophrenia he assumes a neurobiological process underlying the disturbance , without in any way etiologically , diagnostically , or therapeutically reducing the disturbance to the brain process . he was aware of the variable familial occurrence of schizophrenia and convinced of the role of genetic factors , but his work did not focus on biological causes of schizophrenia but on symptoms , their meanings and their personal and social consequences . this suggests that he embraced a kind of non - eliminative naturalism , whilst in his later work he seems to have turned , or to have been in search of , some form of mind - body identity theory . although these theoretical questions remain unresolved , bleuler 's clinical position was clear . in his patient - oriented attitude he displayed an early understanding of mental illness as bio - psychosocially constituted , and thus needing to be therapeutically addressed on all three levels . such understanding has proven clinically useful and therefore become common sense in medicine generally , even though a theoretically satisfying explanatory model for this bio - psychosocial understanding is still lacking . to return to the question posed earlier , namely , why was bleuler 's construct of schizophrenia as accessible to understanding and therapy not taken up from the outset ? might the sheer multidimensionality of his conception of schizophrenia , which remains hard to grasp when compared with kraepelin 's clear - cut dementia praecox , have impeded acceptance ? or is it , that regardless of the theoretical conception , the phenomena observed in schizophrenia point to something so utterly unfamiliar that despite all knowledge to the contrary , a pessimistic prognosis is easily assumed ? or again , has the difficulty in overcoming therapeutic nihilism and stigmatization less to do with the phenomenon itself and more with society 's general tendency to search for and define the other ? these remain open questions , but ones that need to be taken into account when considering and reconsidering the nosological status of schizophrenia and psychiatric nosology more broadly .

the introduction of the term and concept schizophrenia earned its inventor , swiss psychiatrist eugen bleuler , worldwide fame . prompted by the rejection of the main principle of kraepelinian nosology , namely prognosis , bleuler 's belief in the clinical unity of what kraepelin had described as dementia praecox required him to search for alternative characterizing features that would allow scientific description and classification . this led him to consider psychological , and to a lesser degree , social factors alongside an assumed underlying neurobiological disease process as constitutive of what he then termed schizophrenia , thus making him an early proponent of a bio - psycho - social understanding of mental illness . reviewing bleuler 's conception of schizophrenia against the background of his overall clinical and theoretical work , this paper provides a critical overview of bleuler 's key nosological principles and links his work with present - day debates about naturalism , essentialism , and stigma .

Introduction Bleuler's contribution to nosology Bleuler's intellectual background Introducing the group of schizophrenias Bleuler's later work Discussion and conclusion

eugen bleuler ( 1857 - 1939 ) was born and raised in the village of zollikon near zurich in switzerland . after graduating in medicine study trips took him to paris to work with jean - martin charcot , to munich where he trained under bernhard von gudden , and to london . he completed his residential training at the university hospital of psychiatry in zurich , known as burghlzli , and was appointed director of the mental asylum of rheinau in 1886 . whilst today bleuler is perhaps best known for the introduction of the term and concept schizophrenia , or more precisely the group of schizophrenias , this paper considers his work on schizophrenia principally in terms of its relationship to long - standing and complex theoretical debates in psychiatric nosology . these concern the very nature of mental illness , in particular the relationships between nature and mind and the individual and society . debates about how nature , ie , the brain and body , is related to the mind were highly topical with regard to mental illness in bleuler 's time and remain so today , as nicely captured in lipowski 's interrogative : psychiatry : mindless or brainless , both or neither ? he can thus be considered an early proponent of a bio - psychosocial model in psychiatry . reviewing bleuler 's conception of schizophrenia against the background of his overall clinical and theoretical work and contrasting it with kraepelin 's earlier concept dementia praecox , this paper provides a critical overview of bleuler 's key nosological principles and links his work with present day debates about naturalism , essentialism , and stigma . bleuler 's work stands in the conceptually rich tradition of turn - of - the - century psychiatry and psychology and can be considered a synthesis of various contemporary concepts and theories ( see ref 5 for an overview ) . acknowledging that science at the time did not yet allow identification of these posited disease entities , he based his preliminary nosology on an alternative principle , namely following karl ludwig kahibaum ( 1828 - 1899)on the course and prognosis of a given illness . this led to a dichotomous nosology based on the distinction between the concept of dementia praecox , characterized by a fatal course , and the concept of manic - depressive insanity , characterized by a positive course . this nosological system had gained wide acceptance amongst psychiatrists by the turn of the century . also rooted in a biological tradition , yet offering a new psychological explanation of mental illness as the unfolding of unconscious intrapsychic conflicts , freud 's theory was met with skepticism , if not outright rejection , as a dogmatic world - view . bleuler 's thinking was further shaped by the german philosopher and psychologist johann friedrich herbart ( 1776 - 1841 ) . as will be shown in the following section , bleuler , like the viennese psychiatrist erwin stransky ( 1877 - 1962 ) , integrated aspects of this atomistic approach to psycho(patho)logy in his work on dementia praecox and its reconceptualization as schizophrenia . in 1908 , bleuler publicly introduced the term and concept schizophrenia in a lecture given at the meeting of the deutscher verein fur psychatrie ( german psychiatric association ) in berlin . in the opening paragraph , he summarized his reasons for abandoning kraepelin 's earlier concept dementia praecox : i wish to emphasize that in kraepelin 's , dementia praecox it is neither a question of an essential dementia nor of a necessary precociousness . for this reason , and because from the expression dementia praecox one can not form further adjectives nor substantives , i am taking the liberty of employing the word schizophrenia for revising the kraepelinian concept . in my opinion the breaking up or splitting of psychic functioning is an excellent symptom of the whole group [ ... ] ( translation from ref 20 ) whilst this passage also underscores the importance of linguistic labels in psychiatry , the wish to rename kraepelin 's dementia praecox is only a secondary motive in bleuler 's introduction of schizophrenia . having gathered epidemiological data on the prognosis and end states of patients admitted with a diagnosis of dementia praecox , he came to the conclusion that this group of patients could not be coherently defined by a specific prognosis , ie , that this kraepelinian nosological principle had to be rejected . importantly , however , bleuler wanted to maintain the nosological unity of the group of patients that kraepelin identified by dementia praecox and believed that there was something specifically schizophrenic behind the general manifestations of the disease . he shared kraepelin 's assumption of an underlying physical disease process that the sciences of his day could not yet identify , and having rejected kraepelin 's principle of prognosis he set about searching for alternative criteria to define the essence of schizophrenia . to this purpose , bleuler turned to psychology , where , influenced by herbart 's atomistic view of the mind , he identified the alteration of associations , ie , of the way in which basic mental acts meaningfully combine into more complex units , as schizophrenia 's most fundamental feature . this idea is more fully developed in bleuler 's 1911 volume dementia praecox or the group of schizophrenias : the connections between associations are lost . thus , the result of the thought process is rendered unusual , and often logically incorrect , bleuler develops a symptomatology organized around two dichotomous distinctions : that between basic and accessory and that between primary and secondary symptoms . the distinction of primary and secondary refers to both etiology and pathogenesis , with primary symptoms being caused directly by the assumed neurobiological disease process , whilst secondary symptoms are seen as the potentially understandable reactions of the psyche to the disturbing primary symptoms . the alteration of associations is the only symptom that bleuler regarded as both basic and primary , and can thus be described as the core disturbance in the bleulerian conception of schizophrenia . also , bleuler was keen to stress that the alteration of associations had been identified by empirical observation , not by theoretical speculation , and that it was accessible to experimental testing . by ambivalence , he understood the simultaneous presence of contradictory ideas and emotions : autism described the phenomenon of a patient 's getting lost in personal ideas , emotions , and intentions without being able to adapt to the external reality , resulting in a reduction of communication . linking bleuler 's implementation of psychoanalytic , or more generally psychodynamic , ideas , with his distinction between primary and secondary symptoms and thus to the interaction between brain / body and mind , a further distinction has to be introduced , namely that between form and content . as the following quotation makes clear , he thus assumed a neurobiological disease process giving rise to a primary symptom , the alteration of associations , to which understandably the psyche reacts giving rise to secondary symptoms , with individually meaningful content : it goes without saying that the disease process can not give rise to the complex psychological symptoms which we are accustomed to consider first and foremost . the characterization of schizophrenic symptoms as bearing individually meaningful content is one of the important novelties in bleuler 's understanding of schizophrenia . in the words of his son manfred bleuler : one of bleuler 's main aims in choosing and following his career was to arrive at an understanding of schizophrenic symptoms as expressions of an inner psychodynamic life . [ ... ] he studied the schizophrenic 's life essentially in the same way as we study the inner life of neurotics , of healthy men , and of ourselves . unlike kraepelin , then , bleuler saw no contradiction between the assumption of an underlying neurobiological disease process and the assumption of psychological understandability , but integrated both in his conception of schizophrenia . in the light of his keen awareness of social factors in the course of the disease and his efforts to address those therapeutically , bleuler can thus be seen as an early proponent of a bio - psychosocial understanding of mental illness . whilst the belief in a specifically schizophrenic feature spurred bleuler 's reconceptualization , he was anxious to leave his concept open for scientific revision and to accommodate the possibility that such a unifying feature might indeed not exist . bleuler 's nosological changes were met with enthusiasm by some , with criticism by others , and the reception of his work varied between countries ie , psychiatric cultures . in britain , where kraepelin 's prognosis - based nosology had been criticized early on for promoting therapeutic nihilism and unjustified counsels of despair , the reception of bleuler 's schizophrenia was impeded by its perceived close connection with kraepelin 's dementia praecox . once the break with kraepelinian nosology implicit in the new concept was recognized , this latter aspect was especially welcomed . this may be due to the rather speculative , and in parts obscure , nature of works like psychoids : organizing principle of organic development or in these works , bleuler proposes a comprehensive life science in which physical , mental , and social phenomena are not seen as separate or even opposed , but as equal aspects of a single integrative life principle . in the light of the portrayal of bleuler as a proponent of a bio - psychosocial understanding of mental illness given above , these works might be interpreted as his attempt to theorize the relation and interaction between brain / body , mind and the social sphere . bleuler 's work stands in the conceptually rich tradition of turn - of - the - century psychiatry and psychology and can be considered a synthesis of various contemporary concepts and theories ( see ref 5 for an overview ) . acknowledging that science at the time did not yet allow identification of these posited disease entities , he based his preliminary nosology on an alternative principle , namely following karl ludwig kahibaum ( 1828 - 1899)on the course and prognosis of a given illness . this led to a dichotomous nosology based on the distinction between the concept of dementia praecox , characterized by a fatal course , and the concept of manic - depressive insanity , characterized by a positive course . this nosological system had gained wide acceptance amongst psychiatrists by the turn of the century . bleuler 's thinking was further shaped by the german philosopher and psychologist johann friedrich herbart ( 1776 - 1841 ) . as will be shown in the following section , bleuler , like the viennese psychiatrist erwin stransky ( 1877 - 1962 ) , integrated aspects of this atomistic approach to psycho(patho)logy in his work on dementia praecox and its reconceptualization as schizophrenia . in 1908 , bleuler publicly introduced the term and concept schizophrenia in a lecture given at the meeting of the deutscher verein fur psychatrie ( german psychiatric association ) in berlin . in the opening paragraph , he summarized his reasons for abandoning kraepelin 's earlier concept dementia praecox : i wish to emphasize that in kraepelin 's , dementia praecox it is neither a question of an essential dementia nor of a necessary precociousness . for this reason , and because from the expression dementia praecox one can not form further adjectives nor substantives , i am taking the liberty of employing the word schizophrenia for revising the kraepelinian concept . in my opinion the breaking up or splitting of psychic functioning is an excellent symptom of the whole group [ ... ] ( translation from ref 20 ) whilst this passage also underscores the importance of linguistic labels in psychiatry , the wish to rename kraepelin 's dementia praecox is only a secondary motive in bleuler 's introduction of schizophrenia . having gathered epidemiological data on the prognosis and end states of patients admitted with a diagnosis of dementia praecox , he came to the conclusion that this group of patients could not be coherently defined by a specific prognosis , ie , that this kraepelinian nosological principle had to be rejected . importantly , however , bleuler wanted to maintain the nosological unity of the group of patients that kraepelin identified by dementia praecox and believed that there was something specifically schizophrenic behind the general manifestations of the disease . he shared kraepelin 's assumption of an underlying physical disease process that the sciences of his day could not yet identify , and having rejected kraepelin 's principle of prognosis he set about searching for alternative criteria to define the essence of schizophrenia . to this purpose , bleuler turned to psychology , where , influenced by herbart 's atomistic view of the mind , he identified the alteration of associations , ie , of the way in which basic mental acts meaningfully combine into more complex units , as schizophrenia 's most fundamental feature . this idea is more fully developed in bleuler 's 1911 volume dementia praecox or the group of schizophrenias : the connections between associations are lost . thus , the result of the thought process is rendered unusual , and often logically incorrect , bleuler develops a symptomatology organized around two dichotomous distinctions : that between basic and accessory and that between primary and secondary symptoms . the distinction of primary and secondary refers to both etiology and pathogenesis , with primary symptoms being caused directly by the assumed neurobiological disease process , whilst secondary symptoms are seen as the potentially understandable reactions of the psyche to the disturbing primary symptoms . the alteration of associations is the only symptom that bleuler regarded as both basic and primary , and can thus be described as the core disturbance in the bleulerian conception of schizophrenia . by ambivalence , he understood the simultaneous presence of contradictory ideas and emotions : autism described the phenomenon of a patient 's getting lost in personal ideas , emotions , and intentions without being able to adapt to the external reality , resulting in a reduction of communication . linking bleuler 's implementation of psychoanalytic , or more generally psychodynamic , ideas , with his distinction between primary and secondary symptoms and thus to the interaction between brain / body and mind , a further distinction has to be introduced , namely that between form and content . as the following quotation makes clear , he thus assumed a neurobiological disease process giving rise to a primary symptom , the alteration of associations , to which understandably the psyche reacts giving rise to secondary symptoms , with individually meaningful content : it goes without saying that the disease process can not give rise to the complex psychological symptoms which we are accustomed to consider first and foremost . the characterization of schizophrenic symptoms as bearing individually meaningful content is one of the important novelties in bleuler 's understanding of schizophrenia . in the words of his son manfred bleuler : one of bleuler 's main aims in choosing and following his career was to arrive at an understanding of schizophrenic symptoms as expressions of an inner psychodynamic life . [ ... ] he studied the schizophrenic 's life essentially in the same way as we study the inner life of neurotics , of healthy men , and of ourselves . unlike kraepelin , then , bleuler saw no contradiction between the assumption of an underlying neurobiological disease process and the assumption of psychological understandability , but integrated both in his conception of schizophrenia . in the light of his keen awareness of social factors in the course of the disease and his efforts to address those therapeutically , bleuler can thus be seen as an early proponent of a bio - psychosocial understanding of mental illness . whilst the belief in a specifically schizophrenic feature spurred bleuler 's reconceptualization , he was anxious to leave his concept open for scientific revision and to accommodate the possibility that such a unifying feature might indeed not exist . bleuler 's nosological changes were met with enthusiasm by some , with criticism by others , and the reception of his work varied between countries ie , psychiatric cultures . in britain , where kraepelin 's prognosis - based nosology had been criticized early on for promoting therapeutic nihilism and unjustified counsels of despair , the reception of bleuler 's schizophrenia was impeded by its perceived close connection with kraepelin 's dementia praecox . once the break with kraepelinian nosology implicit in the new concept was recognized , this latter aspect was especially welcomed . this may be due to the rather speculative , and in parts obscure , nature of works like psychoids : organizing principle of organic development or in these works , bleuler proposes a comprehensive life science in which physical , mental , and social phenomena are not seen as separate or even opposed , but as equal aspects of a single integrative life principle . in the light of the portrayal of bleuler as a proponent of a bio - psychosocial understanding of mental illness given above , these works might be interpreted as his attempt to theorize the relation and interaction between brain / body , mind and the social sphere . when we examine bleuler 's intellectual background and the major theoretical debates in psychiatry around the turn of the century , it is hard not to notice important similarities with ongoing debates today . born of the rejection of kraepelin 's principle of prognosis , on which his dementia praecox was based , the view of a variety of possible courses of schizophrenia , often summarized in a simplified manner as a rule of thirds , belongs to the stock - in - trade of present - day psychiatry . yet current debates about renaming schizophrenia would seem to suggest that the kraepelinian understanding still lingers amongst professionals and lay persons alike : one of the declared aims of renaming schizophrenia in japan , for instance , was to replace the view of an incurable condition by one associated with therapeutic optimism , in other words , to replace a kraepelinian by a bleulerian understanding . we shall return to this question later after first reconsidering other features of bleuler 's concept . stemming from the search for something specifically schizophrenic , bleuler 's approach stands in the tradition of essentialist views of mental illness , to which it further contributes through the identification of the alteration of associations as schizophrenia 's clinical core . efforts to naturalize mental illness come in many forms , and are observable not only in psychiatry but in all the mind sciences , including psychology and philosophy ( of mind ) . despite these efforts , the authors of the latest edition of the diagnostic and statistical manual of mental disorders ( dsm-5 ) have not included biomarkers , regarding them as still incapable of carving nature at its joints , ie , correctly and reliably identifying natural disease entities . this leaves current psychiatry in a position very much akin to bleuler 's : whilst a neurobiological basis of mental illness is generally taken for granted , it is not considered to provide a sufficiently firm foundation for a psychiatric nosology . he was aware of the variable familial occurrence of schizophrenia and convinced of the role of genetic factors , but his work did not focus on biological causes of schizophrenia but on symptoms , their meanings and their personal and social consequences . in his patient - oriented attitude he displayed an early understanding of mental illness as bio - psychosocially constituted , and thus needing to be therapeutically addressed on all three levels . to return to the question posed earlier , namely , why was bleuler 's construct of schizophrenia as accessible to understanding and therapy not taken up from the outset ? might the sheer multidimensionality of his conception of schizophrenia , which remains hard to grasp when compared with kraepelin 's clear - cut dementia praecox , have impeded acceptance ? or again , has the difficulty in overcoming therapeutic nihilism and stigmatization less to do with the phenomenon itself and more with society 's general tendency to search for and define the other ?

this cross - sectional study compared risk factors associated with type 2 diabetes between overweight and obese adolescents stratified by the presence or absence of hepatic steatosis and healthy weight adolescents without hepatic steatosis . through local advertising , we recruited 108 adolescents between age 13 and 18 years considered overweight or obese according to age- and sex - specific bmi cut points established by the international obesity task force ( 9 ) . we also recruited 11 healthy weight normoglycemic adolescents to provide an estimate of expected values for insulin sensitivity and hepatic triglyceride in the absence of obesity . exclusion criteria included diagnosis of type 2 diabetes or impaired glucose tolerance ( igt ) , treatment with antipsychotics or corticosteroids , orthopedic injury or illness preventing participation in the exercise test , and enrolment in a weight loss program in the previous 6 months . a standard 2-h 75-g oral glucose tolerance test was used to screen for type 2 diabetes . youth with type 2 diabetes or igt ( 10 ) were excluded ( n = 8) . youth unable to complete the magnetic resonance imaging ( mri ) as a result of size restriction were subsequently excluded from the study ( n = 2 ) . because type 2 diabetes is more prevalent in the first nations population in our region ( 11 ) , ethnicity was self - reported for inclusion as a confounding variable . the study was approved by the university of manitoba biomedical research ethics board and performed according to the declaration of helsinki . whole - body insulin sensitivity was determined from a modified frequently sampled intravenous glucose tolerance test ( 12 ) . fasting blood samples were collected prior to an intravenous bolus of a 25% glucose solution ( 0.3 g / kg body wt to a maximum dose of 34 g glucose ) at time 0 , followed by an intravenous bolus of regular human insulin ( 0.03 units / kg body wt ) at 20 min . serum glucose and insulin were collected at 1 , 2 , 3 , 4 , 5 , 6 , 8 , 10 , 14 , 19 , 22 , 25 , 30 , 40 , 50 , 70 , 100 , 140 , and 180 min . glucose and insulin kinetics were modeled using the bergman minimal model provided in customized software ( minmod ) to estimate whole - body insulin sensitivity ( 13 ) . the acute insulin response and disposition index were also determined from this method as measures of glucose - stimulated insulin secretion . metabolic syndrome was treated as a binary outcome using cut points for systolic blood pressure , serum triglycerides , waist circumference , fasting glucose , and hdl cholesterol ( hdl - c ) that were statistically derived to reflect cut points in adults ( 14 ) . adolescents were classified as having metabolic syndrome if they had three or more of five comorbidities ( 14 ) . automated blood pressure ( dynamap ) was measured in triplicate on the left arm with participants seated . waist circumference was measured at the level of the iliac crest in duplicate with a flexible tape measure . waist circumference was expressed as z scores according to canadian reference data for between - group comparisons ( 15 ) . glucose area under the curve ( auc ) was determined by summing the area under serum glucose measures between each 15- or 30-min segment after a 75-g oral glucose challenge using the trapezium rule ( 16 ) . plasma glucose was measured on a roche modular p analyzer with an ultraviolet test principle ( hexokinase method ) . insulin was measured on an immulite solid - phase , two - site chemiluminescent immunometric assay . serum lipoproteins , triglycerides , alanine aminotransferase ( alt ) , and aspartate aminotransferase ( ast ) were measured on a roche modular p analyzer . ldl cholesterol ( ldl - c ) was calculated using the friedewald equation ( ldl - c = total cholesterol [ hdl - c ( triglyceride/2.2 ) ] ) . mri and spectroscopy were performed using a 1.5 t whole - body magnet ( ge medical systems , milwaukee , wi ) as previously described ( 17,18 ) . high - resolution images of the liver were obtained in three planes using standard clinical techniques , and a voxel was placed in an area devoid of subcutaneous or visceral fat for the acquisition of proton spectra . a total of 64 spectra were averaged for the determination of intracellular water and lipid content . lcmodel software ( 19 ) was used to isolate and quantify lipid and water peaks . previous population - based studies show that 5.5% fat / water approximates the 95th percentile for healthy normoglycemic adults and is equivalent to biopsy - derived lipid concentration of 5.5 mg / g ( 20 ) . visceral fat mass was quantified from high - resolution magnetic resonance images at a level between the third and fifth lumbar vertebrae as previously described ( 17,21 ) using slicer3 software ( version 3.21 ; boston , ma ) . cardiorespiratory fitness was determined using indirect calorimetry with a graded maximal exercise test to exhaustion on a cycle ergometer ( 22 ) . physical activity was measured as the average of step counts during 7 consecutive days using hip pedometers . dual - energy x - ray absorptiometry ( hologic , bedford , ma ) was used to quantify fat mass ( kilograms ) , fat - free mass ( kilograms ) , and percent body fat . nonesterified fatty acids ( nefas ) were measured at 30-min intervals during the oral glucose challenge to determine insulin - mediated suppression of lipolysis . adolescents were stratified into one of three categories according to weight status and hepatic triglyceride content : 1 ) healthy weight normoglycemic adolescents without hepatic steatosis ( n = 11 ) , 2 ) overweight adolescents without hepatic steatosis ( triglyceride < 5.5% fat / water , n = 68 ) , and 3 ) overweight adolescents with hepatic steatosis we controlled for potential confounding effects of age , sex , and visceral adiposity ( within 20% ) by pair matching overweight youth with hepatic steatosis to overweight youth without hepatic steatosis . to validate the associations between hepatic steatosis and type 2 diabetes risk factors , we conducted a reciprocal matching study with a separate cohort of overweight youth discrepant for visceral fat mass ( < 70.0 vs. > for nonnormally distributed variables , mann - whitney u tests were used to examine differences between groups of pair - matched subjects . comparisons of normally distributed variables between subjects within each pair - matched grouping were performed using student t test . levene test was used to assess the equality of group variances on the dependant variables . multiple stepwise linear regression analysis was used to determine if hepatic triglyceride was associated with various metabolic risk factors independent of age , ethnicity , sex , bmi z score , cardiorespiratory fitness , and visceral obesity . all analyses were performed with spss version 16.0 ( sas , chicago , il ) . this cross - sectional study compared risk factors associated with type 2 diabetes between overweight and obese adolescents stratified by the presence or absence of hepatic steatosis and healthy weight adolescents without hepatic steatosis . through local advertising , we recruited 108 adolescents between age 13 and 18 years considered overweight or obese according to age- and sex - specific bmi cut points established by the international obesity task force ( 9 ) . we also recruited 11 healthy weight normoglycemic adolescents to provide an estimate of expected values for insulin sensitivity and hepatic triglyceride in the absence of obesity . exclusion criteria included diagnosis of type 2 diabetes or impaired glucose tolerance ( igt ) , treatment with antipsychotics or corticosteroids , orthopedic injury or illness preventing participation in the exercise test , and enrolment in a weight loss program in the previous 6 months . a standard 2-h 75-g oral glucose tolerance test was used to screen for type 2 diabetes . youth with type 2 diabetes or igt ( 10 ) were excluded ( n = 8) . youth unable to complete the magnetic resonance imaging ( mri ) as a result of size restriction were subsequently excluded from the study ( n = 2 ) . because type 2 diabetes is more prevalent in the first nations population in our region ( 11 ) , ethnicity was self - reported for inclusion as a confounding variable . the study was approved by the university of manitoba biomedical research ethics board and performed according to the declaration of helsinki . whole - body insulin sensitivity was determined from a modified frequently sampled intravenous glucose tolerance test ( 12 ) . fasting blood samples were collected prior to an intravenous bolus of a 25% glucose solution ( 0.3 g / kg body wt to a maximum dose of 34 g glucose ) at time 0 , followed by an intravenous bolus of regular human insulin ( 0.03 units / kg body wt ) at 20 min . serum glucose and insulin were collected at 1 , 2 , 3 , 4 , 5 , 6 , 8 , 10 , 14 , 19 , 22 , 25 , 30 , 40 , 50 , 70 , 100 , 140 , and 180 min . glucose and insulin kinetics were modeled using the bergman minimal model provided in customized software ( minmod ) to estimate whole - body insulin sensitivity ( 13 ) . the acute insulin response and disposition index were also determined from this method as measures of glucose - stimulated insulin secretion . metabolic syndrome was treated as a binary outcome using cut points for systolic blood pressure , serum triglycerides , waist circumference , fasting glucose , and hdl cholesterol ( hdl - c ) that were statistically derived to reflect cut points in adults ( 14 ) . adolescents were classified as having metabolic syndrome if they had three or more of five comorbidities ( 14 ) . automated blood pressure ( dynamap ) was measured in triplicate on the left arm with participants seated . waist circumference was measured at the level of the iliac crest in duplicate with a flexible tape measure . waist circumference was expressed as z scores according to canadian reference data for between - group comparisons ( 15 ) . glucose area under the curve ( auc ) was determined by summing the area under serum glucose measures between each 15- or 30-min segment after a 75-g oral glucose challenge using the trapezium rule ( 16 ) . plasma glucose was measured on a roche modular p analyzer with an ultraviolet test principle ( hexokinase method ) . insulin was measured on an immulite solid - phase , two - site chemiluminescent immunometric assay . serum lipoproteins , triglycerides , alanine aminotransferase ( alt ) , and aspartate aminotransferase ( ast ) were measured on a roche modular p analyzer . ldl cholesterol ( ldl - c ) was calculated using the friedewald equation ( ldl - c = total cholesterol [ hdl - c ( triglyceride/2.2 ) ] ) . mri and spectroscopy were performed using a 1.5 t whole - body magnet ( ge medical systems , milwaukee , wi ) as previously described ( 17,18 ) . high - resolution images of the liver were obtained in three planes using standard clinical techniques , and a voxel was placed in an area devoid of subcutaneous or visceral fat for the acquisition of proton spectra . a total of 64 spectra were averaged for the determination of intracellular water and lipid content . lcmodel software ( 19 ) was used to isolate and quantify lipid and water peaks . previous population - based studies show that 5.5% fat / water approximates the 95th percentile for healthy normoglycemic adults and is equivalent to biopsy - derived lipid concentration of 5.5 mg / g ( 20 ) . visceral fat mass was quantified from high - resolution magnetic resonance images at a level between the third and fifth lumbar vertebrae as previously described ( 17,21 ) using slicer3 software ( version 3.21 ; boston , ma ) . cardiorespiratory fitness was determined using indirect calorimetry with a graded maximal exercise test to exhaustion on a cycle ergometer ( 22 ) . physical activity was measured as the average of step counts during 7 consecutive days using hip pedometers . dual - energy x - ray absorptiometry ( hologic , bedford , ma ) was used to quantify fat mass ( kilograms ) , fat - free mass ( kilograms ) , and percent body fat . nonesterified fatty acids ( nefas ) were measured at 30-min intervals during the oral glucose challenge to determine insulin - mediated suppression of lipolysis . adolescents were stratified into one of three categories according to weight status and hepatic triglyceride content : 1 ) healthy weight normoglycemic adolescents without hepatic steatosis ( n = 11 ) , 2 ) overweight adolescents without hepatic steatosis ( triglyceride < 5.5% fat / water , n = 68 ) , and 3 ) overweight adolescents with hepatic steatosis ( triglyceride 5.5% fat / water , n = 30 ) . we controlled for potential confounding effects of age , sex , and visceral adiposity ( within 20% ) by pair matching overweight youth with hepatic steatosis to overweight youth without hepatic steatosis . to validate the associations between hepatic steatosis and type 2 diabetes risk factors , we conducted a reciprocal matching study with a separate cohort of overweight youth discrepant for visceral fat mass ( < 70.0 vs. > for nonnormally distributed variables , mann - whitney u tests were used to examine differences between groups of pair - matched subjects . comparisons of normally distributed variables between subjects within each pair - matched grouping were performed using student t test . levene test was used to assess the equality of group variances on the dependant variables . multiple stepwise linear regression analysis was used to determine if hepatic triglyceride was associated with various metabolic risk factors independent of age , ethnicity , sex , bmi z score , cardiorespiratory fitness , and visceral obesity . all analyses were performed with spss version 16.0 ( sas , chicago , il ) . hepatic steatosis was evident in 31% ( n = 30 ) of overweight and obese adolescents and none of the healthy weight adolescents . overweight and obese adolescents with or without hepatic steatosis displayed lower insulin sensitivity , higher rates of metabolic syndrome , and higher serum glucose after the oral glucose challenge compared with healthy weight adolescents ( table 1 ) . bmi z score ( 2.1 vs. 1.8 , p < 0.001 ) and waist circumference z score ( 6.09 vs. 4.62 , p = 0.004 ) were higher among overweight and obese adolescents with hepatic steatosis compared with those without , despite similar visceral fat mass and percent body fat ( table 1 ) . the proportion of male participants was higher in the cohort of youth with hepatic steatosis relative to overweight and obese youth without ( 43 vs. 25% , p = 0.04 ) ( table 1 ) . a greater proportion of first nations youth composed the overweight and obese groups , compared with the healthy weight group ( 24 vs. 9% , p < 0.01 ) . a higher proportion of youth with hepatic steatosis met the criteria for obesity compared with youth without hepatic steatosis ( 90 vs. 65% , p < 0.05 ) . physical activity levels ( steps per day ) were not significantly different between groups of youth . characteristics of study participants insulin sensitivity was 55% lower , the presence of metabolic syndrome was approximately twofold higher ( 50 vs. 25% , p = 0.001 ) , and serum glucose during the oral glucose challenge was 10% higher among youth with hepatic steatosis compared with healthy weight , overweight , and obese youth without hepatic steatosis . none of the adolescents had clinically relevant elevations ( greater than two times upper reference range ) in liver aminotransferases ( ast or alt ) . the nefa auc after a 75-g oral glucose challenge was not different between youth with hepatic steatosis compared with overweight and obese youth without hepatic steatosis ( table 1 ) . as hepatic triglyceride content and visceral fat mass are strongly associated , we used a reciprocal matching strategy similar to that used by fabbrini et al . this complementary research design permits the isolation of each aspect of adiposity on metabolic risk controlling for the collinearity in the exposure variables . after matching for age , sex , and visceral fat mass , we found that insulin sensitivity remained 25% lower ( 3.5 vs. 4.5 mu kg min , p = 0.03 ) , the presence of metabolic syndrome remained greater than twofold higher ( 48 vs. 20% , p = 0.03 ) , and the glucose auc was 20% higher in overweight and obese adolescents with hepatic steatosis compared with overweight and obese adolescents without hepatic steatosis ( table 2 ) . in the reciprocal comparison , when youth were matched for hepatic triglyceride content , no differences in metabolic syndrome or glucose auc were observed , despite significant differences in visceral fat mass ( 63.7 vs. 102.1 cm ) ( table 2 ) . insulin sensitivity remained significantly different between groups ( 3.2 vs. 5.5 mu kg min , p = 0.02 ) . characteristics of pair - matched overweight and obese youth for age , sex , and visceral fat or hepatic triglyceride content hepatic triglyceride content was positively associated with glucose auc ( = 0.44 , p < 0.001 ) , bmi z score ( = 0.47 , p < 0.001 ) , waist circumference z score ( = 0.34 , p < 0.001 ) , systolic blood pressure ( = 0.16 , p = 0.005 ) , serum triglycerides ( = 0.43 , p < 0.001 ) , and alt ( = 0.36 , p < 0.001 ) and negatively associated with insulin sensitivity ( = 0.40 , p < 0.001 ) and hdl - c ( = 0.31 , p < 0.001 ) . cardiorespiratory fitness was not associated with insulin sensitivity or hepatic triglyceride content . multiple regression analysis revealed that hepatic triglyceride content was associated with the presence of metabolic syndrome ( r = 0.57 , p < 0.001 ) , insulin sensitivity ( r = 0.36 , p = 0.04 ) , and glucose auc ( r = 0.24 , p = 0.02 ) after adjusting for age , sex , bmi z score , waist circumference z score , visceral fat , whole - body fat mass , fitness level , and nefa auc . hepatic steatosis was evident in 31% ( n = 30 ) of overweight and obese adolescents and none of the healthy weight adolescents . overweight and obese adolescents with or without hepatic steatosis displayed lower insulin sensitivity , higher rates of metabolic syndrome , and higher serum glucose after the oral glucose challenge compared with healthy weight adolescents ( table 1 ) . bmi z score ( 2.1 vs. 1.8 , p < 0.001 ) and waist circumference z score ( 6.09 vs. 4.62 , p = 0.004 ) were higher among overweight and obese adolescents with hepatic steatosis compared with those without , despite similar visceral fat mass and percent body fat ( table 1 ) . the proportion of male participants was higher in the cohort of youth with hepatic steatosis relative to overweight and obese youth without ( 43 vs. 25% , p = 0.04 ) ( table 1 ) . a greater proportion of first nations youth composed the overweight and obese groups , compared with the healthy weight group ( 24 vs. 9% , p < 0.01 ) . a higher proportion of youth with hepatic steatosis met the criteria for obesity compared with youth without hepatic steatosis ( 90 vs. 65% , p < 0.05 ) . physical activity levels ( steps per day ) were not significantly different between groups of youth . characteristics of study participants insulin sensitivity was 55% lower , the presence of metabolic syndrome was approximately twofold higher ( 50 vs. 25% , p = 0.001 ) , and serum glucose during the oral glucose challenge was 10% higher among youth with hepatic steatosis compared with healthy weight , overweight , and obese youth without hepatic steatosis . none of the adolescents had clinically relevant elevations ( greater than two times upper reference range ) in liver aminotransferases ( ast or alt ) . the nefa auc after a 75-g oral glucose challenge was not different between youth with hepatic steatosis compared with overweight and obese youth without hepatic steatosis ( table 1 ) . as hepatic triglyceride content and visceral fat mass are strongly associated , we used a reciprocal matching strategy similar to that used by fabbrini et al . this complementary research design permits the isolation of each aspect of adiposity on metabolic risk controlling for the collinearity in the exposure variables . after matching for age , sex , and visceral fat mass , we found that insulin sensitivity remained 25% lower ( 3.5 vs. 4.5 mu kg min , p = 0.03 ) , the presence of metabolic syndrome remained greater than twofold higher ( 48 vs. 20% , p = 0.03 ) , and the glucose auc was 20% higher in overweight and obese adolescents with hepatic steatosis compared with overweight and obese adolescents without hepatic steatosis ( table 2 ) . in the reciprocal comparison , when youth were matched for hepatic triglyceride content , no differences in metabolic syndrome or glucose auc were observed , despite significant differences in visceral fat mass ( 63.7 vs. 102.1 cm ) ( table 2 ) . insulin sensitivity remained significantly different between groups ( 3.2 vs. 5.5 mu kg min , p = 0.02 ) . characteristics of pair - matched overweight and obese youth for age , sex , and visceral fat or hepatic triglyceride content hepatic triglyceride content was positively associated with glucose auc ( = 0.44 , p < 0.001 ) , bmi z score ( = 0.47 , p < 0.001 ) , waist circumference z score ( = 0.34 , p < 0.001 ) , systolic blood pressure ( = 0.16 , p = 0.005 ) , serum triglycerides ( = 0.43 , p < 0.001 ) , and alt ( = 0.36 , p < 0.001 ) and negatively associated with insulin sensitivity ( = 0.40 , p < 0.001 ) and hdl - c ( = 0.31 , p < 0.001 ) . multiple regression analysis revealed that hepatic triglyceride content was associated with the presence of metabolic syndrome ( r = 0.57 , p < 0.001 ) , insulin sensitivity ( r = 0.36 , p = 0.04 ) , and glucose auc ( r = 0.24 , p = 0.02 ) after adjusting for age , sex , bmi z score , waist circumference z score , visceral fat , whole - body fat mass , fitness level , and nefa auc . hepatic triglyceride content was positively associated with glucose auc ( = 0.44 , p < 0.001 ) , bmi z score ( = 0.47 , p < 0.001 ) , waist circumference z score ( = 0.34 , p < 0.001 ) , systolic blood pressure ( = 0.16 , p = 0.005 ) , serum triglycerides ( = 0.43 , p < 0.001 ) , and alt ( = 0.36 , p < 0.001 ) and negatively associated with insulin sensitivity ( = 0.40 , p < 0.001 ) and hdl - c ( = 0.31 , p < 0.001 ) . cardiorespiratory fitness was not associated with insulin sensitivity or hepatic triglyceride content . multiple regression analysis revealed that hepatic triglyceride content was associated with the presence of metabolic syndrome ( r = 0.57 , p < 0.001 ) , insulin sensitivity ( r = 0.36 , p = 0.04 ) , and glucose auc ( r = 0.24 , p = 0.02 ) after adjusting for age , sex , bmi z score , waist circumference z score , visceral fat , whole - body fat mass , fitness level , and nefa auc . this study reveals a novel combination of findings with clinical implications for predicting type 2 diabetes risk in youth . first , hepatic steatosis is associated with 1 ) increased prevalence of metabolic syndrome , 2 ) elevated serum glucose in response to an oral glucose challenge , and 3 ) dyslipidemia , independent of visceral fat mass and cardiorespiratory fitness . second , several metabolic characteristics associated with visceral adiposity in adolescents , including metabolic syndrome and preclinical dysglycemia , can be attributed to the presence of elevated hepatic triglyceride levels . finally , cardiorespiratory fitness was not associated with hepatic triglyceride content , nor did it mediate the association between hepatic steatosis and type 2 diabetes risk factors . our data support the concept that hepatic steatosis is an early precursor to glucose intolerance and cardiometabolic risk independent of fitness in overweight and obese youth . previous cross - sectional studies show that both visceral fat mass and hepatic triglyceride content are associated with metabolic abnormalities in overweight adolescents ( 23,24 ) . recent studies showing hepatic steatosis as a determinant of insulin resistance in overweight youth failed to include a cohort matched for hepatic triglyceride content with disparate visceral fat mass as a positive control ( 4,7 ) . by effectively isolating hepatic steatosis from visceral obesity , we demonstrated that hepatic steatosis is the dominant predictor of metabolic syndrome among overweight or obese adolescents . several case control studies show that metabolic risk factor clustering is associated with the presence of hepatic steatosis ( 4,5 ) . using biopsy - proven or mri - derived measures of hepatic triglyceride , previous reports show that 50% of adolescents with hepatic steatosis meet diagnostic criteria for metabolic syndrome , a rate approximately two- to threefold higher than in adolescents without hepatic steatosis ( 5,25 ) . conventional wisdom suggests that the association between hepatic steatosis and metabolic risk factor clustering is mediated by whole - body insulin resistance ( 4,26 ) . in contrast , we found no differences in the prevalence of metabolic syndrome in youth with discrepant visceral fat mass , matched for hepatic triglyceride content , despite significant differences in insulin sensitivity . thus , insulin resistance may not be the sole factor linking hepatic steatosis to metabolic risk in overweight youth . these data imply that hepatic steatosis is a sensitive biomarker and potential mediating factor for metabolic risk factor clustering and glucose intolerance in overweight and obese adolescents independent of insulin resistance . prior evidence suggests hepatic steatosis contributes to the loss of glucose tolerance among overweight and obese adolescents . first , hepatic steatosis is a common feature of igt and type 2 diabetes ( 24 ) . second , hepatic steatosis is associated with a loss of insulin - induced suppression of hepatic glucose output among overweight and obese individuals ( 7 ) . finally , glucose - stimulated insulin secretion is impaired among obese adolescents with hepatic steatosis ( 4,26 ) . our study extends these findings by showing that glucose excursion after an oral glucose challenge is higher among overweight and obese adolescents with hepatic steatosis . in contrast to others ( 4 ) , we did not observe a difference in the acute insulin response to glucose expressed in absolute terms or relative to insulin sensitivity between adolescents with and without hepatic steatosis . however , considering that differences in serum glucose occurred later during the 2-h challenge , the earliest defects in -cell function may occur in the second phase of insulin secretion . defects in mitochondrial oxidative capacity are purported to render individuals susceptible to ectopic lipid accumulation . previous cross - sectional studies of hepatic steatosis in adolescents have failed to provide a measure of mitochondrial function or fitness levels . because recent studies show that the association between cardiorespiratory fitness and insulin resistance appears to be mediated in part by hepatic triglyceride content , we felt it was important to assess fitness in this cohort ( 8) . furthermore , recent intervention studies in adults ( 27 ) suggest that increased physical activity or elevated cardiorespiratory fitness may attenuate triglyceride accumulation in the liver . in contrast to these studies , our data do not support a role for cardiorespiratory fitness in the clustering of cardiometabolic disease risk among overweight or obese adolescents . specifically , cardiorespiratory fitness was not associated with any of the risk factors for type 2 diabetes , nor was it different between overweight and obese youth with and without hepatic steatosis . it is possible that hepatic steatosis in youth is predominantly a function of diet and/or factors related to fatty acid uptake and synthesis . alternatively , fitness levels achieved in the convenience sample of youth studied here may have been too low to detect associations with cardiometabolic risk . prospective intervention studies are needed to determine if increasing cardiorespiratory fitness can reduce hepatic triglyceride content in adolescents . the cross - sectional design precludes determination of a causal nature for the associations described . however , the strategy of reciprocal matching permitted the dissection of the independent roles of hepatic steatosis and visceral adiposity in metabolic risk factor clustering in overweight and obese adolescents . differences in sex and ethnicity existed between groups ; however , after matching for sex and including ethnicity as a covariate in multiple linear regression models , group - wise differences in insulin sensitivity , presence of metabolic syndrome , or glucose auc were unchanged . finally , in the absence of data on dietary intake , we were unable to study this covariate . hepatic steatosis is a prevalent comorbidity in overweight and obese adolescents that exists prior to clinically elevated serum aminotransferases . the presence of hepatic steatosis is accompanied by a clustering of factors associated with type 2 diabetes and cardiovascular disease , independent of visceral fat mass , whole - body adiposity , and cardiorespiratory fitness . these results support the growing body of evidence that hepatic steatosis is a potentially modifiable biomarker of risk for type 2 diabetes and metabolic syndrome in overweight and obese adolescents .

objectiveto test the hypothesis that hepatic steatosis is associated with risk factors for type 2 diabetes in overweight and obese youth , mediated by cardiorespiratory fitness.research design and methodsthis was a cross - sectional study comparing insulin sensitivity between 30 overweight and obese adolescents with hepatic steatosis , 68 overweight and obese adolescents without hepatic steatosis , and 11 healthy weight adolescents without hepatic steatosis . cardiorespiratory fitness was determined by a graded maximal exercise test on a cycle ergometer . secondary outcomes included presence of metabolic syndrome and glucose response to a 75-g oral glucose challenge.resultsthe presence of hepatic steatosis was associated with 55% lower insulin sensitivity ( p = 0.02 ) and a twofold greater prevalence of metabolic syndrome ( p = 0.001 ) . differences in insulin sensitivity ( 3.5 vs. 4.5 mu kg1 min1 , p = 0.03 ) , prevalence of metabolic syndrome ( 48 vs. 20% , p = 0.03 ) , and glucose area under the curve ( 816 vs. 710 , p = 0.04 ) remained between groups after matching for age , sex , and visceral fat . the association between hepatic steatosis and insulin sensitivity ( = 0.24 , t = 2.29 , p < 0.025 ) , metabolic syndrome ( = 0.54 , t = 5.8 , p < 0.001 ) , and glucose area under the curve ( = 0.33 , t = 3.3 , p < 0.001 ) was independent of visceral and whole - body adiposity . cardiorespiratory fitness was not associated with hepatic steatosis , insulin sensitivity , or presence of metabolic syndrome.conclusionshepatic steatosis is associated with type 2 diabetes risk factors independent of cardiorespiratory fitness , whole - body adiposity , and visceral fat mass .

RESEARCH DESIGN AND METHODS Study design and study population Primary outcome measureinsulin sensitivity Secondary outcome measuresmetabolic syndrome and serum glucose levels in response to an oral glucose challenge Biochemical measurements Primary exposure variableshepatic triglyceride content and visceral fat mass Confounding/exploratory variablescardiorespiratory fitness, physical activity, adiposity, and adipocyte lipolysis Statistical analyses RESULTS Participant characteristics Metabolic characteristics of overweight adolescents with hepatic steatosis Matched comparisons Associations. CONCLUSIONS

this cross - sectional study compared risk factors associated with type 2 diabetes between overweight and obese adolescents stratified by the presence or absence of hepatic steatosis and healthy weight adolescents without hepatic steatosis . exclusion criteria included diagnosis of type 2 diabetes or impaired glucose tolerance ( igt ) , treatment with antipsychotics or corticosteroids , orthopedic injury or illness preventing participation in the exercise test , and enrolment in a weight loss program in the previous 6 months . a standard 2-h 75-g oral glucose tolerance test was used to screen for type 2 diabetes . whole - body insulin sensitivity was determined from a modified frequently sampled intravenous glucose tolerance test ( 12 ) . serum glucose and insulin were collected at 1 , 2 , 3 , 4 , 5 , 6 , 8 , 10 , 14 , 19 , 22 , 25 , 30 , 40 , 50 , 70 , 100 , 140 , and 180 min . glucose and insulin kinetics were modeled using the bergman minimal model provided in customized software ( minmod ) to estimate whole - body insulin sensitivity ( 13 ) . glucose area under the curve ( auc ) was determined by summing the area under serum glucose measures between each 15- or 30-min segment after a 75-g oral glucose challenge using the trapezium rule ( 16 ) . cardiorespiratory fitness was determined using indirect calorimetry with a graded maximal exercise test to exhaustion on a cycle ergometer ( 22 ) . dual - energy x - ray absorptiometry ( hologic , bedford , ma ) was used to quantify fat mass ( kilograms ) , fat - free mass ( kilograms ) , and percent body fat . adolescents were stratified into one of three categories according to weight status and hepatic triglyceride content : 1 ) healthy weight normoglycemic adolescents without hepatic steatosis ( n = 11 ) , 2 ) overweight adolescents without hepatic steatosis ( triglyceride < 5.5% fat / water , n = 68 ) , and 3 ) overweight adolescents with hepatic steatosis we controlled for potential confounding effects of age , sex , and visceral adiposity ( within 20% ) by pair matching overweight youth with hepatic steatosis to overweight youth without hepatic steatosis . to validate the associations between hepatic steatosis and type 2 diabetes risk factors , we conducted a reciprocal matching study with a separate cohort of overweight youth discrepant for visceral fat mass ( < 70.0 vs. > for nonnormally distributed variables , mann - whitney u tests were used to examine differences between groups of pair - matched subjects . multiple stepwise linear regression analysis was used to determine if hepatic triglyceride was associated with various metabolic risk factors independent of age , ethnicity , sex , bmi z score , cardiorespiratory fitness , and visceral obesity . this cross - sectional study compared risk factors associated with type 2 diabetes between overweight and obese adolescents stratified by the presence or absence of hepatic steatosis and healthy weight adolescents without hepatic steatosis . exclusion criteria included diagnosis of type 2 diabetes or impaired glucose tolerance ( igt ) , treatment with antipsychotics or corticosteroids , orthopedic injury or illness preventing participation in the exercise test , and enrolment in a weight loss program in the previous 6 months . a standard 2-h 75-g oral glucose tolerance test was used to screen for type 2 diabetes . whole - body insulin sensitivity was determined from a modified frequently sampled intravenous glucose tolerance test ( 12 ) . serum glucose and insulin were collected at 1 , 2 , 3 , 4 , 5 , 6 , 8 , 10 , 14 , 19 , 22 , 25 , 30 , 40 , 50 , 70 , 100 , 140 , and 180 min . glucose and insulin kinetics were modeled using the bergman minimal model provided in customized software ( minmod ) to estimate whole - body insulin sensitivity ( 13 ) . glucose area under the curve ( auc ) was determined by summing the area under serum glucose measures between each 15- or 30-min segment after a 75-g oral glucose challenge using the trapezium rule ( 16 ) . cardiorespiratory fitness was determined using indirect calorimetry with a graded maximal exercise test to exhaustion on a cycle ergometer ( 22 ) . dual - energy x - ray absorptiometry ( hologic , bedford , ma ) was used to quantify fat mass ( kilograms ) , fat - free mass ( kilograms ) , and percent body fat . adolescents were stratified into one of three categories according to weight status and hepatic triglyceride content : 1 ) healthy weight normoglycemic adolescents without hepatic steatosis ( n = 11 ) , 2 ) overweight adolescents without hepatic steatosis ( triglyceride < 5.5% fat / water , n = 68 ) , and 3 ) overweight adolescents with hepatic steatosis ( triglyceride 5.5% fat / water , n = 30 ) . we controlled for potential confounding effects of age , sex , and visceral adiposity ( within 20% ) by pair matching overweight youth with hepatic steatosis to overweight youth without hepatic steatosis . to validate the associations between hepatic steatosis and type 2 diabetes risk factors , we conducted a reciprocal matching study with a separate cohort of overweight youth discrepant for visceral fat mass ( < 70.0 vs. > for nonnormally distributed variables , mann - whitney u tests were used to examine differences between groups of pair - matched subjects . multiple stepwise linear regression analysis was used to determine if hepatic triglyceride was associated with various metabolic risk factors independent of age , ethnicity , sex , bmi z score , cardiorespiratory fitness , and visceral obesity . hepatic steatosis was evident in 31% ( n = 30 ) of overweight and obese adolescents and none of the healthy weight adolescents . overweight and obese adolescents with or without hepatic steatosis displayed lower insulin sensitivity , higher rates of metabolic syndrome , and higher serum glucose after the oral glucose challenge compared with healthy weight adolescents ( table 1 ) . bmi z score ( 2.1 vs. 1.8 , p < 0.001 ) and waist circumference z score ( 6.09 vs. 4.62 , p = 0.004 ) were higher among overweight and obese adolescents with hepatic steatosis compared with those without , despite similar visceral fat mass and percent body fat ( table 1 ) . the proportion of male participants was higher in the cohort of youth with hepatic steatosis relative to overweight and obese youth without ( 43 vs. 25% , p = 0.04 ) ( table 1 ) . a greater proportion of first nations youth composed the overweight and obese groups , compared with the healthy weight group ( 24 vs. 9% , p < 0.01 ) . a higher proportion of youth with hepatic steatosis met the criteria for obesity compared with youth without hepatic steatosis ( 90 vs. 65% , p < 0.05 ) . characteristics of study participants insulin sensitivity was 55% lower , the presence of metabolic syndrome was approximately twofold higher ( 50 vs. 25% , p = 0.001 ) , and serum glucose during the oral glucose challenge was 10% higher among youth with hepatic steatosis compared with healthy weight , overweight , and obese youth without hepatic steatosis . the nefa auc after a 75-g oral glucose challenge was not different between youth with hepatic steatosis compared with overweight and obese youth without hepatic steatosis ( table 1 ) . after matching for age , sex , and visceral fat mass , we found that insulin sensitivity remained 25% lower ( 3.5 vs. 4.5 mu kg min , p = 0.03 ) , the presence of metabolic syndrome remained greater than twofold higher ( 48 vs. 20% , p = 0.03 ) , and the glucose auc was 20% higher in overweight and obese adolescents with hepatic steatosis compared with overweight and obese adolescents without hepatic steatosis ( table 2 ) . in the reciprocal comparison , when youth were matched for hepatic triglyceride content , no differences in metabolic syndrome or glucose auc were observed , despite significant differences in visceral fat mass ( 63.7 vs. 102.1 cm ) ( table 2 ) . insulin sensitivity remained significantly different between groups ( 3.2 vs. 5.5 mu kg min , p = 0.02 ) . characteristics of pair - matched overweight and obese youth for age , sex , and visceral fat or hepatic triglyceride content hepatic triglyceride content was positively associated with glucose auc ( = 0.44 , p < 0.001 ) , bmi z score ( = 0.47 , p < 0.001 ) , waist circumference z score ( = 0.34 , p < 0.001 ) , systolic blood pressure ( = 0.16 , p = 0.005 ) , serum triglycerides ( = 0.43 , p < 0.001 ) , and alt ( = 0.36 , p < 0.001 ) and negatively associated with insulin sensitivity ( = 0.40 , p < 0.001 ) and hdl - c ( = 0.31 , p < 0.001 ) . cardiorespiratory fitness was not associated with insulin sensitivity or hepatic triglyceride content . multiple regression analysis revealed that hepatic triglyceride content was associated with the presence of metabolic syndrome ( r = 0.57 , p < 0.001 ) , insulin sensitivity ( r = 0.36 , p = 0.04 ) , and glucose auc ( r = 0.24 , p = 0.02 ) after adjusting for age , sex , bmi z score , waist circumference z score , visceral fat , whole - body fat mass , fitness level , and nefa auc . hepatic steatosis was evident in 31% ( n = 30 ) of overweight and obese adolescents and none of the healthy weight adolescents . overweight and obese adolescents with or without hepatic steatosis displayed lower insulin sensitivity , higher rates of metabolic syndrome , and higher serum glucose after the oral glucose challenge compared with healthy weight adolescents ( table 1 ) . bmi z score ( 2.1 vs. 1.8 , p < 0.001 ) and waist circumference z score ( 6.09 vs. 4.62 , p = 0.004 ) were higher among overweight and obese adolescents with hepatic steatosis compared with those without , despite similar visceral fat mass and percent body fat ( table 1 ) . the proportion of male participants was higher in the cohort of youth with hepatic steatosis relative to overweight and obese youth without ( 43 vs. 25% , p = 0.04 ) ( table 1 ) . a greater proportion of first nations youth composed the overweight and obese groups , compared with the healthy weight group ( 24 vs. 9% , p < 0.01 ) . a higher proportion of youth with hepatic steatosis met the criteria for obesity compared with youth without hepatic steatosis ( 90 vs. 65% , p < 0.05 ) . characteristics of study participants insulin sensitivity was 55% lower , the presence of metabolic syndrome was approximately twofold higher ( 50 vs. 25% , p = 0.001 ) , and serum glucose during the oral glucose challenge was 10% higher among youth with hepatic steatosis compared with healthy weight , overweight , and obese youth without hepatic steatosis . the nefa auc after a 75-g oral glucose challenge was not different between youth with hepatic steatosis compared with overweight and obese youth without hepatic steatosis ( table 1 ) . after matching for age , sex , and visceral fat mass , we found that insulin sensitivity remained 25% lower ( 3.5 vs. 4.5 mu kg min , p = 0.03 ) , the presence of metabolic syndrome remained greater than twofold higher ( 48 vs. 20% , p = 0.03 ) , and the glucose auc was 20% higher in overweight and obese adolescents with hepatic steatosis compared with overweight and obese adolescents without hepatic steatosis ( table 2 ) . in the reciprocal comparison , when youth were matched for hepatic triglyceride content , no differences in metabolic syndrome or glucose auc were observed , despite significant differences in visceral fat mass ( 63.7 vs. 102.1 cm ) ( table 2 ) . insulin sensitivity remained significantly different between groups ( 3.2 vs. 5.5 mu kg min , p = 0.02 ) . characteristics of pair - matched overweight and obese youth for age , sex , and visceral fat or hepatic triglyceride content hepatic triglyceride content was positively associated with glucose auc ( = 0.44 , p < 0.001 ) , bmi z score ( = 0.47 , p < 0.001 ) , waist circumference z score ( = 0.34 , p < 0.001 ) , systolic blood pressure ( = 0.16 , p = 0.005 ) , serum triglycerides ( = 0.43 , p < 0.001 ) , and alt ( = 0.36 , p < 0.001 ) and negatively associated with insulin sensitivity ( = 0.40 , p < 0.001 ) and hdl - c ( = 0.31 , p < 0.001 ) . multiple regression analysis revealed that hepatic triglyceride content was associated with the presence of metabolic syndrome ( r = 0.57 , p < 0.001 ) , insulin sensitivity ( r = 0.36 , p = 0.04 ) , and glucose auc ( r = 0.24 , p = 0.02 ) after adjusting for age , sex , bmi z score , waist circumference z score , visceral fat , whole - body fat mass , fitness level , and nefa auc . hepatic triglyceride content was positively associated with glucose auc ( = 0.44 , p < 0.001 ) , bmi z score ( = 0.47 , p < 0.001 ) , waist circumference z score ( = 0.34 , p < 0.001 ) , systolic blood pressure ( = 0.16 , p = 0.005 ) , serum triglycerides ( = 0.43 , p < 0.001 ) , and alt ( = 0.36 , p < 0.001 ) and negatively associated with insulin sensitivity ( = 0.40 , p < 0.001 ) and hdl - c ( = 0.31 , p < 0.001 ) . cardiorespiratory fitness was not associated with insulin sensitivity or hepatic triglyceride content . multiple regression analysis revealed that hepatic triglyceride content was associated with the presence of metabolic syndrome ( r = 0.57 , p < 0.001 ) , insulin sensitivity ( r = 0.36 , p = 0.04 ) , and glucose auc ( r = 0.24 , p = 0.02 ) after adjusting for age , sex , bmi z score , waist circumference z score , visceral fat , whole - body fat mass , fitness level , and nefa auc . first , hepatic steatosis is associated with 1 ) increased prevalence of metabolic syndrome , 2 ) elevated serum glucose in response to an oral glucose challenge , and 3 ) dyslipidemia , independent of visceral fat mass and cardiorespiratory fitness . second , several metabolic characteristics associated with visceral adiposity in adolescents , including metabolic syndrome and preclinical dysglycemia , can be attributed to the presence of elevated hepatic triglyceride levels . finally , cardiorespiratory fitness was not associated with hepatic triglyceride content , nor did it mediate the association between hepatic steatosis and type 2 diabetes risk factors . our data support the concept that hepatic steatosis is an early precursor to glucose intolerance and cardiometabolic risk independent of fitness in overweight and obese youth . previous cross - sectional studies show that both visceral fat mass and hepatic triglyceride content are associated with metabolic abnormalities in overweight adolescents ( 23,24 ) . recent studies showing hepatic steatosis as a determinant of insulin resistance in overweight youth failed to include a cohort matched for hepatic triglyceride content with disparate visceral fat mass as a positive control ( 4,7 ) . by effectively isolating hepatic steatosis from visceral obesity , we demonstrated that hepatic steatosis is the dominant predictor of metabolic syndrome among overweight or obese adolescents . several case control studies show that metabolic risk factor clustering is associated with the presence of hepatic steatosis ( 4,5 ) . using biopsy - proven or mri - derived measures of hepatic triglyceride , previous reports show that 50% of adolescents with hepatic steatosis meet diagnostic criteria for metabolic syndrome , a rate approximately two- to threefold higher than in adolescents without hepatic steatosis ( 5,25 ) . conventional wisdom suggests that the association between hepatic steatosis and metabolic risk factor clustering is mediated by whole - body insulin resistance ( 4,26 ) . in contrast , we found no differences in the prevalence of metabolic syndrome in youth with discrepant visceral fat mass , matched for hepatic triglyceride content , despite significant differences in insulin sensitivity . these data imply that hepatic steatosis is a sensitive biomarker and potential mediating factor for metabolic risk factor clustering and glucose intolerance in overweight and obese adolescents independent of insulin resistance . prior evidence suggests hepatic steatosis contributes to the loss of glucose tolerance among overweight and obese adolescents . first , hepatic steatosis is a common feature of igt and type 2 diabetes ( 24 ) . second , hepatic steatosis is associated with a loss of insulin - induced suppression of hepatic glucose output among overweight and obese individuals ( 7 ) . finally , glucose - stimulated insulin secretion is impaired among obese adolescents with hepatic steatosis ( 4,26 ) . our study extends these findings by showing that glucose excursion after an oral glucose challenge is higher among overweight and obese adolescents with hepatic steatosis . in contrast to others ( 4 ) , we did not observe a difference in the acute insulin response to glucose expressed in absolute terms or relative to insulin sensitivity between adolescents with and without hepatic steatosis . previous cross - sectional studies of hepatic steatosis in adolescents have failed to provide a measure of mitochondrial function or fitness levels . because recent studies show that the association between cardiorespiratory fitness and insulin resistance appears to be mediated in part by hepatic triglyceride content , we felt it was important to assess fitness in this cohort ( 8) . specifically , cardiorespiratory fitness was not associated with any of the risk factors for type 2 diabetes , nor was it different between overweight and obese youth with and without hepatic steatosis . however , the strategy of reciprocal matching permitted the dissection of the independent roles of hepatic steatosis and visceral adiposity in metabolic risk factor clustering in overweight and obese adolescents . differences in sex and ethnicity existed between groups ; however , after matching for sex and including ethnicity as a covariate in multiple linear regression models , group - wise differences in insulin sensitivity , presence of metabolic syndrome , or glucose auc were unchanged . hepatic steatosis is a prevalent comorbidity in overweight and obese adolescents that exists prior to clinically elevated serum aminotransferases . the presence of hepatic steatosis is accompanied by a clustering of factors associated with type 2 diabetes and cardiovascular disease , independent of visceral fat mass , whole - body adiposity , and cardiorespiratory fitness . these results support the growing body of evidence that hepatic steatosis is a potentially modifiable biomarker of risk for type 2 diabetes and metabolic syndrome in overweight and obese adolescents .

six - month mri becomes base line appearance for further follow - up or monitoring . in 2012 , with 417,000 new cases and 92,200 deaths , prostate cancer was the third most common cancer in europe overall after female breast cancer and colorectal cancer . in men only it was the most common cancer , followed by lung and colorectal cancers . the incidence of prostate cancer , especially for localised disease , has been growing since the 1990s , a dynamic that may be attributable to the widespread use of prostate - specific antigen ( psa ) tests and increasing life expectancies . radical prostatectomy , external beam radiotherapy or brachytherapy are still considered gold - standard treatments , but carry with them well - known side effects ( urinary incontinence , erectile dysfunction , intestinal toxicity ) . active surveillance and watchful waiting are attractive options for localised tumours . various focal treatments , such as phototherapy , cryotherapy , interstitial thermotherapy and dynamic high - frequency ultrasound ( hifu ) , are being evaluated [ 4 , 5 ] . photodynamic therapy is a recent technology wherein a photosensitising agent ( wst11 ) is administered intravenously , then activated by light to induce tumour necrosis . the light is applied using laser optical fibres positioned within the prostate as determined by magnetic resonance imaging ( mri ) planning . hollow , transparent , plastic brachytherapy catheters were positioned into the prostate , using trus - image guidance in accordance with a previously devised mri - based treatment plan ( fig laser light was delivered to the prostate , using a multichannel diode laser . the single tookad soluble intravenous ( i.v . ) dose was a 10-min infusion , followed by continuous illumination of the prostate gland for between 20 and 25 min . the total duration of the whole procedure was ~2 h. the light activates the photosensitiser in the prostate , generating reactive oxygen species that activate thrombosis within the vessels . this results in obliteration of the microvessel anatomy with resultant deprivation of oxygen and nutrients to the tumour cells and surrounding prostate tissue in the treated area [ 68 ] . the treatment appears promising for localised prostate cancer and benefits from a reasonably low rate of complications . currently , mri is the reference imaging technique for the diagnosis of disease and follow - up after local treatment [ 1113].fig . catheters were positioned into the prostate , using trus - image guidance in accordance with a previously devised mri - based treatment plan . cylindrically diffusing optical fibres were inserted into the catheters ( b ) procedure performed under general anaesthesia , in the darkness ( a ) . catheters were positioned into the prostate , using trus - image guidance in accordance with a previously devised mri - based treatment plan . cylindrically diffusing optical fibres were inserted into the catheters ( b ) however , the changes induced by photodynamic therapy are poorly reported in the literature . thus in the present pictorial review , we illustrate various mri features in the follow - up of patients who underwent photodynamic therapy for localised prostate cancer . we studied early and late normal aspects , local complications and aspects of recurrence , with histopathological correlation . the iconography presented in this paper comes from a group of 77 patients enrolled by our institution in a prospective international multicentric study ( phase ii / iii ) , and who received photodynamic therapy with wst11 ( tookad soluble ) for localised prostate cancer . inclusion criteria are : men over 18 years old ; diagnosed prostate cancer histologically proved on biopsies ( systematic biopsies most of the time , rarely targeted biopsies ) ; eligible for active surveillance ; no prior treatment for prostate cancer ; tnm stage up to ct2b- n0/nx- m0/mx ; gleason score 3 + 3 , 3 + 4 accepted in certain conditions ; psa < 10 ng / ml . all patients had a pre - treatment mri then post - treatment mris at 1 week ( early post - treatment ) and 6 months ( late post - treatment ) . some of the patients also had a post - treatment mri at 3 months . systematic biopsies with trus ( trans - rectal ultrasound ) guidance were performed at 6 months to explore correlations to mri features . in case of positive biopsy at 6 months , function of the gleason score , different options were proposed to the patients : treatment of the other lobe , retreatment on the same lobe , active surveillance or surgery . all mris were performed with a 1.5-t mri system ( ge excite , general electrics , milwaukee , il ) . an external body surface coil ( eight channels ) was used , with a field of view ( fov ) equal to 24 cm , except for the dynamic contrast - enhanced sequences for which fov was 42 cm . the imaging protocol included the following sequences : axial and coronal frfse t2 with fat saturation ( 4-mm slice thickness ) , axial fse t1 ( 4-mm slice thickness ) , axial diffusion - weighted ( b = 600 s / mm ) ; a three - dimensional ( 3d ) dynamic contrast - enhanced t1 sequence ( lava ) with fat saturation after intravenous injection of 20 ml gadoterate meglumine ( dotarem , guerbet , france ) , followed by axial and sagittal fat saturation contrast - enhanced fse t1-weighted images . these last sequences were systematically realised , to better define boundaries of necrosis , especially in case of extra prostatic necrosis . pre- and post - treatment mri exams were analysed on an imaging work station ( fujifilm synapse 3d ) . we analysed mri modifications for the areas treated with photodynamic therapy ( optical fibre disposition map ) and the histopathological results of systematic 6-month biopsies . some patients underwent prostatectomy after the phototherapy treatment , permitting a more accurate radiological - pathological correlation . pre - treatment mri served only to plan the anatomic distribution of the optic fibres into the lobe to be treated , targeting the zones of positive biopsy . pre - treatment mri was performed as a planning procedure to determine the position and the number of optical fibres and to exclude any loco - regional extension that would have contraindicated the treatment . only small tumours with low aggressiveness ( gleason 6 ) were considered for photodynamic therapy and thus not all of them were visible . when tumours were visible , the classical aspect was a hypointense nodule in t2-weighted mr images , with restriction on the apparent diffusion coefficient map and early enhancement on dynamic contrast - enhanced mr images ( fig . 2).fig . 2pre - treatment mri of a 65-year - old patient with a psa level of 9.7 ng / ml and a positive biopsy in the right prostate apex ( adenocarcinoma gleason 6 ) . axial and sagittal t2-weighted images ( a , b ) show a hypointense nodule in the right peripheral prostate with restriction on the apparent diffusion coefficient map of the diffusion - weighted image ( c , low signal intensity ) and early enhancement on the axial dynamic contrast - enhanced mr image ( d , arrowhead ) pre - treatment mri of a 65-year - old patient with a psa level of 9.7 ng / ml and a positive biopsy in the right prostate apex ( adenocarcinoma gleason 6 ) . axial and sagittal t2-weighted images ( a , b ) show a hypointense nodule in the right peripheral prostate with restriction on the apparent diffusion coefficient map of the diffusion - weighted image ( c , low signal intensity ) and early enhancement on the axial dynamic contrast - enhanced mr image ( d , arrowhead ) one - week mri examinations show t2 heterogeneous signals in the treated lobes ( fig . 3 ) that are related to the oedema and ischaemic modifications induced by phototherapy . these modifications are secondary to local intravascular coagulation triggered by the photosensitising agent when exposed to laser light.fig . 3oedema and necrosis . left lobe photodynamic therapy in a 65-year - old patient with a psa level of 6.2 ng / ml and two positive biopsies in the left lobe gleason 6 ( 3 + 3 ) adenocarcinoma . a pre - treatment axial t2-weighted image shows diffuse bilateral hyposignal of the peripheral prostate without focal nodularity . b day-7 post - treatment axial t2-weighted image shows a heterogeneous signal of the treated lobe extending to the peripheral and transitional zone ( arrowhead ) . note that the signal of the right lobe peripheral zone is unchanged compared with the pre - treatment image oedema and necrosis . left lobe photodynamic therapy in a 65-year - old patient with a psa level of 6.2 ng / ml and two positive biopsies in the left lobe gleason 6 ( 3 + 3 ) adenocarcinoma . a pre - treatment axial t2-weighted image shows diffuse bilateral hyposignal of the peripheral prostate without focal nodularity . b day-7 post - treatment axial t2-weighted image shows a heterogeneous signal of the treated lobe extending to the peripheral and transitional zone ( arrowhead ) . note that the signal of the right lobe peripheral zone is unchanged compared with the pre - treatment image mri sequences show an increased volume of the treated lobe . when the procedure is unilateral , the volume variation is obvious and particularly well visualised in t2-weighted sequences ( fig . 4volume increase of the treated lobe . a 65-year - old patient with a middle right lobe positive biopsy . axial ( b ) and coronal ( c ) day-7 post - treatment t2-weighted images show an asymmetrical increase of the volume of the treated lobe ( star ) volume increase of the treated lobe . a 65-year - old patient with a middle right lobe positive biopsy . pre - treatment axial t2 sequence ( a ) shows a normal prostate . axial ( b ) and coronal ( c ) day-7 post - treatment t2-weighted images show an asymmetrical increase of the volume of the treated lobe ( star ) among all heterogeneities within the treated lobe , besides the loss of physiological distinction between transitional and peripheral prostate tissue in t2 images , the paths of the optical fibres can be observed as small hyperintense spots in the axial plane and linear bands in the sagittal and coronal planes ( fig . right prostate lobe treatment for adenocarcinoma gleason 6 ( 3 + 3 ) in a 62-year - old patient with a psa level of 9 ng / ml . tumour was not visible on pre - treatment mri . the paths of the optical fibres in the treated area are visible on the day-7 post - treatment axial t2-weighted image ( a ) as small hyperintense spots corresponding to thin linear bands ( star ) in the coronal view ( b ) path of optical fibres . right prostate lobe treatment for adenocarcinoma gleason 6 ( 3 + 3 ) in a 62-year - old patient with a psa level of 9 ng / ml . tumour was not visible on pre - treatment mri . the paths of the optical fibres in the treated area are visible on the day-7 post - treatment axial t2-weighted image ( a ) as small hyperintense spots corresponding to thin linear bands ( star ) in the coronal view ( b ) on t1-weighted images before gadolinium injection , hyperintense , poorly defined areas can be identified . . 6 ) and make t1-weighted contrast - enhanced images difficult to analyse ; in this situation the use of image subtraction may be helpful.fig . a day-7 post - treatment axial t1 mr image in a 64-year - old patient with right lobe photodynamic therapy shows an asymmetric prostate ( star ) with increased volume of the treated lobe but without any spontaneous high signal intensity evoking haemorrhage in the treated area . day-7 post - treatment axial t1 shows a hyperintense area ( arrowhead ) corresponding to post - treatment haemorrhagic suffusions . day-7 post - treatment axial t1-weighted mr image shows a spontaneous high - signal - intensity area in the right lobe ( arrowhead ) , but also an extended spontaneous high signal intensity in the left ( untreated ) lobe ( star ) haemorrhage within treated lobes is variable . a day-7 post - treatment axial t1 mr image in a 64-year - old patient with right lobe photodynamic therapy shows an asymmetric prostate ( star ) with increased volume of the treated lobe but without any spontaneous high signal intensity evoking haemorrhage in the treated area . day-7 post - treatment axial t1 shows a hyperintense area ( arrowhead ) corresponding to post - treatment haemorrhagic suffusions . day-7 post - treatment axial t1-weighted mr image shows a spontaneous high - signal - intensity area in the right lobe ( arrowhead ) , but also an extended spontaneous high signal intensity in the left ( untreated ) lobe ( star ) necrosis is best defined on t1-weighted fat - sat contrast - enhanced images . it appears as non - enhancing areas with irregular but well - defined and highly enhanced edges ( fig . a treated lobe presents a single , large , homogeneous necrosis area on t1 contrast - enhanced sequences . however , in some cases , islands of spared tissue may be observed with high early enhancement ( fig . a 72-year - old patient with a psa level of 4.5 ng / ml treated for gleason 6 ( 3 + 3 ) left lobe adenocarcinoma . axial dynamic contrast - enhanced images before treatment ( a ) show a zone of post - biopsy haemorrhage on the right lobe without any suspicious enhancement . day-7 post - treatment left mri ( b ) shows no enhancement of the left treated lobe ( necrosis ) . right lobe treatment for gleason 6 adenocarcinoma : day-7 post - treatment axial ( c ) and sagittal ( d ) fs contrast - enhanced images show a large homogenous hypointense non - enhanced area in the treated lobe ( necrosis ) with well - defined and highly enhanced boundariesfig . axial t1 ( a ) and contrast - enhanced axial t1 fs ( b ) images show islands of spared tissue ( arrowhead ) that enhance within the treated area well - defined necrosis boundaries . a 72-year - old patient with a psa level of 4.5 ng / ml treated for gleason 6 ( 3 + 3 ) left lobe adenocarcinoma . axial dynamic contrast - enhanced images before treatment ( a ) show a zone of post - biopsy haemorrhage on the right lobe without any suspicious enhancement . day-7 post - treatment left mri ( b ) shows no enhancement of the left treated lobe ( necrosis ) . right lobe treatment for gleason 6 adenocarcinoma : day-7 post - treatment axial ( c ) and sagittal ( d ) fs contrast - enhanced images show a large homogenous hypointense non - enhanced area in the treated lobe ( necrosis ) with well - defined and highly enhanced boundaries focal spared tissue . left lobe photodynamic therapy in a 66-year - old patient . axial t1 ( a ) and contrast - enhanced axial t1 fs ( b ) images show islands of spared tissue ( arrowhead ) that enhance within the treated area another pitfall to be avoided is focal inflammatory enhancements in the treated lobe presenting a nodular feature ( fig . these lesions are usually no longer present on 3-month post - treatment mris.fig . 9suspected residual tumour . day-7 post - treatment dynamic contrast - enhanced fat - saturated axial t1-weighted mr image showing early nodular enhancement in the prostate apex suggestive of residual tumour away from the treated area . the nodule was no longer visible on the 3-month post - treatment and 6-month post - treatment mris , and the 6-month post - treatment biopsy was negative suspected residual tumour . day-7 post - treatment dynamic contrast - enhanced fat - saturated axial t1-weighted mr image showing early nodular enhancement in the prostate apex suggestive of residual tumour away from the treated area . the nodule was no longer visible on the 3-month post - treatment and 6-month post - treatment mris , and the 6-month post - treatment biopsy was negative in cases with concomitant bilateral treatment , the 1-week mri shows the same types of modifications but they are symmetrical . normally , the intraprostatic urethra exhibits a continuous ring of peripheral enhancement . a discontinuous ring ( fig . 10 ) on the 1-week post - photodynamic therapy mri is thus suggestive of urethral wall necrosis . in this situation , despite earlier animal studies reporting structural and functional resistance of the urethra to photodynamic therapy , close clinical monitoring is essential.fig . day-7 post - treatment axial t1 fs contrast - enhanced mr images for a bilaterally treated patient show necrosis in both lobes . a rupture of this ring enhancement ( arrowhead ) after photodynamic therapy is considered suspicious of urethral wall necrosis ( b ) . no urinary complications were identified in this patient during close clinical follow - up periurethral enhancement ring . day-7 post - treatment axial t1 fs contrast - enhanced mr images for a bilaterally treated patient show necrosis in both lobes . a rupture of this ring enhancement ( arrowhead ) after photodynamic therapy is considered suspicious of urethral wall necrosis ( b ) . no urinary complications were identified in this patient during close clinical follow - up finally , it is important to note that on the 1-week post - therapy images the surroundings of the gland may also show enhancement due to local inflammation . at 6 months after photodynamic therapy , important changes of the prostate shape and signal are found . small areas of residual necrosis may still be present in the treated lobe , corresponding to coagulation necrosis ( fig six - month post - treatment mri in a patient treated by left photodynamic therapy . axial t1 fs contrast - enhanced ( a ) and axial t2-weighted images ( b ) show persistence of a low signal intensity area with no enhancement ( arrowhead ) corresponding to a small area of residual necrosis ( coagulation necrosis ) . pathology slides from biopsy at low ( c ) and high magnification ( d ) show loose connective tissue coreless , with cell ghosts , characteristic of coagulation necrosis residual necrosis . six - month post - treatment mri in a patient treated by left photodynamic therapy . axial t1 fs contrast - enhanced ( a ) and axial t2-weighted images ( b ) show persistence of a low signal intensity area with no enhancement ( arrowhead ) corresponding to a small area of residual necrosis ( coagulation necrosis ) . pathology slides from biopsy at low ( c ) and high magnification ( d ) show loose connective tissue coreless , with cell ghosts , characteristic of coagulation necrosis classically , the volume of the treated lobe decreases , sometimes to less than the volume of the lobe before treatment . axial t2-weighted mr ( a ) and axial t1 fs contrast - enhanced ( b ) images , in a case of bilateral photodynamic therapy show a post - treatment pattern , with a small area of low signal intensity , without enhancement , corresponding to fibrous scar tissue ( arrowhead ) . low power ( c , d ) images from radical prostatectomy show a focus of sclerotic and hyaline necrosis on the left lobe suggesting a 7 5-mm therapy scar fibrous scar . axial t2-weighted mr ( a ) and axial t1 fs contrast - enhanced ( b ) images , in a case of bilateral photodynamic therapy show a post - treatment pattern , with a small area of low signal intensity , without enhancement , corresponding to fibrous scar tissue ( arrowhead ) . low power ( c , d ) images from radical prostatectomy show a focus of sclerotic and hyaline necrosis on the left lobe suggesting a 7 5-mm therapy scar residual , irregularly shaped fluid cavities related to atrophy in the treated lobe are frequently seen ( fig . on t2-weighted images , the normal hypersignal of the peripheral prostate is replaced by the irregular hyposignal of the scar.fig . six - month post - treatment follow - up mri in a patient with left photodynamic therapy . axial t2-weighted ( a ) and t1-weighted ( b ) mr images show an irregularly shaped residual fluid cavity in the treated lobe , with a low signal on t1 ( arrowhead ) and a high signal on t2 residual fluid cavity . six - month post - treatment follow - up mri in a patient with left photodynamic therapy . axial t2-weighted ( a ) and t1-weighted ( b ) mr images show an irregularly shaped residual fluid cavity in the treated lobe , with a low signal on t1 ( arrowhead ) and a high signal on t2 unilateral treatment induces an asymmetric prostate , whereas bilateral treatment results in a small , heterogeneous , but symmetrical prostate ( fig . axial t2-weighted mr images from before treatment ( a ) and 6 months after treatment ( b ) in a 65-year - old patient with left unilateral photodynamic therapy illustrate post - treatment asymmetric prostate due to unilateral scar tissue . however , pre - treatment ( c ) and 6-month post - treatment ( d ) mris in a case of bilateral treatment ( 71-year - old patient ) illustrate a symmetrical aspect . note the disappearance of the peripheral zone of the treated lobes on the 6-month mris asymmetry / symmetry . axial t2-weighted mr images from before treatment ( a ) and 6 months after treatment ( b ) in a 65-year - old patient with left unilateral photodynamic therapy illustrate post - treatment asymmetric prostate due to unilateral scar tissue . however , pre - treatment ( c ) and 6-month post - treatment ( d ) mris in a case of bilateral treatment ( 71-year - old patient ) illustrate a symmetrical aspect . note the disappearance of the peripheral zone of the treated lobes on the 6-month mris furthermore , the aspect observed at 6 months is definitive ( figs . we thus underline that the 6-month mri is very important as it will become the new base line appearance for any further follow - up or monitoring.fig . six - month post - treatment axial t2 images in a 66-year - old left photodynamic therapy patient show left lobe atrophy ( particularly for the peripheral prostate ) as well as a small fluid cavity ( arrowhead ) in the middle of the scar . six - month ( a ) and 3-year ( b ) post - treatment mri for right photodynamic therapy . the 6-month and 3 year axial t2-weighted images show the same aspect : atrophy of the right lobe with attraction of the urethra , and the disappearance of normal peripheral prostate hyperintensity . treatment resulted in a lesser modification of the transitional zone compared with the contralateral lobe unchanged scar appearance over time . six - month post - treatment axial t2 images in a 66-year - old left photodynamic therapy patient show left lobe atrophy ( particularly for the peripheral prostate ) as well as a small fluid cavity ( arrowhead ) in the middle of the scar . there was no change in this aspect at 1 year ( b ) unchanged scar appearance over time . six - month ( a ) and 3-year ( b ) post - treatment mri for right photodynamic therapy . the 6-month and 3 year axial t2-weighted images show the same aspect : atrophy of the right lobe with attraction of the urethra , and the disappearance of normal peripheral prostate hyperintensity . treatment resulted in a lesser modification of the transitional zone compared with the contralateral lobe three - month mris are not pertinent for treatment follow - up as they tend to show aspects that are between early necrosis and late fibrosis and thus difficult to analyse ( fig . 17).fig . 17three - month post - treatment mri in a 69-year - old patient with concomitant bilateral treatment for prostate adenocarcinoma , gleason 6 ( psa level of 8.14 ng / ml , 1/12 positive biopsy at right apex ) . axial t1 fs contrast - enhanced image ( a ) shows the persistence of two small areas of residual necrosis ( arrowhead ) three - month post - treatment mri in a 69-year - old patient with concomitant bilateral treatment for prostate adenocarcinoma , gleason 6 ( psa level of 8.14 ng / ml , 1/12 positive biopsy at right apex ) . axial t1 fs contrast - enhanced image ( a ) shows the persistence of two small areas of residual necrosis ( arrowhead ) the indication criteria of retreatment are positive biopsies in treated or contralateral lobe during the follow - up , with gleason score 3 + 3 or 3 + 4 in certain conditions . when ipsilateral retreatment is needed , the modifications seen after 1 week are similar to those seen 1 week after the initial treatment ( fig . 18 ) . after 6 months , 18retreatment of the same lobe . a 60-year - old patient with retreatment of the right lobe . day-7 post - retreatment axial t2-weighted mr image ( a ) and axial t1 fs contrast - enhanced mr image ( b ) show a slight volume increase of the treated lobe compared with the first procedure ; the area of prostate necrosis presents two different intensity levels : the residual fluid cavity ( arrowhead ) and tissue necrosis ( arrow ) , with less low intensity . note , 6 months post first treatment axial t2-weighted mr image ( c ) corresponding to pre - retreatment aspect retreatment of the same lobe . a 60-year - old patient with retreatment of the right lobe . day-7 post - retreatment axial t2-weighted mr image ( a ) and axial t1 fs contrast - enhanced mr image ( b ) show a slight volume increase of the treated lobe compared with the first procedure ; the area of prostate necrosis presents two different intensity levels : the residual fluid cavity ( arrowhead ) and tissue necrosis ( arrow ) , with less low intensity . note , 6 months post first treatment axial t2-weighted mr image ( c ) corresponding to pre - retreatment aspect when a renewed intervention is for the contralateral lobe ( fig . 19 ) the asymmetric aspect is more pronounced at 1 week because of the atrophy of the previously treated lobe , but at 6 months , this asymmetry disappears and the prostate takes on the same appearance as a concomitant bilateral treatment.fig . 19contralateral treatment . a 56-year - old patient , with initial right photodynamic therapy , then left therapy in a second intervention . pre - left therapy ( a ) and day-7 post - left therapy ( b ) axial t2-weighted images ; day-7 post - left therapy ( c ) axial t1 fs contrast - enhanced mr image . on the left lobe , a heterogeneous t2 area with low signal in t1-weighted image , unenhanced , corresponding to the recent tissue necrosis ( star ) . in the right lobe the t2 hyperintense old cystic cavity increased in size ( arrowhead ) contralateral treatment . a 56-year - old patient , with initial right photodynamic therapy , then left therapy in a second intervention . pre - left therapy ( a ) and day-7 post - left therapy ( b ) axial t2-weighted images ; day-7 post - left therapy ( c ) axial t1 fs contrast - enhanced mr image . on the left lobe , a heterogeneous t2 area with low signal in t1-weighted image , unenhanced , corresponding to the recent tissue necrosis ( star ) . in the right lobe the t2 hyperintense old cystic cavity increased in size ( arrowhead ) photodynamic therapy appears to have a reasonably low rate of complications . in a study on different types of focal therapies performed in a cohort of men with low - risk prostate cancer , the overall complication rate was 13 % , with only two clavien - dindo grade 3 complications . ( clavien dindo is a grading system from 1 to 5 , used for the classification of surgical complications ; grade 3 or more are serious complications : grade 3complication which requires surgical , endoscopic or radiological intervention up to grade 5 , which corresponds to the death of the patient ) . in that series . found that most treatment - emergent adverse events ( teaes ) post photodynamic therapy were mild or moderate and only 9 % of patients reported serious teaes ( table 1).table 1treatment - emergent adverse events ( teaes ) of photodynamic therapy . study mild teaeserious teaedysuriaprostatitisuti ( urinary tractus infection)haematuriaurinary retentionepididymo - orchitisconstipationcystoprostatitisperineal painischaemic optic neuropathyinflammatory prostatic cyst treatment - emergent adverse events ( teaes ) of photodynamic therapy . study the main post - photodynamic therapy complication is extraprostatic necrosis , defined as a lack of enhancement on t1-weighted contrast - enhanced mr images involving the different structures surrounding the prostate . when present , necrosis most frequently extends to periprostatic fatty tissue ( fig . the muscles close to the prostate can also be affected , especially the levator ani ( fig . however , in our experience with this situation , anal functional abnormalities are not observed and a normal aspect is recovered after 6 months ( fig . a 62-year - old patient who had underwent left photodynamic therapy 1 year earlier , and right treatment 7 days earlier . axial t1-weighted ( a ) , axial t1 fs unenhanced ( b ) and contrast - enhanced ( c ) images show a nodular lesion ( arrowhead ) located in the right rectoprostatic angle , with a high intensity t1 fs signal , unenhanced , corresponding to focal haemorrhagic necrosis of periprostatic fatty tissuefig . right photodynamic therapy in a 65-year - old patient for gleason 6 adenocarcinoma ( two positive biopsies in right prostate ) . day-7 post - treatment axial ( a ) and coronal ( b ) t2-weighted mr images visualise a heterogeneous high signal of the right puborectalis muscle ( arrowheads ) ; axial and sagittal t1 fs contrast - enhanced images ( c , d ) show a low - signal well - defined area surrounded by a hyperintense enhanced border after gadolinium injection ( arrows ) corresponding to extraprostatic necrosis affecting the right levator ani musclefig . day-7 post - treatment axial t1 fs contrast - enhanced ( a ) and axial t2-weighted ( b ) mr images after right photodynamic therapy show extension of necrosis to puborectalis muscle ( star ) ; at the 6-month post - treatment mri follow - up , the axial t2 image ( c ) illustrated the complete disappearance of the area of necrosis ( arrowhead ) . no anal functional abnormalities were seen in this patient periprostatic fatty tissue . a 62-year - old patient who had underwent left photodynamic therapy 1 year earlier , and right treatment 7 days earlier . axial t1-weighted ( a ) , axial t1 fs unenhanced ( b ) and contrast - enhanced ( c ) images show a nodular lesion ( arrowhead ) located in the right rectoprostatic angle , with a high intensity t1 fs signal , unenhanced , corresponding to focal haemorrhagic necrosis of periprostatic fatty tissue levator ani muscle . right photodynamic therapy in a 65-year - old patient for gleason 6 adenocarcinoma ( two positive biopsies in right prostate ) . day-7 post - treatment axial ( a ) and coronal ( b ) t2-weighted mr images visualise a heterogeneous high signal of the right puborectalis muscle ( arrowheads ) ; axial and sagittal t1 fs contrast - enhanced images ( c , d ) show a low - signal well - defined area surrounded by a hyperintense enhanced border after gadolinium injection ( arrows ) corresponding to extraprostatic necrosis affecting the right levator ani muscle levator ani muscle . day-7 post - treatment axial t1 fs contrast - enhanced ( a ) and axial t2-weighted ( b ) mr images after right photodynamic therapy show extension of necrosis to puborectalis muscle ( star ) ; at the 6-month post - treatment mri follow - up , the axial t2 image ( c ) illustrated the complete disappearance of the area of necrosis ( arrowhead ) . no anal functional abnormalities were seen in this patient extraprostatic necrosis may also affect the internal obturator muscle ( fig . 23 ) and rarely it may extend to the anterior rectal wall ( fig . , fistulisation does not seem to occur and the rectal wall recovers its normal aspect on the 6-month follow - up mri.fig . 23internal obturator muscle . left photodynamic therapy in a 66-year - old patient with gleason 6 adenocarcinoma ( pre - treatment / day-7 post - treatment/6-month post - treatment ) . axial ( a ) and coronal ( b ) t2-weighted mr images show high signals of the left internal obturator muscle ( arrowhead ) and the puborectalis muscle ( arrow ) . extraprostatic necrosis extending to muscles is well defined on the t1 fs contrast - enhanced image ( c , flash)fig . day-7 post - treatment ( a ) and 6-month post - treatment ( b ) axial t1 fs contrast - enhanced mr images show the presence of a thin line of necrosis extending to the anterior rectal wall ( arrow ) , limited to the muscularis propria . note the complete recovery of the rectal wall at 6 months . also no fistulae were clinically identified internal obturator muscle . left photodynamic therapy in a 66-year - old patient with gleason 6 adenocarcinoma ( pre - treatment / day-7 post - treatment/6-month post - treatment ) . axial ( a ) and coronal ( b ) t2-weighted mr images show high signals of the left internal obturator muscle ( arrowhead ) and the puborectalis muscle ( arrow ) . extraprostatic necrosis extending to muscles is well defined on the t1 fs contrast - enhanced image ( c , flash ) anterior rectal wall . day-7 post - treatment ( a ) and 6-month post - treatment ( b ) axial t1 fs contrast - enhanced mr images show the presence of a thin line of necrosis extending to the anterior rectal wall ( arrow ) , limited to the muscularis propria . note the complete recovery of the rectal wall at 6 months . also no fistulae were clinically identified sliding of the optical fibre may result in an extension to a seminal vesicle ( fig . day-7 post - treatment axial ( a ) and sagittal ( b ) t1 fs contrast - enhanced mr images show a small extension of necrosis to the left seminal vesicle ( arrowhead ) by extraprostatic sliding of an optical fibre seminal vesicle day-7 post - treatment axial ( a ) and sagittal ( b ) t1 fs contrast - enhanced mr images show a small extension of necrosis to the left seminal vesicle ( arrowhead ) by extraprostatic sliding of an optical fibre as mentioned earlier , necrosis may affect the intraprostatic urethra ; a break of the periurethral enhancement ring or its complete disappearance at 1 week is an interesting sign suggestive of urethral parietal necrosis ( fig . right photodynamic therapy in a 60-year - old patient with a psa level of 12.9 ng / ml and gleason 6 adenocarcinoma ( positive base biopsies , right middle prostate ) . day-7 post - treatment axial ( a ) and sagittal ( b ) t1 fs contrast - enhanced mr images do not show the normal periurethral ring of enhancement ; the patient developed acute urinary retention treated by urinary catheter urethral necrosis . right photodynamic therapy in a 60-year - old patient with a psa level of 12.9 ng / ml and gleason 6 adenocarcinoma ( positive base biopsies , right middle prostate ) . day-7 post - treatment axial ( a ) and sagittal ( b ) t1 fs contrast - enhanced mr images do not show the normal periurethral ring of enhancement ; the patient developed acute urinary retention treated by urinary catheter mri detection of tumour recurrence in the treated area is made difficult by the loss of the normal hyperintensity of the peripheral prostate in t2-weighted images and the signal of the scar . thus , after photodynamic therapy , the diagnosis of recurrence is mainly based on psa levels and systematic biopsy . there is no reliable feature of recurrence and cases of small unaggressive recurrence are usually not visible on mri ( fig . early dynamic contrast - enhanced images and apparent diffusion coefficient mapping may be useful for detecting recurrence ( fig . therefore , any suspicious nodule seen on follow - up mri should be biopsied ( fig . 29 ) . targeted biopsies with trus are performed in nodules presenting pirads criteria on follow - up mri.fig . six - month post - treatment axial t2-weighted ( a ) and axial dynamic contrast - enhanced ( b ) images show atrophy of the right lobe including the peripheral prostate ; no suspicious nodules are visible . pathology slide at high magnification ( c ) of a right base prostate biopsy , shows a small glandular focus ( circled ) between normal glands , lined by a single layer of cellsfig . six - month post - treatment axial t2 ( a ) image shows a hypointense nodule of the right peripheral prostate and the axial dynamic contrast - enhanced ( b ) image illustrates an early enhancement after gadolinium injection of the nodule suggestive of recurrence ( arrowhead ) . low - power ( c ) and high - power ( d ) pathology slides of a right base prostate biopsy show small irregular glands lined by a single layer of cells , confirming the presence of an infiltrating gleason 7 adenocarcinoma with perineural invasionfig . mri was performed : axial t2 ( a ) , axial adc ( b ) and axial t1 dynamic contrast - enhanced ( c ) mr images show a suspicious left median focus with t2 hyposignal , early enhancement and mild diffusion restriction on adc map ( arrowhead ) . the location of suspicious lesion seen on mri is indicated on a prostate map . pathology slide at high magnification ( d ) shows a 1-mm tumour focus in a scar tissue . crushed appearance of several glands ( circled ) , suspect , confirmed by immunohistochemical study , and surrounded by thick collagen fibres ( fibrous scar ) . the nodule on mri corresponds to the scar tissue ; the tumour focus is too small to be seen on mri small recurrence not seen on mri . six - month post - treatment axial t2-weighted ( a ) and axial dynamic contrast - enhanced ( b ) images show atrophy of the right lobe including the peripheral prostate ; no suspicious nodules are visible . pathology slide at high magnification ( c ) of a right base prostate biopsy , shows a small glandular focus ( circled ) between normal glands , lined by a single layer of cells recurrence seen on mri . six - month post - treatment axial t2 ( a ) image shows a hypointense nodule of the right peripheral prostate and the axial dynamic contrast - enhanced ( b ) image illustrates an early enhancement after gadolinium injection of the nodule suggestive of recurrence ( arrowhead ) . low - power ( c ) and high - power ( d ) pathology slides of a right base prostate biopsy show small irregular glands lined by a single layer of cells , confirming the presence of an infiltrating gleason 7 adenocarcinoma with perineural invasion mismatch . mri was performed : axial t2 ( a ) , axial adc ( b ) and axial t1 dynamic contrast - enhanced ( c ) mr images show a suspicious left median focus with t2 hyposignal , early enhancement and mild diffusion restriction on adc map ( arrowhead ) . the location of suspicious lesion seen on mri is indicated on a prostate map . pathology slide at high magnification ( d ) shows a 1-mm tumour focus in a scar tissue . crushed appearance of several glands ( circled ) , suspect , confirmed by immunohistochemical study , and surrounded by thick collagen fibres ( fibrous scar ) . the nodule on mri corresponds to the scar tissue ; the tumour focus is too small to be seen on mri pre - treatment mri was performed as a planning procedure to determine the position and the number of optical fibres and to exclude any loco - regional extension that would have contraindicated the treatment . only small tumours with low aggressiveness ( gleason 6 ) were considered for photodynamic therapy and thus not all of them were visible . when tumours were visible , the classical aspect was a hypointense nodule in t2-weighted mr images , with restriction on the apparent diffusion coefficient map and early enhancement on dynamic contrast - enhanced mr images ( fig . 2).fig . 2pre - treatment mri of a 65-year - old patient with a psa level of 9.7 ng / ml and a positive biopsy in the right prostate apex ( adenocarcinoma gleason 6 ) . axial and sagittal t2-weighted images ( a , b ) show a hypointense nodule in the right peripheral prostate with restriction on the apparent diffusion coefficient map of the diffusion - weighted image ( c , low signal intensity ) and early enhancement on the axial dynamic contrast - enhanced mr image ( d , arrowhead ) pre - treatment mri of a 65-year - old patient with a psa level of 9.7 ng / ml and a positive biopsy in the right prostate apex ( adenocarcinoma gleason 6 ) . axial and sagittal t2-weighted images ( a , b ) show a hypointense nodule in the right peripheral prostate with restriction on the apparent diffusion coefficient map of the diffusion - weighted image ( c , low signal intensity ) and early enhancement on the axial dynamic contrast - enhanced mr image ( d , arrowhead ) one - week mri examinations show t2 heterogeneous signals in the treated lobes ( fig . 3 ) that are related to the oedema and ischaemic modifications induced by phototherapy . these modifications are secondary to local intravascular coagulation triggered by the photosensitising agent when exposed to laser light.fig . 3oedema and necrosis . left lobe photodynamic therapy in a 65-year - old patient with a psa level of 6.2 ng / ml and two positive biopsies in the left lobe gleason 6 ( 3 + 3 ) adenocarcinoma . a pre - treatment axial t2-weighted image shows diffuse bilateral hyposignal of the peripheral prostate without focal nodularity . b day-7 post - treatment axial t2-weighted image shows a heterogeneous signal of the treated lobe extending to the peripheral and transitional zone ( arrowhead ) . note that the signal of the right lobe peripheral zone is unchanged compared with the pre - treatment image oedema and necrosis . left lobe photodynamic therapy in a 65-year - old patient with a psa level of 6.2 ng / ml and two positive biopsies in the left lobe gleason 6 ( 3 + 3 ) adenocarcinoma . a pre - treatment axial t2-weighted image shows diffuse bilateral hyposignal of the peripheral prostate without focal nodularity . b day-7 post - treatment axial t2-weighted image shows a heterogeneous signal of the treated lobe extending to the peripheral and transitional zone ( arrowhead ) . note that the signal of the right lobe peripheral zone is unchanged compared with the pre - treatment image mri sequences show an increased volume of the treated lobe . when the procedure is unilateral , the volume variation is obvious and particularly well visualised in t2-weighted sequences ( fig . 4volume increase of the treated lobe . a 65-year - old patient with a middle right lobe positive biopsy . pre - treatment axial t2 sequence ( a ) shows a normal prostate . axial ( b ) and coronal ( c ) day-7 post - treatment t2-weighted images show an asymmetrical increase of the volume of the treated lobe ( star ) volume increase of the treated lobe . a 65-year - old patient with a middle right lobe positive biopsy . pre - treatment axial t2 sequence ( a ) shows a normal prostate . axial ( b ) and coronal ( c ) day-7 post - treatment t2-weighted images show an asymmetrical increase of the volume of the treated lobe ( star ) among all heterogeneities within the treated lobe , besides the loss of physiological distinction between transitional and peripheral prostate tissue in t2 images , the paths of the optical fibres can be observed as small hyperintense spots in the axial plane and linear bands in the sagittal and coronal planes ( fig . right prostate lobe treatment for adenocarcinoma gleason 6 ( 3 + 3 ) in a 62-year - old patient with a psa level of 9 ng / ml . tumour was not visible on pre - treatment mri . the paths of the optical fibres in the treated area are visible on the day-7 post - treatment axial t2-weighted image ( a ) as small hyperintense spots corresponding to thin linear bands ( star ) in the coronal view ( b ) path of optical fibres . right prostate lobe treatment for adenocarcinoma gleason 6 ( 3 + 3 ) in a 62-year - old patient with a psa level of 9 ng / ml . tumour was not visible on pre - treatment mri . the paths of the optical fibres in the treated area are visible on the day-7 post - treatment axial t2-weighted image ( a ) as small hyperintense spots corresponding to thin linear bands ( star ) in the coronal view ( b ) on t1-weighted images before gadolinium injection , hyperintense , poorly defined areas can be identified . 6 ) and make t1-weighted contrast - enhanced images difficult to analyse ; in this situation the use of image subtraction may be helpful.fig . a day-7 post - treatment axial t1 mr image in a 64-year - old patient with right lobe photodynamic therapy shows an asymmetric prostate ( star ) with increased volume of the treated lobe but without any spontaneous high signal intensity evoking haemorrhage in the treated area . day-7 post - treatment axial t1 shows a hyperintense area ( arrowhead ) corresponding to post - treatment haemorrhagic suffusions . day-7 post - treatment axial t1-weighted mr image shows a spontaneous high - signal - intensity area in the right lobe ( arrowhead ) , but also an extended spontaneous high signal intensity in the left ( untreated ) lobe ( star ) haemorrhage within treated lobes is variable . a day-7 post - treatment axial t1 mr image in a 64-year - old patient with right lobe photodynamic therapy shows an asymmetric prostate ( star ) with increased volume of the treated lobe but without any spontaneous high signal intensity evoking haemorrhage in the treated area . day-7 post - treatment axial t1 shows a hyperintense area ( arrowhead ) corresponding to post - treatment haemorrhagic suffusions . day-7 post - treatment axial t1-weighted mr image shows a spontaneous high - signal - intensity area in the right lobe ( arrowhead ) , but also an extended spontaneous high signal intensity in the left ( untreated ) lobe ( star ) necrosis is best defined on t1-weighted fat - sat contrast - enhanced images . it appears as non - enhancing areas with irregular but well - defined and highly enhanced edges ( fig . a treated lobe presents a single , large , homogeneous necrosis area on t1 contrast - enhanced sequences . however , in some cases , islands of spared tissue may be observed with high early enhancement ( fig . a 72-year - old patient with a psa level of 4.5 ng / ml treated for gleason 6 ( 3 + 3 ) left lobe adenocarcinoma . axial dynamic contrast - enhanced images before treatment ( a ) show a zone of post - biopsy haemorrhage on the right lobe without any suspicious enhancement . day-7 post - treatment left mri ( b ) shows no enhancement of the left treated lobe ( necrosis ) . right lobe treatment for gleason 6 adenocarcinoma : day-7 post - treatment axial ( c ) and sagittal ( d ) fs contrast - enhanced images show a large homogenous hypointense non - enhanced area in the treated lobe ( necrosis ) with well - defined and highly enhanced boundariesfig . axial t1 ( a ) and contrast - enhanced axial t1 fs ( b ) images show islands of spared tissue ( arrowhead ) that enhance within the treated area well - defined necrosis boundaries . a 72-year - old patient with a psa level of 4.5 ng / ml treated for gleason 6 ( 3 + 3 ) left lobe adenocarcinoma . axial dynamic contrast - enhanced images before treatment ( a ) show a zone of post - biopsy haemorrhage on the right lobe without any suspicious enhancement . day-7 post - treatment left mri ( b ) shows no enhancement of the left treated lobe ( necrosis ) . right lobe treatment for gleason 6 adenocarcinoma : day-7 post - treatment axial ( c ) and sagittal ( d ) fs contrast - enhanced images show a large homogenous hypointense non - enhanced area in the treated lobe ( necrosis ) with well - defined and highly enhanced boundaries focal spared tissue . axial t1 ( a ) and contrast - enhanced axial t1 fs ( b ) images show islands of spared tissue ( arrowhead ) that enhance within the treated area another pitfall to be avoided is focal inflammatory enhancements in the treated lobe presenting a nodular feature ( fig . day-7 post - treatment dynamic contrast - enhanced fat - saturated axial t1-weighted mr image showing early nodular enhancement in the prostate apex suggestive of residual tumour away from the treated area . the nodule was no longer visible on the 3-month post - treatment and 6-month post - treatment mris , and the 6-month post - treatment biopsy was negative suspected residual tumour . day-7 post - treatment dynamic contrast - enhanced fat - saturated axial t1-weighted mr image showing early nodular enhancement in the prostate apex suggestive of residual tumour away from the treated area . the nodule was no longer visible on the 3-month post - treatment and 6-month post - treatment mris , and the 6-month post - treatment biopsy was negative in cases with concomitant bilateral treatment , the 1-week mri shows the same types of modifications but they are symmetrical . normally , the intraprostatic urethra exhibits a continuous ring of peripheral enhancement . a discontinuous ring ( fig . 10 ) on the 1-week post - photodynamic therapy mri is thus suggestive of urethral wall necrosis . in this situation , despite earlier animal studies reporting structural and functional resistance of the urethra to photodynamic therapy , close clinical monitoring is essential.fig . day-7 post - treatment axial t1 fs contrast - enhanced mr images for a bilaterally treated patient show necrosis in both lobes . the intraprostatic urethra presents a continuous enhancement ring ( a ) . a rupture of this ring enhancement ( arrowhead ) after photodynamic therapy no urinary complications were identified in this patient during close clinical follow - up periurethral enhancement ring . day-7 post - treatment axial t1 fs contrast - enhanced mr images for a bilaterally treated patient show necrosis in both lobes . the intraprostatic urethra presents a continuous enhancement ring ( a ) . a rupture of this ring enhancement ( arrowhead ) after photodynamic therapy no urinary complications were identified in this patient during close clinical follow - up finally , it is important to note that on the 1-week post - therapy images the surroundings of the gland may also show enhancement due to local inflammation . at 6 months after photodynamic therapy , important changes of the prostate shape and signal are found . small areas of residual necrosis may still be present in the treated lobe , corresponding to coagulation necrosis ( fig . six - month post - treatment mri in a patient treated by left photodynamic therapy . axial t1 fs contrast - enhanced ( a ) and axial t2-weighted images ( b ) show persistence of a low signal intensity area with no enhancement ( arrowhead ) corresponding to a small area of residual necrosis ( coagulation necrosis ) . pathology slides from biopsy at low ( c ) and high magnification ( d ) show loose connective tissue coreless , with cell ghosts , characteristic of coagulation necrosis residual necrosis . six - month post - treatment mri in a patient treated by left photodynamic therapy . axial t1 fs contrast - enhanced ( a ) and axial t2-weighted images ( b ) show persistence of a low signal intensity area with no enhancement ( arrowhead ) corresponding to a small area of residual necrosis ( coagulation necrosis ) . pathology slides from biopsy at low ( c ) and high magnification ( d ) show loose connective tissue coreless , with cell ghosts , characteristic of coagulation necrosis classically , the volume of the treated lobe decreases , sometimes to less than the volume of the lobe before treatment . axial t2-weighted mr ( a ) and axial t1 fs contrast - enhanced ( b ) images , in a case of bilateral photodynamic therapy show a post - treatment pattern , with a small area of low signal intensity , without enhancement , corresponding to fibrous scar tissue ( arrowhead ) . low power ( c , d ) images from radical prostatectomy show a focus of sclerotic and hyaline necrosis on the left lobe suggesting a 7 5-mm therapy scar fibrous scar . axial t2-weighted mr ( a ) and axial t1 fs contrast - enhanced ( b ) images , in a case of bilateral photodynamic therapy show a post - treatment pattern , with a small area of low signal intensity , without enhancement , corresponding to fibrous scar tissue ( arrowhead ) . low power ( c , d ) images from radical prostatectomy show a focus of sclerotic and hyaline necrosis on the left lobe suggesting a 7 5-mm therapy scar residual , irregularly shaped fluid cavities related to atrophy in the treated lobe are frequently seen ( fig . the normal hypersignal of the peripheral prostate is replaced by the irregular hyposignal of the scar.fig . six - month post - treatment follow - up mri in a patient with left photodynamic therapy . axial t2-weighted ( a ) and t1-weighted ( b ) mr images show an irregularly shaped residual fluid cavity in the treated lobe , with a low signal on t1 ( arrowhead ) and a high signal on t2 residual fluid cavity . six - month post - treatment follow - up mri in a patient with left photodynamic therapy . axial t2-weighted ( a ) and t1-weighted ( b ) mr images show an irregularly shaped residual fluid cavity in the treated lobe , with a low signal on t1 ( arrowhead ) and a high signal on t2 unilateral treatment induces an asymmetric prostate , whereas bilateral treatment results in a small , heterogeneous , but symmetrical prostate ( fig . axial t2-weighted mr images from before treatment ( a ) and 6 months after treatment ( b ) in a 65-year - old patient with left unilateral photodynamic therapy illustrate post - treatment asymmetric prostate due to unilateral scar tissue . however , pre - treatment ( c ) and 6-month post - treatment ( d ) mris in a case of bilateral treatment ( 71-year - old patient ) illustrate a symmetrical aspect . note the disappearance of the peripheral zone of the treated lobes on the 6-month mris asymmetry / symmetry . axial t2-weighted mr images from before treatment ( a ) and 6 months after treatment ( b ) in a 65-year - old patient with left unilateral photodynamic therapy illustrate post - treatment asymmetric prostate due to unilateral scar tissue . however , pre - treatment ( c ) and 6-month post - treatment ( d ) mris in a case of bilateral treatment ( 71-year - old patient ) illustrate a symmetrical aspect . note the disappearance of the peripheral zone of the treated lobes on the 6-month mris furthermore , the aspect observed at 6 months is definitive ( figs . we thus underline that the 6-month mri is very important as it will become the new base line appearance for any further follow - up or monitoring.fig . six - month post - treatment axial t2 images in a 66-year - old left photodynamic therapy patient show left lobe atrophy ( particularly for the peripheral prostate ) as well as a small fluid cavity ( arrowhead ) in the middle of the scar . six - month ( a ) and 3-year ( b ) post - treatment mri for right photodynamic therapy . the 6-month and 3 year axial t2-weighted images show the same aspect : atrophy of the right lobe with attraction of the urethra , and the disappearance of normal peripheral prostate hyperintensity . treatment resulted in a lesser modification of the transitional zone compared with the contralateral lobe unchanged scar appearance over time . six - month post - treatment axial t2 images in a 66-year - old left photodynamic therapy patient show left lobe atrophy ( particularly for the peripheral prostate ) as well as a small fluid cavity ( arrowhead ) in the middle of the scar . there was no change in this aspect at 1 year ( b ) unchanged scar appearance over time . six - month ( a ) and 3-year ( b ) post - treatment mri for right photodynamic therapy . the 6-month and 3 year axial t2-weighted images show the same aspect : atrophy of the right lobe with attraction of the urethra , and the disappearance of normal peripheral prostate hyperintensity . treatment resulted in a lesser modification of the transitional zone compared with the contralateral lobe three - month mris are not pertinent for treatment follow - up as they tend to show aspects that are between early necrosis and late fibrosis and thus difficult to analyse ( fig . 17).fig . 17three - month post - treatment mri in a 69-year - old patient with concomitant bilateral treatment for prostate adenocarcinoma , gleason 6 ( psa level of 8.14 ng / ml , 1/12 positive biopsy at right apex ) . axial t1 fs contrast - enhanced image ( a ) shows the persistence of two small areas of residual necrosis ( arrowhead ) three - month post - treatment mri in a 69-year - old patient with concomitant bilateral treatment for prostate adenocarcinoma , gleason 6 ( psa level of 8.14 ng / ml , 1/12 positive biopsy at right apex ) . axial t1 fs contrast - enhanced image ( a ) shows the persistence of two small areas of residual necrosis ( arrowhead ) the indication criteria of retreatment are positive biopsies in treated or contralateral lobe during the follow - up , with gleason score 3 + 3 or 3 + 4 in certain conditions . when ipsilateral retreatment is needed , the modifications seen after 1 week are similar to those seen 1 week after the initial treatment ( fig . 18 ) . after 6 months , the atrophy in the retreated lobe is more marked than after the first treatment.fig . 18retreatment of the same lobe . a 60-year - old patient with retreatment of the right lobe . day-7 post - retreatment axial t2-weighted mr image ( a ) and axial t1 fs contrast - enhanced mr image ( b ) show a slight volume increase of the treated lobe compared with the first procedure ; the area of prostate necrosis presents two different intensity levels : the residual fluid cavity ( arrowhead ) and tissue necrosis ( arrow ) , with less low intensity . note , 6 months post first treatment axial t2-weighted mr image ( c ) corresponding to pre - retreatment aspect retreatment of the same lobe . a 60-year - old patient with retreatment of the right lobe . day-7 post - retreatment axial t2-weighted mr image ( a ) and axial t1 fs contrast - enhanced mr image ( b ) show a slight volume increase of the treated lobe compared with the first procedure ; the area of prostate necrosis presents two different intensity levels : the residual fluid cavity ( arrowhead ) and tissue necrosis ( arrow ) , with less low intensity . note , 6 months post first treatment axial t2-weighted mr image ( c ) corresponding to pre - retreatment aspect when a renewed intervention is for the contralateral lobe ( fig . 19 ) the asymmetric aspect is more pronounced at 1 week because of the atrophy of the previously treated lobe , but at 6 months , this asymmetry disappears and the prostate takes on the same appearance as a concomitant bilateral treatment.fig . a 56-year - old patient , with initial right photodynamic therapy , then left therapy in a second intervention . pre - left therapy ( a ) and day-7 post - left therapy ( b ) axial t2-weighted images ; day-7 post - left therapy ( c ) axial t1 fs contrast - enhanced mr image . on the left lobe , a heterogeneous t2 area with low signal in t1-weighted image , unenhanced , corresponding to the recent tissue necrosis ( star ) . in the right lobe a 56-year - old patient , with initial right photodynamic therapy , then left therapy in a second intervention . pre - left therapy ( a ) and day-7 post - left therapy ( b ) axial t2-weighted images ; day-7 post - left therapy ( c ) axial t1 fs contrast - enhanced mr image . on the left lobe , a heterogeneous t2 area with low signal in t1-weighted image , unenhanced , corresponding to the recent tissue necrosis ( star ) . in the right lobe photodynamic therapy appears to have a reasonably low rate of complications . in a study on different types of focal therapies performed in a cohort of men with low - risk prostate cancer , the overall complication rate was 13 % , with only two clavien - dindo grade 3 complications . ( clavien dindo is a grading system from 1 to 5 , used for the classification of surgical complications ; grade 3 or more are serious complications : grade 3complication which requires surgical , endoscopic or radiological intervention up to grade 5 , which corresponds to the death of the patient ) . in that series . found that most treatment - emergent adverse events ( teaes ) post photodynamic therapy were mild or moderate and only 9 % of patients reported serious teaes ( table 1).table 1treatment - emergent adverse events ( teaes ) of photodynamic therapy . study mild teaeserious teaedysuriaprostatitisuti ( urinary tractus infection)haematuriaurinary retentionepididymo - orchitisconstipationcystoprostatitisperineal painischaemic optic neuropathyinflammatory prostatic cyst treatment - emergent adverse events ( teaes ) of photodynamic therapy . study the main post - photodynamic therapy complication is extraprostatic necrosis , defined as a lack of enhancement on t1-weighted contrast - enhanced mr images involving the different structures surrounding the prostate . when present , necrosis most frequently extends to periprostatic fatty tissue ( fig . however , in our experience with this situation , anal functional abnormalities are not observed and a normal aspect is recovered after 6 months ( fig . a 62-year - old patient who had underwent left photodynamic therapy 1 year earlier , and right treatment 7 days earlier . axial t1-weighted ( a ) , axial t1 fs unenhanced ( b ) and contrast - enhanced ( c ) images show a nodular lesion ( arrowhead ) located in the right rectoprostatic angle , with a high intensity t1 fs signal , unenhanced , corresponding to focal haemorrhagic necrosis of periprostatic fatty tissuefig . 21levator ani muscle . right photodynamic therapy in a 65-year - old patient for gleason 6 adenocarcinoma ( two positive biopsies in right prostate ) . day-7 post - treatment axial ( a ) and coronal ( b ) t2-weighted mr images visualise a heterogeneous high signal of the right puborectalis muscle ( arrowheads ) ; axial and sagittal t1 fs contrast - enhanced images ( c , d ) show a low - signal well - defined area surrounded by a hyperintense enhanced border after gadolinium injection ( arrows ) corresponding to extraprostatic necrosis affecting the right levator ani musclefig . day-7 post - treatment axial t1 fs contrast - enhanced ( a ) and axial t2-weighted ( b ) mr images after right photodynamic therapy show extension of necrosis to puborectalis muscle ( star ) ; at the 6-month post - treatment mri follow - up , the axial t2 image ( c ) illustrated the complete disappearance of the area of necrosis ( arrowhead ) . no anal functional abnormalities were seen in this patient periprostatic fatty tissue . a 62-year - old patient who had underwent left photodynamic therapy 1 year earlier , and right treatment 7 days earlier . axial t1-weighted ( a ) , axial t1 fs unenhanced ( b ) and contrast - enhanced ( c ) images show a nodular lesion ( arrowhead ) located in the right rectoprostatic angle , with a high intensity t1 fs signal , unenhanced , corresponding to focal haemorrhagic necrosis of periprostatic fatty tissue levator ani muscle . right photodynamic therapy in a 65-year - old patient for gleason 6 adenocarcinoma ( two positive biopsies in right prostate ) . day-7 post - treatment axial ( a ) and coronal ( b ) t2-weighted mr images visualise a heterogeneous high signal of the right puborectalis muscle ( arrowheads ) ; axial and sagittal t1 fs contrast - enhanced images ( c , d ) show a low - signal well - defined area surrounded by a hyperintense enhanced border after gadolinium injection ( arrows ) corresponding to extraprostatic necrosis affecting the right levator ani muscle levator ani muscle . day-7 post - treatment axial t1 fs contrast - enhanced ( a ) and axial t2-weighted ( b ) mr images after right photodynamic therapy show extension of necrosis to puborectalis muscle ( star ) ; at the 6-month post - treatment mri follow - up , the axial t2 image ( c ) illustrated the complete disappearance of the area of necrosis ( arrowhead ) . no anal functional abnormalities were seen in this patient extraprostatic necrosis may also affect the internal obturator muscle ( fig . 23 ) and rarely it may extend to the anterior rectal wall ( fig . , fistulisation does not seem to occur and the rectal wall recovers its normal aspect on the 6-month follow - up mri.fig . 23internal obturator muscle . left photodynamic therapy in a 66-year - old patient with gleason 6 adenocarcinoma ( pre - treatment / day-7 post - treatment/6-month post - treatment ) . axial ( a ) and coronal ( b ) t2-weighted mr images show high signals of the left internal obturator muscle ( arrowhead ) and the puborectalis muscle ( arrow ) . extraprostatic necrosis extending to muscles is well defined on the t1 fs contrast - enhanced image ( c , flash)fig . day-7 post - treatment ( a ) and 6-month post - treatment ( b ) axial t1 fs contrast - enhanced mr images show the presence of a thin line of necrosis extending to the anterior rectal wall ( arrow ) , limited to the muscularis propria . note the complete recovery of the rectal wall at 6 months . also no fistulae were clinically identified internal obturator muscle . left photodynamic therapy in a 66-year - old patient with gleason 6 adenocarcinoma ( pre - treatment / day-7 post - treatment/6-month post - treatment ) . axial ( a ) and coronal ( b ) t2-weighted mr images show high signals of the left internal obturator muscle ( arrowhead ) and the puborectalis muscle ( arrow ) . extraprostatic necrosis extending to muscles is well defined on the t1 fs contrast - enhanced image ( c , flash ) anterior rectal wall . day-7 post - treatment ( a ) and 6-month post - treatment ( b ) axial t1 fs contrast - enhanced mr images show the presence of a thin line of necrosis extending to the anterior rectal wall ( arrow ) , limited to the muscularis propria . note the complete recovery of the rectal wall at 6 months . also no fistulae were clinically identified sliding of the optical fibre day-7 post - treatment axial ( a ) and sagittal ( b ) t1 fs contrast - enhanced mr images show a small extension of necrosis to the left seminal vesicle ( arrowhead ) by extraprostatic sliding of an optical fibre seminal vesicle day-7 post - treatment axial ( a ) and sagittal ( b ) t1 fs contrast - enhanced mr images show a small extension of necrosis to the left seminal vesicle ( arrowhead ) by extraprostatic sliding of an optical fibre as mentioned earlier , necrosis may affect the intraprostatic urethra ; a break of the periurethral enhancement ring or its complete disappearance at 1 week is an interesting sign suggestive of urethral parietal necrosis ( fig . right photodynamic therapy in a 60-year - old patient with a psa level of 12.9 ng / ml and gleason 6 adenocarcinoma ( positive base biopsies , right middle prostate ) . day-7 post - treatment axial ( a ) and sagittal ( b ) t1 fs contrast - enhanced mr images do not show the normal periurethral ring of enhancement ; the patient developed acute urinary retention treated by urinary catheter urethral necrosis . right photodynamic therapy in a 60-year - old patient with a psa level of 12.9 ng / ml and gleason 6 adenocarcinoma ( positive base biopsies , right middle prostate ) . day-7 post - treatment axial ( a ) and sagittal ( b ) t1 fs contrast - enhanced mr images do not show the normal periurethral ring of enhancement ; the patient developed acute urinary retention treated by urinary catheter mri detection of tumour recurrence in the treated area is made difficult by the loss of the normal hyperintensity of the peripheral prostate in t2-weighted images and the signal of the scar . thus , after photodynamic therapy , the diagnosis of recurrence is mainly based on psa levels and systematic biopsy . there is no reliable feature of recurrence and cases of small unaggressive recurrence are usually not visible on mri ( fig . early dynamic contrast - enhanced images and apparent diffusion coefficient mapping may be useful for detecting recurrence ( fig . therefore , any suspicious nodule seen on follow - up mri should be biopsied ( fig . 29 ) . targeted biopsies with trus are performed in nodules presenting pirads criteria on follow - up mri.fig . six - month post - treatment axial t2-weighted ( a ) and axial dynamic contrast - enhanced ( b ) images show atrophy of the right lobe including the peripheral prostate ; no suspicious nodules are visible . pathology slide at high magnification ( c ) of a right base prostate biopsy , shows a small glandular focus ( circled ) between normal glands , lined by a single layer of cellsfig . six - month post - treatment axial t2 ( a ) image shows a hypointense nodule of the right peripheral prostate and the axial dynamic contrast - enhanced ( b ) image illustrates an early enhancement after gadolinium injection of the nodule suggestive of recurrence ( arrowhead ) . low - power ( c ) and high - power ( d ) pathology slides of a right base prostate biopsy show small irregular glands lined by a single layer of cells , confirming the presence of an infiltrating gleason 7 adenocarcinoma with perineural invasionfig . mri was performed : axial t2 ( a ) , axial adc ( b ) and axial t1 dynamic contrast - enhanced ( c ) mr images show a suspicious left median focus with t2 hyposignal , early enhancement and mild diffusion restriction on adc map ( arrowhead ) . the location of suspicious lesion seen on mri is indicated on a prostate map . pathology slide at high magnification ( d ) shows a 1-mm tumour focus in a scar tissue . crushed appearance of several glands ( circled ) , suspect , confirmed by immunohistochemical study , and surrounded by thick collagen fibres ( fibrous scar ) . the nodule on mri corresponds to the scar tissue ; the tumour focus is too small to be seen on mri small recurrence not seen on mri . six - month post - treatment axial t2-weighted ( a ) and axial dynamic contrast - enhanced ( b ) images show atrophy of the right lobe including the peripheral prostate ; no suspicious nodules are visible . pathology slide at high magnification ( c ) of a right base prostate biopsy , shows a small glandular focus ( circled ) between normal glands , lined by a single layer of cells recurrence seen on mri . six - month post - treatment axial t2 ( a ) image shows a hypointense nodule of the right peripheral prostate and the axial dynamic contrast - enhanced ( b ) image illustrates an early enhancement after gadolinium injection of the nodule suggestive of recurrence ( arrowhead ) . low - power ( c ) and high - power ( d ) pathology slides of a right base prostate biopsy show small irregular glands lined by a single layer of cells , confirming the presence of an infiltrating gleason 7 adenocarcinoma with perineural invasion mismatch . mri was performed : axial t2 ( a ) , axial adc ( b ) and axial t1 dynamic contrast - enhanced ( c ) mr images show a suspicious left median focus with t2 hyposignal , early enhancement and mild diffusion restriction on adc map ( arrowhead ) . the location of suspicious lesion seen on mri is indicated on a prostate map . pathology slide at high magnification ( d ) shows a 1-mm tumour focus in a scar tissue . crushed appearance of several glands ( circled ) , suspect , confirmed by immunohistochemical study , and surrounded by thick collagen fibres ( fibrous scar ) . the nodule on mri corresponds to the scar tissue ; the tumour focus is too small to be seen on mri necrosis is obtained at 1 week and the final post treatment aspect at 6 months . knowledge of these features permits the appreciation of treatment efficacy and the accurate diagnosis of complications or recurrence .

objectivesphotodynamic therapy is a new focal therapy for prostate cancer.methodsin this technique , a photosensitising agent is introduced intravenously , then activated by local laser illumination to induce tumour necrosis . treatment efficacy is assessed by magnetic resonance imaging ( mri).results and conclusionswe illustrate specific post - treatment mri aspects at early and late follow - up with pathological correlations.teaching points dynamic phototherapy is a new and promising focal therapy for prostate cancer. one - week mri shows increased volume of the treated lobe and large , homogeneous necrosis area. six - month mri shows significant changes of the prostate shape and signal. six - month mri becomes base line appearance for further follow - up or monitoring .

Teaching points Introduction Iconography and imaging protocol MRI protocol Pre-treatment MRI Early post-treatment MRI features Late post-treatment MRI features Post-retreatment features Complications Recurrence in the treated area Conclusions

six - month mri becomes base line appearance for further follow - up or monitoring . photodynamic therapy is a recent technology wherein a photosensitising agent ( wst11 ) is administered intravenously , then activated by light to induce tumour necrosis . the light is applied using laser optical fibres positioned within the prostate as determined by magnetic resonance imaging ( mri ) planning . some of the patients also had a post - treatment mri at 3 months . axial and sagittal t2-weighted images ( a , b ) show a hypointense nodule in the right peripheral prostate with restriction on the apparent diffusion coefficient map of the diffusion - weighted image ( c , low signal intensity ) and early enhancement on the axial dynamic contrast - enhanced mr image ( d , arrowhead ) one - week mri examinations show t2 heterogeneous signals in the treated lobes ( fig . b day-7 post - treatment axial t2-weighted image shows a heterogeneous signal of the treated lobe extending to the peripheral and transitional zone ( arrowhead ) . b day-7 post - treatment axial t2-weighted image shows a heterogeneous signal of the treated lobe extending to the peripheral and transitional zone ( arrowhead ) . note that the signal of the right lobe peripheral zone is unchanged compared with the pre - treatment image mri sequences show an increased volume of the treated lobe . axial ( b ) and coronal ( c ) day-7 post - treatment t2-weighted images show an asymmetrical increase of the volume of the treated lobe ( star ) volume increase of the treated lobe . axial ( b ) and coronal ( c ) day-7 post - treatment t2-weighted images show an asymmetrical increase of the volume of the treated lobe ( star ) among all heterogeneities within the treated lobe , besides the loss of physiological distinction between transitional and peripheral prostate tissue in t2 images , the paths of the optical fibres can be observed as small hyperintense spots in the axial plane and linear bands in the sagittal and coronal planes ( fig . the paths of the optical fibres in the treated area are visible on the day-7 post - treatment axial t2-weighted image ( a ) as small hyperintense spots corresponding to thin linear bands ( star ) in the coronal view ( b ) path of optical fibres . the paths of the optical fibres in the treated area are visible on the day-7 post - treatment axial t2-weighted image ( a ) as small hyperintense spots corresponding to thin linear bands ( star ) in the coronal view ( b ) on t1-weighted images before gadolinium injection , hyperintense , poorly defined areas can be identified . a day-7 post - treatment axial t1 mr image in a 64-year - old patient with right lobe photodynamic therapy shows an asymmetric prostate ( star ) with increased volume of the treated lobe but without any spontaneous high signal intensity evoking haemorrhage in the treated area . a day-7 post - treatment axial t1 mr image in a 64-year - old patient with right lobe photodynamic therapy shows an asymmetric prostate ( star ) with increased volume of the treated lobe but without any spontaneous high signal intensity evoking haemorrhage in the treated area . a treated lobe presents a single , large , homogeneous necrosis area on t1 contrast - enhanced sequences . day-7 post - treatment left mri ( b ) shows no enhancement of the left treated lobe ( necrosis ) . right lobe treatment for gleason 6 adenocarcinoma : day-7 post - treatment axial ( c ) and sagittal ( d ) fs contrast - enhanced images show a large homogenous hypointense non - enhanced area in the treated lobe ( necrosis ) with well - defined and highly enhanced boundariesfig . day-7 post - treatment left mri ( b ) shows no enhancement of the left treated lobe ( necrosis ) . day-7 post - treatment dynamic contrast - enhanced fat - saturated axial t1-weighted mr image showing early nodular enhancement in the prostate apex suggestive of residual tumour away from the treated area . day-7 post - treatment dynamic contrast - enhanced fat - saturated axial t1-weighted mr image showing early nodular enhancement in the prostate apex suggestive of residual tumour away from the treated area . at 6 months after photodynamic therapy , important changes of the prostate shape and signal are found . small areas of residual necrosis may still be present in the treated lobe , corresponding to coagulation necrosis ( fig six - month post - treatment mri in a patient treated by left photodynamic therapy . six - month post - treatment mri in a patient treated by left photodynamic therapy . pathology slides from biopsy at low ( c ) and high magnification ( d ) show loose connective tissue coreless , with cell ghosts , characteristic of coagulation necrosis classically , the volume of the treated lobe decreases , sometimes to less than the volume of the lobe before treatment . six - month post - treatment follow - up mri in a patient with left photodynamic therapy . six - month post - treatment follow - up mri in a patient with left photodynamic therapy . we thus underline that the 6-month mri is very important as it will become the new base line appearance for any further follow - up or monitoring.fig . six - month post - treatment axial t2 images in a 66-year - old left photodynamic therapy patient show left lobe atrophy ( particularly for the peripheral prostate ) as well as a small fluid cavity ( arrowhead ) in the middle of the scar . six - month ( a ) and 3-year ( b ) post - treatment mri for right photodynamic therapy . six - month post - treatment axial t2 images in a 66-year - old left photodynamic therapy patient show left lobe atrophy ( particularly for the peripheral prostate ) as well as a small fluid cavity ( arrowhead ) in the middle of the scar . six - month ( a ) and 3-year ( b ) post - treatment mri for right photodynamic therapy . treatment resulted in a lesser modification of the transitional zone compared with the contralateral lobe three - month mris are not pertinent for treatment follow - up as they tend to show aspects that are between early necrosis and late fibrosis and thus difficult to analyse ( fig . 17three - month post - treatment mri in a 69-year - old patient with concomitant bilateral treatment for prostate adenocarcinoma , gleason 6 ( psa level of 8.14 ng / ml , 1/12 positive biopsy at right apex ) . axial t1 fs contrast - enhanced image ( a ) shows the persistence of two small areas of residual necrosis ( arrowhead ) three - month post - treatment mri in a 69-year - old patient with concomitant bilateral treatment for prostate adenocarcinoma , gleason 6 ( psa level of 8.14 ng / ml , 1/12 positive biopsy at right apex ) . day-7 post - retreatment axial t2-weighted mr image ( a ) and axial t1 fs contrast - enhanced mr image ( b ) show a slight volume increase of the treated lobe compared with the first procedure ; the area of prostate necrosis presents two different intensity levels : the residual fluid cavity ( arrowhead ) and tissue necrosis ( arrow ) , with less low intensity . day-7 post - retreatment axial t2-weighted mr image ( a ) and axial t1 fs contrast - enhanced mr image ( b ) show a slight volume increase of the treated lobe compared with the first procedure ; the area of prostate necrosis presents two different intensity levels : the residual fluid cavity ( arrowhead ) and tissue necrosis ( arrow ) , with less low intensity . day-7 post - treatment axial t1 fs contrast - enhanced ( a ) and axial t2-weighted ( b ) mr images after right photodynamic therapy show extension of necrosis to puborectalis muscle ( star ) ; at the 6-month post - treatment mri follow - up , the axial t2 image ( c ) illustrated the complete disappearance of the area of necrosis ( arrowhead ) . day-7 post - treatment axial t1 fs contrast - enhanced ( a ) and axial t2-weighted ( b ) mr images after right photodynamic therapy show extension of necrosis to puborectalis muscle ( star ) ; at the 6-month post - treatment mri follow - up , the axial t2 image ( c ) illustrated the complete disappearance of the area of necrosis ( arrowhead ) . day-7 post - treatment axial ( a ) and sagittal ( b ) t1 fs contrast - enhanced mr images do not show the normal periurethral ring of enhancement ; the patient developed acute urinary retention treated by urinary catheter mri detection of tumour recurrence in the treated area is made difficult by the loss of the normal hyperintensity of the peripheral prostate in t2-weighted images and the signal of the scar . six - month post - treatment axial t2-weighted ( a ) and axial dynamic contrast - enhanced ( b ) images show atrophy of the right lobe including the peripheral prostate ; no suspicious nodules are visible . six - month post - treatment axial t2 ( a ) image shows a hypointense nodule of the right peripheral prostate and the axial dynamic contrast - enhanced ( b ) image illustrates an early enhancement after gadolinium injection of the nodule suggestive of recurrence ( arrowhead ) . six - month post - treatment axial t2-weighted ( a ) and axial dynamic contrast - enhanced ( b ) images show atrophy of the right lobe including the peripheral prostate ; no suspicious nodules are visible . six - month post - treatment axial t2 ( a ) image shows a hypointense nodule of the right peripheral prostate and the axial dynamic contrast - enhanced ( b ) image illustrates an early enhancement after gadolinium injection of the nodule suggestive of recurrence ( arrowhead ) . axial and sagittal t2-weighted images ( a , b ) show a hypointense nodule in the right peripheral prostate with restriction on the apparent diffusion coefficient map of the diffusion - weighted image ( c , low signal intensity ) and early enhancement on the axial dynamic contrast - enhanced mr image ( d , arrowhead ) one - week mri examinations show t2 heterogeneous signals in the treated lobes ( fig . b day-7 post - treatment axial t2-weighted image shows a heterogeneous signal of the treated lobe extending to the peripheral and transitional zone ( arrowhead ) . b day-7 post - treatment axial t2-weighted image shows a heterogeneous signal of the treated lobe extending to the peripheral and transitional zone ( arrowhead ) . note that the signal of the right lobe peripheral zone is unchanged compared with the pre - treatment image mri sequences show an increased volume of the treated lobe . axial ( b ) and coronal ( c ) day-7 post - treatment t2-weighted images show an asymmetrical increase of the volume of the treated lobe ( star ) volume increase of the treated lobe . axial ( b ) and coronal ( c ) day-7 post - treatment t2-weighted images show an asymmetrical increase of the volume of the treated lobe ( star ) among all heterogeneities within the treated lobe , besides the loss of physiological distinction between transitional and peripheral prostate tissue in t2 images , the paths of the optical fibres can be observed as small hyperintense spots in the axial plane and linear bands in the sagittal and coronal planes ( fig . the paths of the optical fibres in the treated area are visible on the day-7 post - treatment axial t2-weighted image ( a ) as small hyperintense spots corresponding to thin linear bands ( star ) in the coronal view ( b ) on t1-weighted images before gadolinium injection , hyperintense , poorly defined areas can be identified . a day-7 post - treatment axial t1 mr image in a 64-year - old patient with right lobe photodynamic therapy shows an asymmetric prostate ( star ) with increased volume of the treated lobe but without any spontaneous high signal intensity evoking haemorrhage in the treated area . a day-7 post - treatment axial t1 mr image in a 64-year - old patient with right lobe photodynamic therapy shows an asymmetric prostate ( star ) with increased volume of the treated lobe but without any spontaneous high signal intensity evoking haemorrhage in the treated area . a treated lobe presents a single , large , homogeneous necrosis area on t1 contrast - enhanced sequences . day-7 post - treatment left mri ( b ) shows no enhancement of the left treated lobe ( necrosis ) . right lobe treatment for gleason 6 adenocarcinoma : day-7 post - treatment axial ( c ) and sagittal ( d ) fs contrast - enhanced images show a large homogenous hypointense non - enhanced area in the treated lobe ( necrosis ) with well - defined and highly enhanced boundariesfig . day-7 post - treatment left mri ( b ) shows no enhancement of the left treated lobe ( necrosis ) . right lobe treatment for gleason 6 adenocarcinoma : day-7 post - treatment axial ( c ) and sagittal ( d ) fs contrast - enhanced images show a large homogenous hypointense non - enhanced area in the treated lobe ( necrosis ) with well - defined and highly enhanced boundaries focal spared tissue . day-7 post - treatment dynamic contrast - enhanced fat - saturated axial t1-weighted mr image showing early nodular enhancement in the prostate apex suggestive of residual tumour away from the treated area . day-7 post - treatment dynamic contrast - enhanced fat - saturated axial t1-weighted mr image showing early nodular enhancement in the prostate apex suggestive of residual tumour away from the treated area . a rupture of this ring enhancement ( arrowhead ) after photodynamic therapy no urinary complications were identified in this patient during close clinical follow - up finally , it is important to note that on the 1-week post - therapy images the surroundings of the gland may also show enhancement due to local inflammation . at 6 months after photodynamic therapy , important changes of the prostate shape and signal are found . six - month post - treatment mri in a patient treated by left photodynamic therapy . six - month post - treatment mri in a patient treated by left photodynamic therapy . pathology slides from biopsy at low ( c ) and high magnification ( d ) show loose connective tissue coreless , with cell ghosts , characteristic of coagulation necrosis classically , the volume of the treated lobe decreases , sometimes to less than the volume of the lobe before treatment . six - month post - treatment follow - up mri in a patient with left photodynamic therapy . six - month post - treatment follow - up mri in a patient with left photodynamic therapy . we thus underline that the 6-month mri is very important as it will become the new base line appearance for any further follow - up or monitoring.fig . six - month post - treatment axial t2 images in a 66-year - old left photodynamic therapy patient show left lobe atrophy ( particularly for the peripheral prostate ) as well as a small fluid cavity ( arrowhead ) in the middle of the scar . six - month ( a ) and 3-year ( b ) post - treatment mri for right photodynamic therapy . six - month post - treatment axial t2 images in a 66-year - old left photodynamic therapy patient show left lobe atrophy ( particularly for the peripheral prostate ) as well as a small fluid cavity ( arrowhead ) in the middle of the scar . six - month ( a ) and 3-year ( b ) post - treatment mri for right photodynamic therapy . treatment resulted in a lesser modification of the transitional zone compared with the contralateral lobe three - month mris are not pertinent for treatment follow - up as they tend to show aspects that are between early necrosis and late fibrosis and thus difficult to analyse ( fig . 17three - month post - treatment mri in a 69-year - old patient with concomitant bilateral treatment for prostate adenocarcinoma , gleason 6 ( psa level of 8.14 ng / ml , 1/12 positive biopsy at right apex ) . axial t1 fs contrast - enhanced image ( a ) shows the persistence of two small areas of residual necrosis ( arrowhead ) three - month post - treatment mri in a 69-year - old patient with concomitant bilateral treatment for prostate adenocarcinoma , gleason 6 ( psa level of 8.14 ng / ml , 1/12 positive biopsy at right apex ) . day-7 post - retreatment axial t2-weighted mr image ( a ) and axial t1 fs contrast - enhanced mr image ( b ) show a slight volume increase of the treated lobe compared with the first procedure ; the area of prostate necrosis presents two different intensity levels : the residual fluid cavity ( arrowhead ) and tissue necrosis ( arrow ) , with less low intensity . day-7 post - retreatment axial t2-weighted mr image ( a ) and axial t1 fs contrast - enhanced mr image ( b ) show a slight volume increase of the treated lobe compared with the first procedure ; the area of prostate necrosis presents two different intensity levels : the residual fluid cavity ( arrowhead ) and tissue necrosis ( arrow ) , with less low intensity . day-7 post - treatment axial t1 fs contrast - enhanced ( a ) and axial t2-weighted ( b ) mr images after right photodynamic therapy show extension of necrosis to puborectalis muscle ( star ) ; at the 6-month post - treatment mri follow - up , the axial t2 image ( c ) illustrated the complete disappearance of the area of necrosis ( arrowhead ) . day-7 post - treatment axial t1 fs contrast - enhanced ( a ) and axial t2-weighted ( b ) mr images after right photodynamic therapy show extension of necrosis to puborectalis muscle ( star ) ; at the 6-month post - treatment mri follow - up , the axial t2 image ( c ) illustrated the complete disappearance of the area of necrosis ( arrowhead ) . six - month post - treatment axial t2-weighted ( a ) and axial dynamic contrast - enhanced ( b ) images show atrophy of the right lobe including the peripheral prostate ; no suspicious nodules are visible . six - month post - treatment axial t2 ( a ) image shows a hypointense nodule of the right peripheral prostate and the axial dynamic contrast - enhanced ( b ) image illustrates an early enhancement after gadolinium injection of the nodule suggestive of recurrence ( arrowhead ) . six - month post - treatment axial t2-weighted ( a ) and axial dynamic contrast - enhanced ( b ) images show atrophy of the right lobe including the peripheral prostate ; no suspicious nodules are visible . six - month post - treatment axial t2 ( a ) image shows a hypointense nodule of the right peripheral prostate and the axial dynamic contrast - enhanced ( b ) image illustrates an early enhancement after gadolinium injection of the nodule suggestive of recurrence ( arrowhead ) .

between march 1998 and may 2012 , a total of 737 patients had surgical interventions for recurrent anterior shoulder instability . of these , 48 patients ( 6.5% ) who met the inclusion criteria underwent the arthroscopic procedure . these patients were randomly divided into two groups : patients who had hill - sachs remplissage in conjunction with an anterior labral and capsule repair ( bankart procedure ) ( group i ) and patients who had an anterior labral and capsule repair only ( group ii ) . the average age at the time of surgery was 29.3 4.12 years ( range , 21 to 39 years ) . all patients had a remarkable history of glenohumeral dislocation , which required reduction at least once in the emergency room , with recurrent shoulder dislocation during sleep at night . the average interval between the onset of shoulder instability and the operation was 79 months ( range , 12 months to 30 years ) . the mean frequency of glenohumeral dislocation was 35 ( range , 10 to 90 ) . preoperative three - dimensional computed tomography ( 3d - ct ) and arthroscopy revealed the presence of humeral head bone defects and recurrent labral tears and preservation of glenoid bone stock of less than 25% in all patients . all 48 patients underwent preoperative 3d - ct scan along with radiography of the affected shoulder to assess the extent of the humeral head bone loss and glenoid bone loss.12 ) complete follow - up was not available for 2 patients in group i and 6 patients in group ii . of the 40 patients , 7 patients ( 17.5% ) there was no statistically significant difference in baseline characteristics between the groups ( table 1 ) . the included patients showed recurrent anterior shoulder instability with a large engaging hill - sachs lesion at any degrees of abduction / external rotation during diagnostic arthroscopy and imaging studies and a minor but substantial glenoid bone loss ( below 25% ) on the en face 3d - ct view ( calanda iii ) . the exclusion criteria included symptomatic recurrent anterior shoulder instability associated with only soft tissue pathologies , major anteroinferior glenoid bone loss of above 25% , primary anterior shoulder instability , multidirectional instability of the shoulder and voluntary posterior shoulder instability , recurrent instability associated with rotator cuff tear , and preexisting severe shoulder arthritis . all patients were informed of the risks and benefits of the surgical procedures before enrollment and signed informed consents . this study was approved by the ulsan university hospital institutional review board ( irb no . the shoulder was examined in abduction and external rotation : dislocation was noted in abduction and external rotation of < 90 , and there was no case of spontaneous reduction . in 30 of posterior tilting of the shoulder , the affected arm was abducted 30 and flexed 15 forward . then , 8 lbs of longitudinal traction and 4 lbs of vertical side - arm traction were applied to the shoulder . huge engaging hill - sachs lesions were identified in group i and ii ( figs . 1 and 2 ) . dynamic arthroscopic examination was performed with the shoulder externally rotated and the arm abducted 45. engagement of the hill - sachs lesion was recognized by this dynamic arthroscopic examination . in group i , the surfaces of hill - sachs lesions were debrided with a motorized - shaver , a burr , and a ring curette , and then the posterior capsule and the inferior capsule were abraded . the number of anchors used for remplissage was 2 ( 2 anchors and 4 sutures were used for the remplissage technique ) . the first bio - cork screw suture anchor ( 5.0 mm ; arthrex , north naples , fl , usa ) was inserted through the posterior portal from the infraspinatus tendon to the posterior articular capsule until it reached the center of a hill - sachs lesion ( fig . the tendon and the posterior articular capsule were penetrated by a 16-gauge needle 1 cm downward and upward from the posterior portal . the surgeon tied the knots at the end of the operation after completing bankart repair ; tying knots earlier would make further procedures more difficult because the posterior portal is closed by the remplissage . the number of anchors was 4 , and the anchor positions were 5 , 4 , 3 , and 2 o'clock for the typical bankart technique . the surgeon used simple stitches with knot - suture - anchors utilizing suture anchor instruments ( 3.5 mm ; arthrex ) . the remplissage technique was completed by making a knot in the subdeltoid space through the posterior portal ( fig . ii , the patient underwent only bankart repair even in the presence of a huge engaging hill - sachs lesion ; however , more capsular plication was done to prevent recurrent dislocation and failure after surgery . the inferior capsule was plicated tightly with 2 or 3 sutures , and the anterior capsule was plicated more tightly with 4 or 5 sutures . the number of anchors was five , and the anchor positions were 6 , 5 , 4 , 3 , and 2 o'clock . capsular shift was greater in patients with bankart repair alone to obtain more stability during shoulder motion . postoperatively , an immobilization abduction brace was applied for about 4 to 6 weeks only allowing gentle daily living activities . patient data were prospectively collected and assessments were performed by two independent clinicians who were blinded to the study at 3 , 6 , 9 , and 12 months after surgery and yearly thereafter . the mean duration of follow - up was 66 months ( range , 24 months to 15 years ) . the shoulder function was evaluated using the american shoulder and elbow surgeons ( ases ) score and rowe6 ) score , and stability was assessed preoperatively and postoperatively using the korean shoulder score for instability ( kssi ) score.7 ) preoperative and postoperative ranges of shoulder motions in active forward elevation and internal and external rotation in abduction were recorded . magnetic resonance imaging ( mri ) was performed to evaluate the operated shoulder at 6 months ( 5.5 to 6.5 months ) postoperatively ( fig . all mri assessments were performed using the same scanner : a 3.0-t closed - type scanner ( achieva 1.5 t a - series mri system ; philips , amsterdam , the netherlands ) and a local small - sized flexible coil . the following sequences were collected : oblique sagittal t1-weighted spin - echo sequence without fat saturation ( repetition time [ tr ] , 419 ms ; echo time [ te ] , 20 ms ; slice thickness , 3 mm ) and fat - saturated oblique coronal t1-weighted spin - echo sequence ( tr , 433 ms ; te , 20 ms ; slice thickness , 3 mm ) , oblique coronal t2-weighted fast spin - echo sequence ( tr , 3,500 ms ; te , 80 ms ; slice thickness , 3 mm ) , and angled axial ( perpendicular to the glenoid joint surface ) t1-weighted spin - echo sequence ( tr , 400 ms ; te , 20 ms ; slice thickness , 3 mm ) . accurate assessment in coronal , parasagittal , and axial images of each plane were performed . the percentage of remplissage defined as the filling of the humeral head defect was classified into four grades8 ) : complete for 100% coverage ; nearly complete for 75% to 99% coverage ; partial coverage for 50% to 74% coverage ; and poor for less than 50% . chi - square test and two - sample t - test were used to compare unpaired results . the included patients showed recurrent anterior shoulder instability with a large engaging hill - sachs lesion at any degrees of abduction / external rotation during diagnostic arthroscopy and imaging studies and a minor but substantial glenoid bone loss ( below 25% ) on the en face 3d - ct view ( calanda iii ) . the exclusion criteria included symptomatic recurrent anterior shoulder instability associated with only soft tissue pathologies , major anteroinferior glenoid bone loss of above 25% , primary anterior shoulder instability , multidirectional instability of the shoulder and voluntary posterior shoulder instability , recurrent instability associated with rotator cuff tear , and preexisting severe shoulder arthritis . all patients were informed of the risks and benefits of the surgical procedures before enrollment and signed informed consents . this study was approved by the ulsan university hospital institutional review board ( irb no . under general anesthesia , the patient 's position was changed from the supine to the lateral decubitus position . the shoulder was examined in abduction and external rotation : dislocation was noted in abduction and external rotation of < 90 , and there was no case of spontaneous reduction . in 30 of posterior tilting of the shoulder then , 8 lbs of longitudinal traction and 4 lbs of vertical side - arm traction were applied to the shoulder . huge engaging hill - sachs lesions were identified in group i and ii ( figs . 1 and 2 ) . dynamic arthroscopic examination was performed with the shoulder externally rotated and the arm abducted 45. engagement of the hill - sachs lesion was recognized by this dynamic arthroscopic examination . in group i , the surfaces of hill - sachs lesions were debrided with a motorized - shaver , a burr , and a ring curette , and then the posterior capsule and the inferior capsule were abraded . the number of anchors used for remplissage was 2 ( 2 anchors and 4 sutures were used for the remplissage technique ) . the first bio - cork screw suture anchor ( 5.0 mm ; arthrex , north naples , fl , usa ) was inserted through the posterior portal from the infraspinatus tendon to the posterior articular capsule until it reached the center of a hill - sachs lesion ( fig . hill - sachs lesions were filled with the posterior capsule and the infraspinatus tendon . the tendon and the posterior articular capsule were penetrated by a 16-gauge needle 1 cm downward and upward from the posterior portal . the surgeon tied the knots at the end of the operation after completing bankart repair ; tying knots earlier would make further procedures more difficult because the posterior portal is closed by the remplissage . the number of anchors was 4 , and the anchor positions were 5 , 4 , 3 , and 2 o'clock for the typical bankart technique . the surgeon used simple stitches with knot - suture - anchors utilizing suture anchor instruments ( 3.5 mm ; arthrex ) . the remplissage technique was completed by making a knot in the subdeltoid space through the posterior portal ( fig . ii , the patient underwent only bankart repair even in the presence of a huge engaging hill - sachs lesion ; however , more capsular plication was done to prevent recurrent dislocation and failure after surgery . the inferior capsule was plicated tightly with 2 or 3 sutures , and the anterior capsule was plicated more tightly with 4 or 5 sutures . the number of anchors was five , and the anchor positions were 6 , 5 , 4 , 3 , and 2 o'clock . capsular shift was greater in patients with bankart repair alone to obtain more stability during shoulder motion . postoperatively , an immobilization abduction brace was applied for about 4 to 6 weeks only allowing gentle daily living activities . patient data were prospectively collected and assessments were performed by two independent clinicians who were blinded to the study at 3 , 6 , 9 , and 12 months after surgery and yearly thereafter . the mean duration of follow - up was 66 months ( range , 24 months to 15 years ) . the shoulder function was evaluated using the american shoulder and elbow surgeons ( ases ) score and rowe6 ) score , and stability was assessed preoperatively and postoperatively using the korean shoulder score for instability ( kssi ) score.7 ) preoperative and postoperative ranges of shoulder motions in active forward elevation and internal and external rotation in abduction were recorded . magnetic resonance imaging ( mri ) was performed to evaluate the operated shoulder at 6 months ( 5.5 to 6.5 months ) postoperatively ( fig . 5 ) . all mri assessments were performed using the same scanner : a 3.0-t closed - type scanner ( achieva 1.5 t a - series mri system ; philips , amsterdam , the netherlands ) and a local small - sized flexible coil . the following sequences were collected : oblique sagittal t1-weighted spin - echo sequence without fat saturation ( repetition time [ tr ] , 419 ms ; echo time [ te ] , 20 ms ; slice thickness , 3 mm ) and fat - saturated oblique coronal t1-weighted spin - echo sequence ( tr , 433 ms ; te , 20 ms ; slice thickness , 3 mm ) , oblique coronal t2-weighted fast spin - echo sequence ( tr , 3,500 ms ; te , 80 ms ; slice thickness , 3 mm ) , and angled axial ( perpendicular to the glenoid joint surface ) t1-weighted spin - echo sequence ( tr , 400 ms ; te , 20 ms ; slice thickness , 3 mm ) . accurate assessment in coronal , parasagittal , and axial images of each plane were performed . the percentage of remplissage defined as the filling of the humeral head defect was classified into four grades8 ) : complete for 100% coverage ; nearly complete for 75% to 99% coverage ; partial coverage for 50% to 74% coverage ; and poor for less than 50% . chi - square test and two - sample t - test were used to compare unpaired results . there were 2 cases of follow - up loss in group i with the remplissage procedure ( group i ) . in the remaining patients of group i , no objective evidence of recurrent dislocation there were 6 cases of follow - up loss in in group ii without the remplissage procedure . there were 5 cases of recurrence in group ii . in the remaining patients of group ii , there was no objective evidence of recurrent dislocation in clinical tests at the last follow - up although severe deficit in external rotation was noted in 1 patient and apprehensive outcome was observed in another patient . the postoperative mean ases score was 87.2 6.57 in group i and 83.5 5.13 in group ii ( p = 0.039 ) . the postoperative mean rowe score was 91.4 3.97 in group i and 84.8 5.83 in group ii ( p = 0.000 ) . the postoperative mean kssi score was 85.8 4.00 in group i and 80.8 4.82 in group ii ( p = 0.000 ) . the postoperative average ases , rowe , and kssi scores and frequency of recurrence were significantly different between the two groups ( table 2 ) . twenty - two of 24 patients ( 91.7% ) in group i , agreed to undergo postoperative mri at 6 months after the operation to evaluate capsulotenodesis healing . mri imaging studies can be useful to assess healing of the capsule and tendon to the bone . there was evidence of healing of the posterior aspect of the capsule and the tendon into the humeral head defect in all 22 patients . of these 22 patients with postoperative mri , 19 ( 86.3% ) had a remplissage of > 75% , 2 had a remplissage of > 50% , and 1 had a remplissage of < 50% . no patient in group i and 5 patients in group ii were disappointed with the outcome of the arthroscopic procedure due to postoperative anterior shoulder dislocation . postoperative shoulder mobility was compared between the groups at 6 and 12 months postoperatively and at the last follow - up . compared with the normal ( contralateral ) side , the mobility of the affected side in group i showed an average loss of 11.3 5.76 in external rotation and 12.1 7.21 in abduction at the last follow - up . none of the patients expressed dissatisfaction with regard to the slight loss in external rotation . in group ii , the affected side exhibited an average loss of 14.4 7.42 in external rotation and 15.2 9.40 in abduction at the last follow - up when compared to the unaffected side . two patients reported dissatisfaction when the arm was externally rotated at maximum 90 abduction and at the side . in group ii , even though only bankart repair was performed in the presence of a huge engaging hill - sachs lesion , more capsular plication was done to prevent recurrent dislocation and postoperative failure . this resulted in the greater loss of abduction and external rotation in the bankart repair only group compared with the remplissage group ( table 3 ) . the frequency of recurrent dislocation was higher in group ii in spite of more capsular plication . therefore , we suggest that the remplissage procedure will be a better option than capsular plication for huge engaging hill - sachs lesions . wound infection or iatrogenic nerve injury were not reported in all patients . recurrence of anterior shoulder instability was noted postoperatively in none of the patients in group i and 5 patients in group ii . the two patients who were lost to the postoperative follow - up in group i were 28 years old and 31 years old , respectively . one had a seizure attack at 14 months after the operation , but the patient did not want further follow - up . another 2 patients had a seizure attack at 6 months and 9 months postoperatively when seizure medications were discontinued . seizure medications were recommenced for these patients and they did not agree to have further follow - up . two patients had a recurrence due to a slip at 6 weeks , and 3 months postoperatively and they did not want a revision operation . the other three patients had a traumatic recurrence of glenohumeral dislocation at 11 , 13 , and 20 postoperative months , respectively , after a fall by landing on the abducted shoulder while playing sports ( 1 during badminton and 2 during a volleyball game ) . the other 2 patients did not want reoperation : this was a single episode and the patient continued to perform activities of daily living until the latest follow - up whereas the other patient had a severe limitation of shoulder motion . there were 2 cases of follow - up loss in group i with the remplissage procedure ( group i ) . in the remaining patients of group i , no objective evidence of recurrent dislocation there were 6 cases of follow - up loss in in group ii without the remplissage procedure . there were 5 cases of recurrence in group ii . in the remaining patients of group ii , there was no objective evidence of recurrent dislocation in clinical tests at the last follow - up although severe deficit in external rotation was noted in 1 patient and apprehensive outcome was observed in another patient . the postoperative mean ases score was 87.2 6.57 in group i and 83.5 5.13 in group ii ( p = 0.039 ) . the postoperative mean rowe score was 91.4 3.97 in group i and 84.8 5.83 in group ii ( p = 0.000 ) . the postoperative mean kssi score was 85.8 4.00 in group i and 80.8 4.82 in group ii ( p = 0.000 ) . the postoperative average ases , rowe , and kssi scores and frequency of recurrence were significantly different between the two groups ( table 2 ) . twenty - two of 24 patients ( 91.7% ) in group i , agreed to undergo postoperative mri at 6 months after the operation to evaluate capsulotenodesis healing . mri imaging studies can be useful to assess healing of the capsule and tendon to the bone . there was evidence of healing of the posterior aspect of the capsule and the tendon into the humeral head defect in all 22 patients . of these 22 patients with postoperative mri , 19 ( 86.3% ) had a remplissage of > 75% , 2 had a remplissage of > 50% , and 1 had a remplissage of < 50% . no patient in group i and 5 patients in group ii were disappointed with the outcome of the arthroscopic procedure due to postoperative anterior shoulder dislocation . postoperative shoulder mobility was compared between the groups at 6 and 12 months postoperatively and at the last follow - up . compared with the normal ( contralateral ) side , the mobility of the affected side in group i showed an average loss of 11.3 5.76 in external rotation and 12.1 7.21 in abduction at the last follow - up . none of the patients expressed dissatisfaction with regard to the slight loss in external rotation . in group ii , the affected side exhibited an average loss of 14.4 7.42 in external rotation and 15.2 9.40 in abduction at the last follow - up when compared to the unaffected side . two patients reported dissatisfaction when the arm was externally rotated at maximum 90 abduction and at the side . in group ii , even though only bankart repair was performed in the presence of a huge engaging hill - sachs lesion , more capsular plication was done to prevent recurrent dislocation and postoperative failure . this resulted in the greater loss of abduction and external rotation in the bankart repair only group compared with the remplissage group ( table 3 ) . the frequency of recurrent dislocation was higher in group ii in spite of more capsular plication . therefore , we suggest that the remplissage procedure will be a better option than capsular plication for huge engaging hill - sachs lesions . recurrence of anterior shoulder instability was noted postoperatively in none of the patients in group i and 5 patients in group ii . the two patients who were lost to the postoperative follow - up in group i were 28 years old and 31 years old , respectively . one had a seizure attack at 14 months after the operation , but the patient did not want further follow - up . another 2 patients had a seizure attack at 6 months and 9 months postoperatively when seizure medications were discontinued . seizure medications were recommenced for these patients and they did not agree to have further follow - up . two patients had a recurrence due to a slip at 6 weeks , and 3 months postoperatively and they did not want a revision operation . the other three patients had a traumatic recurrence of glenohumeral dislocation at 11 , 13 , and 20 postoperative months , respectively , after a fall by landing on the abducted shoulder while playing sports ( 1 during badminton and 2 during a volleyball game ) . the other 2 patients did not want reoperation : this was a single episode and the patient continued to perform activities of daily living until the latest follow - up whereas the other patient had a severe limitation of shoulder motion . our results suggest that the remplissage procedure performed in combination with bankart repair for anterior shoulder instability with a huge engaging hill - sachs lesion can produce excellent functional results with little complications , compared to single bankart repair . an engaging hill - sachs lesion was first described by burkhart and de beer1 ) as a defect in the posterolateral humeral head that was so huge that the edge would drop down over the glenoid rim when the arm was abducted and externally rotated.9 ) boileau et al.8 ) found that the presence of a huge hill - sachs lesion was considerably associated with recurrent instability . even though the exact indications for the treatment of huge humeral head bone loss are as yet ill - defined,9 ) burkhart and de beer1 ) reported that recurrent anterior shoulder dislocation was found in 100% of patients with an engaging hill - sachs lesion , which could be evaluated with the aid of fluoroscopy preoperatively or with aid of arthroscopy intraoperatively . in the current study , we evaluated the engaging huge hill - sachs lesions through physical examination performed under anesthesia and dynamic arthroscopic examination . the management of bone loss was reassessed as part of the anterior shoulder stabilization procedure by bushnell et al.10 ) in 2008 and lynch et al.9 ) in 2009 . various methods including rotational oseotomies1112 ) and filling of the humeral head with allograft,1314 ) soft tissue,35 ) and humeral prostheses have been suggested . weber et al.11 ) designed a humeral rotational osteotomy to increase retroversion of the proximal humerus , which mostly resulted in severe complications according to kronberg and brostrom1215 ) and miniaci and berlet14 ) reported the results of size - matched structural osteochondral allografts , which carried the risk of nonunion , graft resorption , and hardware failure . transhumeral impaction grafting eliminates the potential risks unambiguously associated with the humeral rotational osteotomy and other nonanatomic procedures.9 ) kazel et al.13 ) suggested percutaneous impaction grafting of the humeral head , but the procedure failed to sufficiently reduce the bone defect . chapovsky and kelly17 ) reported an all - arthroscopic procedure to fill the bone loss with an osteoarticular allograft . humeral head resurfacing arthroplasty has also been suggested to manage focal bone loss of the humeral head , and it is considered to be a less prosperous option.18 ) filling the posterolateral humeral head bone loss with transposition of the infraspinatus tendon and posterior capsule is a viable solution for engaging hill - sachs lesions.5 ) remplissage can be performed by open3 ) or arthroscopic45 ) techniques . the procedure described by connolly3 ) is essentially the reverse of the mclaughlin procedure , which was the open transfer of the infraspinatus tendon and posterior capsule into the hill - sachs lesion . purchase et al.5 ) reported a procedure using arthroscopic infraspinatus tenodesis and posterior capsulodesis for huge posterolateral humeral head bone loss . the principles of all arthroscopic remplissage techniques were previously reported by wolf and his associates.45 ) in a study by wolf and pollack,4 ) 22 patients were satisfied with a remplissage procedure and 2 patients had poor outcomes . ko et al.19 ) started to perform a remplissage procedure for recurrent anterior dislocation involving a huge engaging hill - sachs lesion in their patients . in their study , the final decision on the application of the remplissage procedure was made based on intraoperative dynamic arthroscopic evaluation . elkinson et al.20 ) reported that the addition of the remplissage to bankart repair in specimens with a 30% hill - sachs defect prevented engagement of the lesion and enhanced stability in their experimental model . franceschi et al.21 ) described in 2012 that their 25 patients obtained excellent outcomes of arthroscopic remplissage and bankart repair without any redislocation or resubluxation at 2 years postoperatively.22 ) in our study , excellent outcomes were obtained in patients who had huge hill - sachs lesions with medium glenoid bone defects for up to 10 years . on the intergroup comparison , recurrence was not observed in any of the patients in group i , indicating more significant improvement than group ii where five cases of recurrence were noted at the last follow - up . we utilized the rowe6 ) score and the kss score for instability assessment,8 ) which was previously found to be valid , responsive , and reliable to evaluate functional improvement . in group i , the rowe score for stability improved from 43.6 preoperatively to 91.4 at the final follow - up ( p = 0.0021 ) , which was considered good to excellent in 81% of the patients postoperatively . the kss score for instability improved from 46.6 preoperatively to 84.9 at the final follow - up ( p = 0.0015 ) . on the other hand , nourissat et al.23 ) reported that their patients showed no significant difference in different planes of range of motion between groups divided according to the additional application of the remplissage procedure.22 ) patients in the study , however , did not report any posterosuperior pain at the final follow - up . park et al.24 ) described that 3 out of 20 patients reported recurrence of instability . in our study , recurrence of instability was noted in two patients . park et al.25 ) reported that there was evidence of tendon incorporation and filling into the hill - sachs defect following an arthroscopic remplissage , and patients were satisfied with the procedure and demonstrated minimal loss of external rotation ( average , 5.8 ) at an early follow - up . we also observed infraspinatus tendon was incorporated into the hill - sachs defect after the remplissage procedure and there was slight loss of external rotation . boileau et al.8 ) reported that healing of the posterior aspect of the capsule and the infraspinatus tendon into the humeral defect was observed in all patients : 74% of the patients had a remplissage of 75% and 98% of their patients had a stable shoulder at the time of the last follow - up . in our study , except for the 2 patients who did not agree to further follow - up after the remplissage procedure , patients in group i were satisfied or very satisfied with the outcomes . in a study by mccabe et al.,26 ) aggressive capsulolabral reconstruction with a remplissage in traumatic instability patients with moderate bone loss and engaging humeral hill - sachs lesions yielded acceptable outcomes . koo et al.27 ) and haviv et al.18 ) reported that their remplissage procedure was advantageous in that ( 1 ) it was a minimally invasive procedure for changing an intraarticular lesion into an extra - articular one ; ( 2 ) there was no need for any additional graft materials ; ( 3 ) complications associated with an open approach could be avoided ; and ( 4 ) the procedure was simple and easy to perform . patients in group ii underwent bankart repair alone even though there were huge engaging hill - sachs lesions . however , more capsular plication was done to prevent redislocation in these patients , which could be the cause of greater loss of abduction and external rotation compared with the remplissage group . the incidence of redislocation was higher in group ii in spite of the greater capsular plication . therefore , we suggest that the remplissage procedure is a better option than extensive capsular plication in the presence of a huge engaging hill - sachs lesion . in spite of the risk of the transferred infraspinatus and posterior capsules limiting external rotation of the shoulder , it was judged that the benefits of the procedure would outweigh the risk , and none of the patients reported interference with daily living activities . because of the potential abridgement of the arc of motion of the humeral head associated with the remplissage , one could anticipate a greater limitation of motion of the operated shoulder.28 ) however , the extent of restricted motion of the operated shoulder was relatively small in a study of boileau et al.8 ) : an average of 8 in external rotation with the arm at the side and 9 in external rotation with the arm in abduction . furthermore , the study showed a small limitation of below 10 in external rotation did not significantly affect return to overhead activities8 ) : 90% of their patients could return to sports activities involving overhead activities , suggesting that there may be a functional adaptation of the shoulder joint after the remplissage through rebalancing of scapulothoracic and glenohumeral motion.8 ) there were a few limitations to this study . first , the sample size was small although it was determined based on a power analysis . this was because of the relative rarity of huge engaging hill - sachs lesions and the recent introduction of the remplissage technique . second , all procedures were performed by the same surgeon ; therefore , the results of this study may not reflect general surgical outcomes of the procedure . nevertheless , the study included unique postoperative care and follow - up assessments and the same operative indications and consistent surgical technique were applied in all patients . we hope that the results of our study will be confirmed in further studies involving a large cohort of patients . in conclusion , compared to the single arthroscopic bankart repair , the remplissage procedure combined with arthroscopic bankart repair showed superior efficacy for prevention of the recurrence of anterior shoulder instability and had no significant impact on shoulder mobility . therefore , the remplissage procedure performed in combination with arthroscopic bankart repair can be a potential solution for engaging hill - sachs lesions without considerable anteroinferior glenoid bone defects .

backgroundrecurrence of glenohumeral dislocation after arthroscopic bankart repair can be associated with a large osseous defect in the posterosuperior part of the humeral head . our hypothesis is that remplissage is more effective to prevent recurrence of glenohumeral instability without a severe motion deficit.methodsengaging hill - sachs lesions were observed in 48 of 737 patients ( 6.5% ) . twenty - four patients underwent arthroscopic bankart repair combined with remplissage ( group i ) and the other 24 patients underwent arthroscopic bankart repair alone ( group ii ) . clinical outcomes were prospectively evaluated by assessing the range of motion . complications , recurrence rates , and functional results were assessed utilizing the american shoulder and elbow surgeons ( ases ) score , rowe score , and the korean shoulder score for instability ( kssi ) score . capsulotenodesis healing after remplissage was evaluated with magnetic resonance imaging.resultsthe average ases , rowe , and kssi scores were statistically significantly higher in group i than group ii . the frequency of recurrence was statistically significantly higher in group ii . the average loss in external rotation measured with the arm positioned at the side of the trunk was greater in group ii and that in abduction was also higher in group ii.conclusionscompared to single arthroscopic bankart repair , the remplissage procedure combined with arthroscopic bankart repair was more effective to prevent the recurrence of anterior shoulder instability without significant impact on shoulder mobility in patients who had huge hill - sachs lesions .

METHODS Inclusion Criteria Exclusion Criteria Surgical Technique for Remplissage Postoperative Rehabilitation Clinical Follow-ups Postoperative Confirmation of Posterior Capsulotenodesis Healing Statistical Analysis RESULTS Functional and Anatomical Results Subjective Results Postoperative Shoulder Mobility Complications Revisions after the Operations DISCUSSION

between march 1998 and may 2012 , a total of 737 patients had surgical interventions for recurrent anterior shoulder instability . of these , 48 patients ( 6.5% ) who met the inclusion criteria underwent the arthroscopic procedure . these patients were randomly divided into two groups : patients who had hill - sachs remplissage in conjunction with an anterior labral and capsule repair ( bankart procedure ) ( group i ) and patients who had an anterior labral and capsule repair only ( group ii ) . all patients had a remarkable history of glenohumeral dislocation , which required reduction at least once in the emergency room , with recurrent shoulder dislocation during sleep at night . the average interval between the onset of shoulder instability and the operation was 79 months ( range , 12 months to 30 years ) . all 48 patients underwent preoperative 3d - ct scan along with radiography of the affected shoulder to assess the extent of the humeral head bone loss and glenoid bone loss.12 ) complete follow - up was not available for 2 patients in group i and 6 patients in group ii . the included patients showed recurrent anterior shoulder instability with a large engaging hill - sachs lesion at any degrees of abduction / external rotation during diagnostic arthroscopy and imaging studies and a minor but substantial glenoid bone loss ( below 25% ) on the en face 3d - ct view ( calanda iii ) . the exclusion criteria included symptomatic recurrent anterior shoulder instability associated with only soft tissue pathologies , major anteroinferior glenoid bone loss of above 25% , primary anterior shoulder instability , multidirectional instability of the shoulder and voluntary posterior shoulder instability , recurrent instability associated with rotator cuff tear , and preexisting severe shoulder arthritis . the shoulder was examined in abduction and external rotation : dislocation was noted in abduction and external rotation of < 90 , and there was no case of spontaneous reduction . huge engaging hill - sachs lesions were identified in group i and ii ( figs . dynamic arthroscopic examination was performed with the shoulder externally rotated and the arm abducted 45. engagement of the hill - sachs lesion was recognized by this dynamic arthroscopic examination . in group i , the surfaces of hill - sachs lesions were debrided with a motorized - shaver , a burr , and a ring curette , and then the posterior capsule and the inferior capsule were abraded . the surgeon tied the knots at the end of the operation after completing bankart repair ; tying knots earlier would make further procedures more difficult because the posterior portal is closed by the remplissage . ii , the patient underwent only bankart repair even in the presence of a huge engaging hill - sachs lesion ; however , more capsular plication was done to prevent recurrent dislocation and failure after surgery . capsular shift was greater in patients with bankart repair alone to obtain more stability during shoulder motion . the shoulder function was evaluated using the american shoulder and elbow surgeons ( ases ) score and rowe6 ) score , and stability was assessed preoperatively and postoperatively using the korean shoulder score for instability ( kssi ) score.7 ) preoperative and postoperative ranges of shoulder motions in active forward elevation and internal and external rotation in abduction were recorded . the included patients showed recurrent anterior shoulder instability with a large engaging hill - sachs lesion at any degrees of abduction / external rotation during diagnostic arthroscopy and imaging studies and a minor but substantial glenoid bone loss ( below 25% ) on the en face 3d - ct view ( calanda iii ) . the exclusion criteria included symptomatic recurrent anterior shoulder instability associated with only soft tissue pathologies , major anteroinferior glenoid bone loss of above 25% , primary anterior shoulder instability , multidirectional instability of the shoulder and voluntary posterior shoulder instability , recurrent instability associated with rotator cuff tear , and preexisting severe shoulder arthritis . the shoulder was examined in abduction and external rotation : dislocation was noted in abduction and external rotation of < 90 , and there was no case of spontaneous reduction . huge engaging hill - sachs lesions were identified in group i and ii ( figs . dynamic arthroscopic examination was performed with the shoulder externally rotated and the arm abducted 45. engagement of the hill - sachs lesion was recognized by this dynamic arthroscopic examination . in group i , the surfaces of hill - sachs lesions were debrided with a motorized - shaver , a burr , and a ring curette , and then the posterior capsule and the inferior capsule were abraded . hill - sachs lesions were filled with the posterior capsule and the infraspinatus tendon . the surgeon tied the knots at the end of the operation after completing bankart repair ; tying knots earlier would make further procedures more difficult because the posterior portal is closed by the remplissage . ii , the patient underwent only bankart repair even in the presence of a huge engaging hill - sachs lesion ; however , more capsular plication was done to prevent recurrent dislocation and failure after surgery . capsular shift was greater in patients with bankart repair alone to obtain more stability during shoulder motion . the shoulder function was evaluated using the american shoulder and elbow surgeons ( ases ) score and rowe6 ) score , and stability was assessed preoperatively and postoperatively using the korean shoulder score for instability ( kssi ) score.7 ) preoperative and postoperative ranges of shoulder motions in active forward elevation and internal and external rotation in abduction were recorded . the percentage of remplissage defined as the filling of the humeral head defect was classified into four grades8 ) : complete for 100% coverage ; nearly complete for 75% to 99% coverage ; partial coverage for 50% to 74% coverage ; and poor for less than 50% . there were 2 cases of follow - up loss in group i with the remplissage procedure ( group i ) . in the remaining patients of group i , no objective evidence of recurrent dislocation there were 6 cases of follow - up loss in in group ii without the remplissage procedure . in the remaining patients of group ii , there was no objective evidence of recurrent dislocation in clinical tests at the last follow - up although severe deficit in external rotation was noted in 1 patient and apprehensive outcome was observed in another patient . the postoperative mean rowe score was 91.4 3.97 in group i and 84.8 5.83 in group ii ( p = 0.000 ) . the postoperative average ases , rowe , and kssi scores and frequency of recurrence were significantly different between the two groups ( table 2 ) . twenty - two of 24 patients ( 91.7% ) in group i , agreed to undergo postoperative mri at 6 months after the operation to evaluate capsulotenodesis healing . there was evidence of healing of the posterior aspect of the capsule and the tendon into the humeral head defect in all 22 patients . no patient in group i and 5 patients in group ii were disappointed with the outcome of the arthroscopic procedure due to postoperative anterior shoulder dislocation . compared with the normal ( contralateral ) side , the mobility of the affected side in group i showed an average loss of 11.3 5.76 in external rotation and 12.1 7.21 in abduction at the last follow - up . none of the patients expressed dissatisfaction with regard to the slight loss in external rotation . in group ii , the affected side exhibited an average loss of 14.4 7.42 in external rotation and 15.2 9.40 in abduction at the last follow - up when compared to the unaffected side . two patients reported dissatisfaction when the arm was externally rotated at maximum 90 abduction and at the side . in group ii , even though only bankart repair was performed in the presence of a huge engaging hill - sachs lesion , more capsular plication was done to prevent recurrent dislocation and postoperative failure . this resulted in the greater loss of abduction and external rotation in the bankart repair only group compared with the remplissage group ( table 3 ) . the frequency of recurrent dislocation was higher in group ii in spite of more capsular plication . therefore , we suggest that the remplissage procedure will be a better option than capsular plication for huge engaging hill - sachs lesions . recurrence of anterior shoulder instability was noted postoperatively in none of the patients in group i and 5 patients in group ii . the two patients who were lost to the postoperative follow - up in group i were 28 years old and 31 years old , respectively . the other three patients had a traumatic recurrence of glenohumeral dislocation at 11 , 13 , and 20 postoperative months , respectively , after a fall by landing on the abducted shoulder while playing sports ( 1 during badminton and 2 during a volleyball game ) . the other 2 patients did not want reoperation : this was a single episode and the patient continued to perform activities of daily living until the latest follow - up whereas the other patient had a severe limitation of shoulder motion . there were 2 cases of follow - up loss in group i with the remplissage procedure ( group i ) . in the remaining patients of group i , no objective evidence of recurrent dislocation there were 6 cases of follow - up loss in in group ii without the remplissage procedure . in the remaining patients of group ii , there was no objective evidence of recurrent dislocation in clinical tests at the last follow - up although severe deficit in external rotation was noted in 1 patient and apprehensive outcome was observed in another patient . the postoperative mean rowe score was 91.4 3.97 in group i and 84.8 5.83 in group ii ( p = 0.000 ) . the postoperative mean kssi score was 85.8 4.00 in group i and 80.8 4.82 in group ii ( p = 0.000 ) . the postoperative average ases , rowe , and kssi scores and frequency of recurrence were significantly different between the two groups ( table 2 ) . twenty - two of 24 patients ( 91.7% ) in group i , agreed to undergo postoperative mri at 6 months after the operation to evaluate capsulotenodesis healing . there was evidence of healing of the posterior aspect of the capsule and the tendon into the humeral head defect in all 22 patients . no patient in group i and 5 patients in group ii were disappointed with the outcome of the arthroscopic procedure due to postoperative anterior shoulder dislocation . compared with the normal ( contralateral ) side , the mobility of the affected side in group i showed an average loss of 11.3 5.76 in external rotation and 12.1 7.21 in abduction at the last follow - up . none of the patients expressed dissatisfaction with regard to the slight loss in external rotation . in group ii , the affected side exhibited an average loss of 14.4 7.42 in external rotation and 15.2 9.40 in abduction at the last follow - up when compared to the unaffected side . in group ii , even though only bankart repair was performed in the presence of a huge engaging hill - sachs lesion , more capsular plication was done to prevent recurrent dislocation and postoperative failure . this resulted in the greater loss of abduction and external rotation in the bankart repair only group compared with the remplissage group ( table 3 ) . the frequency of recurrent dislocation was higher in group ii in spite of more capsular plication . therefore , we suggest that the remplissage procedure will be a better option than capsular plication for huge engaging hill - sachs lesions . recurrence of anterior shoulder instability was noted postoperatively in none of the patients in group i and 5 patients in group ii . the other three patients had a traumatic recurrence of glenohumeral dislocation at 11 , 13 , and 20 postoperative months , respectively , after a fall by landing on the abducted shoulder while playing sports ( 1 during badminton and 2 during a volleyball game ) . our results suggest that the remplissage procedure performed in combination with bankart repair for anterior shoulder instability with a huge engaging hill - sachs lesion can produce excellent functional results with little complications , compared to single bankart repair . an engaging hill - sachs lesion was first described by burkhart and de beer1 ) as a defect in the posterolateral humeral head that was so huge that the edge would drop down over the glenoid rim when the arm was abducted and externally rotated.9 ) boileau et al.8 ) found that the presence of a huge hill - sachs lesion was considerably associated with recurrent instability . even though the exact indications for the treatment of huge humeral head bone loss are as yet ill - defined,9 ) burkhart and de beer1 ) reported that recurrent anterior shoulder dislocation was found in 100% of patients with an engaging hill - sachs lesion , which could be evaluated with the aid of fluoroscopy preoperatively or with aid of arthroscopy intraoperatively . in the current study , we evaluated the engaging huge hill - sachs lesions through physical examination performed under anesthesia and dynamic arthroscopic examination . the management of bone loss was reassessed as part of the anterior shoulder stabilization procedure by bushnell et al.10 ) in 2008 and lynch et al.9 ) in 2009 . various methods including rotational oseotomies1112 ) and filling of the humeral head with allograft,1314 ) soft tissue,35 ) and humeral prostheses have been suggested . transhumeral impaction grafting eliminates the potential risks unambiguously associated with the humeral rotational osteotomy and other nonanatomic procedures.9 ) kazel et al.13 ) suggested percutaneous impaction grafting of the humeral head , but the procedure failed to sufficiently reduce the bone defect . humeral head resurfacing arthroplasty has also been suggested to manage focal bone loss of the humeral head , and it is considered to be a less prosperous option.18 ) filling the posterolateral humeral head bone loss with transposition of the infraspinatus tendon and posterior capsule is a viable solution for engaging hill - sachs lesions.5 ) remplissage can be performed by open3 ) or arthroscopic45 ) techniques . ko et al.19 ) started to perform a remplissage procedure for recurrent anterior dislocation involving a huge engaging hill - sachs lesion in their patients . in their study , the final decision on the application of the remplissage procedure was made based on intraoperative dynamic arthroscopic evaluation . elkinson et al.20 ) reported that the addition of the remplissage to bankart repair in specimens with a 30% hill - sachs defect prevented engagement of the lesion and enhanced stability in their experimental model . franceschi et al.21 ) described in 2012 that their 25 patients obtained excellent outcomes of arthroscopic remplissage and bankart repair without any redislocation or resubluxation at 2 years postoperatively.22 ) in our study , excellent outcomes were obtained in patients who had huge hill - sachs lesions with medium glenoid bone defects for up to 10 years . on the intergroup comparison , recurrence was not observed in any of the patients in group i , indicating more significant improvement than group ii where five cases of recurrence were noted at the last follow - up . we utilized the rowe6 ) score and the kss score for instability assessment,8 ) which was previously found to be valid , responsive , and reliable to evaluate functional improvement . in group i , the rowe score for stability improved from 43.6 preoperatively to 91.4 at the final follow - up ( p = 0.0021 ) , which was considered good to excellent in 81% of the patients postoperatively . on the other hand , nourissat et al.23 ) reported that their patients showed no significant difference in different planes of range of motion between groups divided according to the additional application of the remplissage procedure.22 ) patients in the study , however , did not report any posterosuperior pain at the final follow - up . park et al.25 ) reported that there was evidence of tendon incorporation and filling into the hill - sachs defect following an arthroscopic remplissage , and patients were satisfied with the procedure and demonstrated minimal loss of external rotation ( average , 5.8 ) at an early follow - up . we also observed infraspinatus tendon was incorporated into the hill - sachs defect after the remplissage procedure and there was slight loss of external rotation . boileau et al.8 ) reported that healing of the posterior aspect of the capsule and the infraspinatus tendon into the humeral defect was observed in all patients : 74% of the patients had a remplissage of 75% and 98% of their patients had a stable shoulder at the time of the last follow - up . in our study , except for the 2 patients who did not agree to further follow - up after the remplissage procedure , patients in group i were satisfied or very satisfied with the outcomes . in a study by mccabe et al.,26 ) aggressive capsulolabral reconstruction with a remplissage in traumatic instability patients with moderate bone loss and engaging humeral hill - sachs lesions yielded acceptable outcomes . patients in group ii underwent bankart repair alone even though there were huge engaging hill - sachs lesions . however , more capsular plication was done to prevent redislocation in these patients , which could be the cause of greater loss of abduction and external rotation compared with the remplissage group . the incidence of redislocation was higher in group ii in spite of the greater capsular plication . therefore , we suggest that the remplissage procedure is a better option than extensive capsular plication in the presence of a huge engaging hill - sachs lesion . because of the potential abridgement of the arc of motion of the humeral head associated with the remplissage , one could anticipate a greater limitation of motion of the operated shoulder.28 ) however , the extent of restricted motion of the operated shoulder was relatively small in a study of boileau et al.8 ) : an average of 8 in external rotation with the arm at the side and 9 in external rotation with the arm in abduction . furthermore , the study showed a small limitation of below 10 in external rotation did not significantly affect return to overhead activities8 ) : 90% of their patients could return to sports activities involving overhead activities , suggesting that there may be a functional adaptation of the shoulder joint after the remplissage through rebalancing of scapulothoracic and glenohumeral motion.8 ) there were a few limitations to this study . this was because of the relative rarity of huge engaging hill - sachs lesions and the recent introduction of the remplissage technique . in conclusion , compared to the single arthroscopic bankart repair , the remplissage procedure combined with arthroscopic bankart repair showed superior efficacy for prevention of the recurrence of anterior shoulder instability and had no significant impact on shoulder mobility . therefore , the remplissage procedure performed in combination with arthroscopic bankart repair can be a potential solution for engaging hill - sachs lesions without considerable anteroinferior glenoid bone defects .

ethical statement : all research was conducted according to the regulations for the care and use of laboratory animals of kitasato university and the national center for global health and medicine . the animal experimentation protocol was approved by the president of kitasato university based on the judgment of the institutional animal care and use committee of kitasato university ( approval i d : no . mouse : crispr / cas9-mediated genome editing in mice was performed as described previously . briefly , sgrna expression vector for the target sequence ( agagacagccattcattcca ) coupled with a t7 promoter was synthesized ( eurofins genomics , brussels , belgium ) , and transcribed in vitro using the megashortscript kit ( thermo fisher scientific , waltham , ma , u.s.a . ) . hcas9 mrna from px330 ( https://www.addgene.org/42230/ ) was synthesized using the mmessage mmachine t7 kit ( thermo fisher scientific ) and was polyadenylated with the poly ( a ) tailing kit ( thermo fisher scientific ) . the purified hcas9 mrnas ( 100 ng/l ) and sgrnas ( 50 ng/l ) were co - injected into the cytoplasm of fertilized eggs derived from bdf1 females ( japan slc , hamamatsu , japan ) . after the injected oocytes were cultured overnight in vitro , two - cell embryos were transferred into pseudo - pregnant female mice . genomic dna was isolated from the offspring from samples taken from the tail , using standard methods . the region around exon 22 of the tns2 locus was amplified by pcr , using two sets of primers : tns2 forward , gcctcagactaaatgttgttccaagt and tns2 reverse , gaaatggcggaccagctgttctga . the resulting founder animals were crossed to fvb / n mice ( clea japan , tokyo , japan ) and then backcrossed to the same for three generations . the tns2 ko heterozygote mice were bred to tns2 ko heterozygote and tns2 ( fvb - tns2 ) mice to produce compound heterozygotes ( tns2/tns2 ) and homozygous knockouts ( tns2/tns2 ) , respectively . the nph genotype derived from the original icgn mice was determined as described previously . measurement of urinary albumin excretion : urine samples were collected by gentle manual compression of the abdomen . a 10- l aliquot [ containing 2% sds , 5% -mercaptoethanol , 10% glycerol , 60 mm tris - hcl ( ph 6.8 ) , bromophenol blue and 5 l of urine ] was heated for 5 min at 95c and subjected to 10% sds - polyacrylamide gel electrophoresis . as a positive control , bovine serum albumin ( bsa ) was loaded simultaneously . the gel was fixed and stained with coomassie brilliant blue ( cbb ; wako , osaka , japan ) according to the manufacturer s instructions . cbb - stained urinary albumin was quantified using the image analysis program imagej ( http://rsb.info.nih.gov/ij/ ) . histology : ten - week - old mice were sacrificed by an overdose of isoflurane , and whole kidneys were dissected out . four - micrometer - thick cryostat sections were cut , transferred to mas - coated slides , air dried and stored at 80c until use . for immunohistochemical analysis , the slides were washed in pbs , fixed with acetone at 4c and incubated for 8 hr at 4c with the primary antibody diluted in 1% bsa in pbs [ rabbit anti - tns2 central portion 1:1,000 ; and rabbit anti - tns2 c - terminal ( sab4200268 , sigma - aldrich , mo , u.s.a . ) 1:1,000 ] . the slides were then washed in pbs and incubated with biotin - conjugated donkey anti - rabbit igg as a secondary antibody ( histofine ; nichirei biosciences , tokyo , japan ) for 30 min at room temperature and treated with horseradish peroxidase - conjugated streptavidin complex ( histofine ; nichirei biosciences ) for 3,3-diaminobenzidine staining . periodic acid - schiff ( pas ) staining and ultrastructural analysis were performed using transmission electron microscopy ( tem ) as described in our previous report . knockdown of tns2 in the podocyte cell line : the conditionally immortalized mouse podocyte cell line mpc5 used in our study was a kind gift from professor peter mundel . briefly , the podocytes were cultured at 33c in rpmi1640 medium ( thermo fisher scientific ) containing 10% fetal bovine serum ( thermo fisher scientific ) and 100 u / ml recombinant mouse interferon- ( sigma - aldrich ) . mpc5 cells were inoculated at a density of 5 10 cells / well in 6-well plates . after 24 hr , cells at 7080% confluence were transfected with stealth select rnai ( ccacucaaagcaacgcaguacucua , uagaguacugcguugcuuugagugg , mss209763 , thermo fisher scientific ) ( nucleotides 321345 of tns2 cdna ; accession no . nm_153533.2 ) or mission sirna universal negative control ( sigma - aldrich ) in serum - free medium using lipofectamine 3000 ( thermo fisher scientific ) according to the manufacturer s instructions . to differentiate mpc5 cells after sirna transfection , the cells were plated on type i collagen dishes and cultured with 1% fbs in a 5% co2 atmosphere at 37c for 3 days . to confirm silencing of tns2 expression , quantitation of tns2 ( nm_153533.2 ) and gapdh ( nm_001289726.1 ) expression total rna was extracted from mpc5 cells using the rneasy mini kit ( qiagen , hilden , germany ) at 72 hr post - transfection . one microgram of total rna was used for cdna synthesis with revertra ace ( toyobo , osaka , japan ) and oligo dt primers . rt - qpcr was performed using the kapa sybr fast qpcr kit ( kapa biosystems , wilmington , ma , u.s.a . ) according to the manufacturer s instructions . the reactions were analyzed using the illumina eco real - time pcr system ( illumina , san diego , ca , u.s.a . ) . primers were designed for tns2 ( aaaggcgacgtcatggtaac and ctccactgaggcttggaaag ) and gapdh ( cgacttcaacagcaactc and gccgtattcattgtcataccag ) . cell adhesion assay : adhesion assays with crystal violet staining were performed according to the method described in a previous report . tns2 knockdown ( kd ) mpc5 cells and control cells were trypsinized and seeded on 6-well plates coated with collagen type iv ( sigma - aldrich , 10 mg / ml ) , laminin ( sigma - aldrich , 10 mg / ml ) , vitronectin ( sigma - aldrich , 10 mg / ml ) or fibronectin ( sigma - aldrich , 10 mg / ml ) at a density of 1.5 10 cells per well . after incubation for 1 hr at 37c , non - adherent cells were removed by gentle washing with pbs , followed by fixation in 100% ethanol . cells were stained in 0.1% crystal violet for 15 min at 25c , washed in water and then counted under a microscope . all experiments were performed in triplicate wells for each condition , and data are expressed as means standard deviation . statistical analyses were performed using student s t - test and dunn s multiple comparison test . transwell migration assays : tns2 kd mpc5 cells and control cells were plated in 8.0-m pore size transwell inserts at a density of 1.5 10 cells per well according to the method described in a previous report . after 24 hr , the cells on the upper side of the insert were removed by scraping , and the cells that had migrated through were fixed on the lower side of the membrane with 100% ethanol , stained with crystal violet and quantified by counting the number of cells in 20 separate fields . all experiments were performed in triplicate wells for each condition , and the data are expressed as means standard deviation . statistical significance was determined using dunn s multiple comparison test , with p - values < 0.05 considered significant . phalloidin staining of tns2 kd cells : tns2 kd mpc5 cells and control cells were seeded onto 24-well collagen i - coated culture glass . to observe actin reorganization , wound gaps were made in the mpc5 monolayers by scratching using a 200-l tip . cells were rinsed twice with pbs , fixed with fresh methanol - free 3.7% pfa for 10 min , permeabilized with 0.1% triton - x for 5 min and incubated with alexa fluor 594 phalloidin ( thermo fisher scientific ) . actin stress fibers were identified and quantified using the evos fl cell imaging system ( thermo fisher scientific ) . statistical significance was determined using dunn s multiple comparison test , and p - values<0.05 were considered significant . generation of tns2 sh2-ptb domain - ko mice : to generate mice carrying mutations that disrupt the tns2 c - terminal sh2-ptb domain , we designed a sgrna targeting exon 22 of tns2 . exon 22 encodes the latter half of sh2 , which binds specically to many intracellular signal - transducing proteins ( fig . 1afig . ( a ) pcr amplicons of the targeted fragment in tensin2 ( tns2 ) in founder mice were sequenced . the target sites of the sgrna are shown in bold font ; the pam sequence is highlighted in grey ; the insertional mutations are underlined and in lower case ; insertions ( + ) or deletions ( ) are shown to the right of each allele . the upper sequence is the amino acid sequence of wt mice , and the lower is the sh2-ptb domain deletion sequence of the m3 , m4 and m5 founders . the frame - shift mutation identified in m3 , m4 and m5 mice is shown in gray font ; it led to changes in the amino acid sequence of the sh2 domain and loss of the entire ptb domain . ) . to verify genetic modication at the target locus , a region of genomic dna including tns2 exon 22 was amplied by pcr and subjected to sequencing analysis . after co - microinjection of sgrna / cas9 mrna into fertilized eggs , five types of progeny were born ( fig . m3 , m4 and m5 mice had 5-bp deletions or a 1-bp insertion close to the pam sequence , which might lead to frame - shift mutations and subsequent protein deletion in both the sh2 and ptb domains ( designated as tns2 ) . all nucleotide changes in these mice were transmitted to the next generation by mating with the fvb strain . ( a ) pcr amplicons of the targeted fragment in tensin2 ( tns2 ) in founder mice were sequenced . the target sites of the sgrna are shown in bold font ; the pam sequence is highlighted in grey ; the insertional mutations are underlined and in lower case ; insertions ( + ) or deletions ( ) are shown to the right of each allele . the upper sequence is the amino acid sequence of wt mice , and the lower is the sh2-ptb domain deletion sequence of the m3 , m4 and m5 founders . the common sequence is underlined . the frame - shift mutation identified in m3 , m4 and m5 mice is shown in gray font ; it led to changes in the amino acid sequence of the sh2 domain and loss of the entire ptb domain . loss of tns2 results in proteinuria and fp effacement of podocytes : because the original icgn mouse is a spontaneous mutant derived from a closed colony of icr mice , there is no control strain . the fvb / n strain has been identified as susceptible to the development of gs and ckd . thus , we previously created fvb.icgn-tns2 ( fvb - tns2 ) congenic mice . next , genetic complementation testing was undertaken using fvb - tns2 and fvb - tns2 . tns2 heterozygous mice ( m3 strain ) were bred to tns2 homozygotes to produce compound heterozygotes ( tns2/tns2 ) in the fvb strain background . to determine whether expression of the tns2 protein remained at a normal level and only the sh2-ptb domain had been deleted , we conducted immunohistochemical analyses on kidney sections of compound heterozygotes ( tns2/tns2 ) compared with fvb - tns2 homozygotes and age - matched wild - type ( wt ) controls ( tns2/tns2 ) . we used two antibodies recognizing the central portion of tns2 and the c - terminal ptb domain , respectively ( fig . 2.immunohistochemical analysis of tns2 expression in the glomeruli of tns2 , tns2 and tns2 mice . the upper domain is tns2 and the lower is tns2 , which lacks the putative sh2-ptb domain . c1 , protein kinase c conserved region 1 domain ; ptpc , protein tyrosine phosphatase catalytic domain ; pten c2 , c2 domain of pten tumor - suppressor protein ; sh2 , src homology 2 domain ; ptb , phospho tyrosine binding . ( b ) immunohistochemical analysis of tns2 expression in the glomeruli of tns2 , tns2 and tns2 mice . 2a ) recognize the central portion of tns2 and the ptb domain , respectively . both abs detected tns2 expression in the glomeruli of tns2 mice , but tns2 protein expression was lost in nph homozygotes ( tns2 ) . in compound heterozygotes ( tns2 ) , tns2 could be detected only by ab1 but not by ab2 , indicating that the sh2-ptb domain had been deleted . scale bars=10 m . ) . the use of the two antibodies revealed that tns2 protein expression was lost in fvb - tns2 mice , which is in agreement with an earlier report . in contrast , tns2 protein expression and localization in glomeruli was normal in the compound heterozygotes . however , tns2 could be detected by ab1 but not by ab2 , indicating that the sh2-ptb domain had been deleted ; deletion of this domain does not affect tns2 protein stability ( fig . immunohistochemical analysis of tns2 expression in the glomeruli of tns2 , tns2 and tns2 mice . the upper domain is tns2 and the lower is tns2 , which lacks the putative sh2-ptb domain . c1 , protein kinase c conserved region 1 domain ; ptpc , protein tyrosine phosphatase catalytic domain ; pten c2 , c2 domain of pten tumor - suppressor protein ; sh2 , src homology 2 domain ; ptb , phospho tyrosine binding . ( b ) immunohistochemical analysis of tns2 expression in the glomeruli of tns2 , tns2 and tns2 mice . 2a ) recognize the central portion of tns2 and the ptb domain , respectively . both abs detected tns2 expression in the glomeruli of tns2 mice , but tns2 protein expression was lost in nph homozygotes ( tns2 ) . in compound heterozygotes ( tns2 ) , tns2 could be detected only by ab1 but not by ab2 , indicating that the sh2-ptb domain had been deleted . proteinuria was detectable at 4 weeks of age using sds - page , and subsequent cbb staining demonstrated remarkable proteinuria ( fig . 3bfig . ( a ) sds - page analysis of representative individuals of heterozygote tns2 and homozygote tns2 mice . 15 ) mice from 4 weeks of age , but not in heterozygote ( tns2 ) mice . ( b ) cbb - stained urinary albumin was quantified using the image analysis program imagej . tns2 ( 4 weeks of age , n=5 ) , tns2 mice ( 10 weeks of age , n=3 ) and tns2 ( 10 weeks of age , n=3 ) mice were used . ) . in contrast , no urinary albumin excretion was detectable in fvb - tns2 mice ( fig . histological analysis with pas staining revealed that almost all glomeruli in the compound heterozygote mice showed entire expansion of the mesangial matrix at 12 weeks of age ( fig . representative light microscopy images with pas staining ( upper panel ) of the kidney from tns2 mice ( left ) and tns2 ( right ) mice at 12 weeks of age . tem analysis ( lower panel ) of tns2 mice ( left ) and tns2 ( right ) mice at 12 weeks of age . ultrastructural analysis revealed fused podocyte fps , loss of slit diaphragms and gbm thickening in all glomeruli in the compound heterozygotes at 12 weeks of age ( fig . in addition , homozygous tns2/tns2 mice displayed massive proteinuria similar to that seen in icgn mice ( fig . compound heterozygotes derived from m1 and m2 founders did not show any abnormalities ( data not shown ) . thus , the tns2 mutation was confirmed to be responsible for gs in icgn mice . further , this result suggests that the sh2-ptb domain of tns2 is required for podocyte integrity . in addition , tns2 homozygote mice did not show any of these other phenotypes , indicating a selective role for tns2 in kidney function . ( a ) sds - page analysis of representative individuals of heterozygote tns2 and homozygote tns2 mice . 15 ) mice from 4 weeks of age , but not in heterozygote ( tns2 ) mice . ( b ) cbb - stained urinary albumin was quantified using the image analysis program imagej . tns2 ( 4 weeks of age , n=5 ) , tns2 mice ( 10 weeks of age , n=3 ) and tns2 ( 10 weeks of age , n=3 ) mice were used . representative light microscopy images with pas staining ( upper panel ) of the kidney from tns2 mice ( left ) and tns2 ( right ) mice at 12 weeks of age . tem analysis ( lower panel ) of tns2 mice ( left ) and tns2 ( right ) mice at 12 weeks of age . scale bar=1 m . loss of podocyte tns2 results in increases in the formation of actin stress fibers and cell migration : cell - cell contact and adherence of podocytes to the extracellular matrix of the gbm are crucial for podocyte function . it is well known that cell - to - cell and cell - to - extracellular matrix ( ecm ) adhesions affect morphological changes involved in cell migration . integrins , a large family of cell adhesion proteins , mediate the adhesion of cells to the ecm and provide traction for cell motility . many proteins present on the cytoplasmic side of focal adhesions , including those in the tensin family , are considered to link transmembrane receptors to the actin cytoskeleton . the actin cytoskeleton is an essential structural and functional element that controls cell shape , cell motility and adhesion . when the extracellular environment is altered , these structures are disassembled and remodeled to meet the new requirements . to examine whether tns2 is involved in adhesion and migration in podocytes , mpc5 cells , a conditionally immortalized podocyte cell line , were transfected with sirnas for the coding sequence of tns2 , and two assays were used to determine the effects of tns2 kd on adhesion and migration . adhesion assays were performed to investigate the effect of tns2 kd on podocyte anchorage to the ecm . reduced levels of tns2 protein ideally should be verified by quantitative western blotting . however , because good tns2 antibodies are not available , we used rt - qpcr to detect knockdown of tns2 mrna . in kd cells , tns2 transcripts were significantly decreased compared to in control cells ( fig . 5afig . 5.decreased tns2 mrna expression results in increased formation of actin stress fibers . ( a ) real - time pcr analysis of tns2 mrna . three days after sirna transfection , tns2 mrna expression significantly decreased in tns2 kd cells ( n=3 ) . cells were seeded on culture dishes coated with collagen type iv ( c ) , fibronectin ( f ) , laminin ( l ) or vitronectin ( v ) . after incubation for 1 hr , non - adherent cells were removed by gentle washing with pbs and then counted ( n=3 ) . the degree of actin stress fiber formation was classified into three categories ( high to low : a , b and c ) , depending on the thickness and length of the actin stress fibers in the cytoplasm . in tns2 kd podocytes , cells in the stress fiber - rich category , with thick cables , ( type a ) increased , whereas cells lacking stress fibers , without thick cables , ( type c ) decreased , suggesting that tns2 suppression significantly enhanced actin stress fiber formation ( fig . 5c and 5d ) . tns2-kd podocytes were compared with control cells in 5 separate fields ( n=5 ) . ) . we then examined the influence on adhesion to several types of ecm ( collagen type iv , fibronectin , laminin and vitronectin ) . figure 5b demonstrates that attachment of tns2-kd podocytes did not differ from that of the negative control for any ecm . thus , tns2 suppression does not signicantly affect the adherence of podocytes under these conditions . the tns paralog , tns1 , has also been demonstrated to interact with and regulate the actin cytoskeleton or integrin . three days after sirna transfection , tns2 mrna expression significantly decreased in tns2 kd cells ( n=3 ) . cells were seeded on culture dishes coated with collagen type iv ( c ) , fibronectin ( f ) , laminin ( l ) or vitronectin ( v ) . after incubation for 1 hr , non - adherent cells were removed by gentle washing with pbs and then counted ( n=3 ) . the degree of actin stress fiber formation was classified into three categories ( high to low : a , b and c ) , depending on the thickness and length of the actin stress fibers in the cytoplasm . in tns2 kd podocytes , cells in the stress fiber - rich category , with thick cables , ( type a ) increased , whereas cells lacking stress fibers , without thick cables , ( type c ) decreased , suggesting that tns2 suppression significantly enhanced actin stress fiber formation ( fig . 5c and 5d ) . tns2-kd podocytes were compared with control cells in 5 separate fields ( n=5 ) . to observe actin reorganization , we created wound gaps in cell monolayers by scratching a straight line with a 200-l tip . tns2 kd produced a tendency toward increased migration in the scratch assay ( data not shown ) . we monitored the actin architecture in tns2 kd podocytes migrating towards the center of the gap by staining with conjugated phalloidin - alexa fluor 594 ( fig . the degree of actin stress fiber formation was classified into three categories ( high to low : a , b and c ) , depending on the thickness and length of the actin stress fibers in the cytoplasm . in tns2-kd podocytes , cells in category a ( rich in stress fibers , with thick cables ) increased , but the number of those in category b was similar to control cells . in contrast , cells in category c ( with few stress fibers ) decreased , suggesting that tns2 suppression significantly enhanced actin stress fiber formation ( fig . 5c and 5d ) . next , to quantify cell migration accurately , we performed a transwell migration assay , which is widely used for studying the motility of different types of cells . cells that migrated across the transwell membrane were quantified by fixing and counting . in general , migrating cells have thicker stress fibers than non - motile cells . as expected , tns2 kd in the podocyte cell line significantly enhanced cell migration ( fig . after incubation for 24 hr , migrated cells were fixed and stained with crystal violet and then quantified by counting the number of cells in each well ( n=3 ) . the number of migrated cells was counted in each group . in tns2-kd podocytes , the number of migrating cells significantly increased . ) . decreased tns2 mrna expression results in increased cell migration . after incubation for 24 hr , migrated cells were fixed and stained with crystal violet and then quantified by counting the number of cells in each well ( n=3 ) . the number of migrated cells was counted in each group . in tns2-kd podocytes , in this study , to test whether tns2 might cause the mutant phenotype , we crossed nph / nph mice with mice that carried a sh2-ptb domain deletion . compound heterozygotes that inherited both tns2 and tns2 displayed marked gs and proteinuria , indicating that these two mutations are allelic , and confirming that tns2 deficiency is responsible for the gs phenotype . further , kd of tns2 expression in the podocyte cell line increased both actin stress fiber formation and migration speed . there are several basic types of proteinuria , including glomerular , tubular , overflow and exercise - induced proteinuria . the currently available evidence suggests that podocytes act as the main component of this barrier , as mutations in a number of podocyte - specific genes have been identified to be responsible for gs [ 23 , 36 ] . thus , podocyte dysfunction is a common determining factor for progression toward many types of kidney diseases . a study of several inherited diseases in humans and of ko mouse models revealed that mutations in several podocyte genes ( actn4 , cd2ap , synpo , myh9 , arhgdia and arhgap24 ) lead to gs [ 1 , 7 , 9 , 15,16,17 ] . these proteins are involved in actin organization in podocytes and the mutations results in fp effacement . thus , it has become ever clearer that the precise organization and regulation of the actin cytoskeleton in podocytes is essential for the maintenance of normal structure and function and the actin cytoskeleton serves as the common final pathway organizing fp effacement , independent of the cause of podocyte damage [ 12 , 22 ] . the function of tns2 can be predicted based on its interactions with proteins of known function . tensins are a family of proteins that are localized to integrin - linked focal adhesions . all isoforms contain a ptb that allows them to interact with the cytoplasmic tail of integrin . the n - terminal region of tns1 interacts with actin at multiple sites , thereby linking the actin cytoskeleton to integrin . both 3 integrin- and 1 integrin - ko mice show podocyte abnormalities and proteinuria similar to that of tns2-deficient mutants . although no tns2 mutation has been found to be associated with human disease , expression of tns2 and tns3 at the mrna and protein levels was found to be largely absent in a panel of diverse human cancer cell lines . the loss of tns3 leads to greater tumor cell motility and consequent metastasis , similar to our in vitro results . thus , it appears that tns2 might anchor integrins to the cytoskeleton or integrins to the ecm , rendering podocytes stable . in contrast , deleted in liver cancer 1 ( dlc1 ) is a recently identified tumor suppressor gene that is frequently underexpressed in hepatocellular carcinomas ( hccs ) . dlc1 encodes a rho gtpase - activating protein domain that exhibits growth - suppressive activity in hcc cell lines . through its rhogap domain , dlc1 inhibits the activity of rhoa gtpase , which regulates the actin cytoskeleton network . it has been reported that the tns2 sh2-ptb domain binds to the dlc1 protein . human dlc1 and tns2 interact and co - localize to punctate structures at focal adhesions , and their interaction is required for tumor suppressive function . in addition , dlc1 kd or ko increases actin stress fiber formation , similar to our result for tns2 kd [ 2 , 37 ] . since dlc1-ko embryos did not survive beyond 10.5 days post coitum , it is unclear whether dlc1 is essential to maintain podocyte viability and function . the mouse podocyte mrna expression database contains mrna expression data from facs - sorted mouse podocytes , as analyzed by rna sequencing . this database shows that both tns2 and dlc1 mrna are highly expressed in podocytes . these results suggest that both proteins may play roles in the regulation of actin reorganization in podocytes . our tns2 mice will contribute to determination of which of the tns2-integrin and the tns2-dlc1 signaling axes is essential for the precise organization and regulation of the actin cytoskeleton in podocytes . in conclusion , we show here that tns2 regulates the podocyte cytoskeleton . further analysis of tns2 should provide a better understanding of the molecular mechanisms of podocyte cytoskeleton regulation . moreover , these studies may lead to the development of podocyte - specific drugs for restoration of the actin cytoskeleton in podocytes .

podocytes are terminally differentiated and highly specialized cells in the glomerulus , and they form a crucial component of the glomerular filtration barrier . the icgn mouse is a model of glomerular dysfunction that shows gross morphological changes in the podocyte foot process , accompanied by proteinuria . previously , we demonstrated that proteinuria in icr - derived glomerulonephritis mouse icgn mice might be caused by a deletion mutation in the tensin2 ( tns2 ) gene ( designated tns2nph ) . to test whether this mutation causes the mutant phenotype , we created knockout ( ko ) mice carrying a tns2 protein deletion in the c - terminal src homology and phosphotyrosine binding ( sh2-ptb ) domains ( designated tns2c ) via crispr / cas9-mediated genome editing . tns2nph / tns2c compound heterozygotes and tns2c / tns2c homozygous ko mice displayed podocyte abnormalities and massive proteinuria similar to icgn mice , indicating that these two mutations are allelic . further , this result suggests that the sh2-ptb domain of tns2 is required for podocyte integrity . tns2 knockdown in a mouse podocyte cell line significantly enhanced actin stress fiber formation and cell migration . thus , this study provides evidence that alteration of actin remodeling resulting from tns2 deficiency causes morphological changes in podocytes and subsequent proteinuria .

MATERIALS AND METHODS RESULTS DISCUSSION

the animal experimentation protocol was approved by the president of kitasato university based on the judgment of the institutional animal care and use committee of kitasato university ( approval i d : no . mouse : crispr / cas9-mediated genome editing in mice was performed as described previously . briefly , sgrna expression vector for the target sequence ( agagacagccattcattcca ) coupled with a t7 promoter was synthesized ( eurofins genomics , brussels , belgium ) , and transcribed in vitro using the megashortscript kit ( thermo fisher scientific , waltham , ma , u.s.a . ) the tns2 ko heterozygote mice were bred to tns2 ko heterozygote and tns2 ( fvb - tns2 ) mice to produce compound heterozygotes ( tns2/tns2 ) and homozygous knockouts ( tns2/tns2 ) , respectively . histology : ten - week - old mice were sacrificed by an overdose of isoflurane , and whole kidneys were dissected out . for immunohistochemical analysis , the slides were washed in pbs , fixed with acetone at 4c and incubated for 8 hr at 4c with the primary antibody diluted in 1% bsa in pbs [ rabbit anti - tns2 central portion 1:1,000 ; and rabbit anti - tns2 c - terminal ( sab4200268 , sigma - aldrich , mo , u.s.a . ) knockdown of tns2 in the podocyte cell line : the conditionally immortalized mouse podocyte cell line mpc5 used in our study was a kind gift from professor peter mundel . after 24 hr , cells at 7080% confluence were transfected with stealth select rnai ( ccacucaaagcaacgcaguacucua , uagaguacugcguugcuuugagugg , mss209763 , thermo fisher scientific ) ( nucleotides 321345 of tns2 cdna ; accession no . cell adhesion assay : adhesion assays with crystal violet staining were performed according to the method described in a previous report . tns2 knockdown ( kd ) mpc5 cells and control cells were trypsinized and seeded on 6-well plates coated with collagen type iv ( sigma - aldrich , 10 mg / ml ) , laminin ( sigma - aldrich , 10 mg / ml ) , vitronectin ( sigma - aldrich , 10 mg / ml ) or fibronectin ( sigma - aldrich , 10 mg / ml ) at a density of 1.5 10 cells per well . after 24 hr , the cells on the upper side of the insert were removed by scraping , and the cells that had migrated through were fixed on the lower side of the membrane with 100% ethanol , stained with crystal violet and quantified by counting the number of cells in 20 separate fields . to observe actin reorganization , wound gaps were made in the mpc5 monolayers by scratching using a 200-l tip . statistical significance was determined using dunn s multiple comparison test , and p - values<0.05 were considered significant . generation of tns2 sh2-ptb domain - ko mice : to generate mice carrying mutations that disrupt the tns2 c - terminal sh2-ptb domain , we designed a sgrna targeting exon 22 of tns2 . ( a ) pcr amplicons of the targeted fragment in tensin2 ( tns2 ) in founder mice were sequenced . the target sites of the sgrna are shown in bold font ; the pam sequence is highlighted in grey ; the insertional mutations are underlined and in lower case ; insertions ( + ) or deletions ( ) are shown to the right of each allele . the upper sequence is the amino acid sequence of wt mice , and the lower is the sh2-ptb domain deletion sequence of the m3 , m4 and m5 founders . the frame - shift mutation identified in m3 , m4 and m5 mice is shown in gray font ; it led to changes in the amino acid sequence of the sh2 domain and loss of the entire ptb domain . ) m3 , m4 and m5 mice had 5-bp deletions or a 1-bp insertion close to the pam sequence , which might lead to frame - shift mutations and subsequent protein deletion in both the sh2 and ptb domains ( designated as tns2 ) . ( a ) pcr amplicons of the targeted fragment in tensin2 ( tns2 ) in founder mice were sequenced . the target sites of the sgrna are shown in bold font ; the pam sequence is highlighted in grey ; the insertional mutations are underlined and in lower case ; insertions ( + ) or deletions ( ) are shown to the right of each allele . the upper sequence is the amino acid sequence of wt mice , and the lower is the sh2-ptb domain deletion sequence of the m3 , m4 and m5 founders . the frame - shift mutation identified in m3 , m4 and m5 mice is shown in gray font ; it led to changes in the amino acid sequence of the sh2 domain and loss of the entire ptb domain . loss of tns2 results in proteinuria and fp effacement of podocytes : because the original icgn mouse is a spontaneous mutant derived from a closed colony of icr mice , there is no control strain . thus , we previously created fvb.icgn-tns2 ( fvb - tns2 ) congenic mice . tns2 heterozygous mice ( m3 strain ) were bred to tns2 homozygotes to produce compound heterozygotes ( tns2/tns2 ) in the fvb strain background . to determine whether expression of the tns2 protein remained at a normal level and only the sh2-ptb domain had been deleted , we conducted immunohistochemical analyses on kidney sections of compound heterozygotes ( tns2/tns2 ) compared with fvb - tns2 homozygotes and age - matched wild - type ( wt ) controls ( tns2/tns2 ) . we used two antibodies recognizing the central portion of tns2 and the c - terminal ptb domain , respectively ( fig . 2.immunohistochemical analysis of tns2 expression in the glomeruli of tns2 , tns2 and tns2 mice . the upper domain is tns2 and the lower is tns2 , which lacks the putative sh2-ptb domain . c1 , protein kinase c conserved region 1 domain ; ptpc , protein tyrosine phosphatase catalytic domain ; pten c2 , c2 domain of pten tumor - suppressor protein ; sh2 , src homology 2 domain ; ptb , phospho tyrosine binding . ( b ) immunohistochemical analysis of tns2 expression in the glomeruli of tns2 , tns2 and tns2 mice . 2a ) recognize the central portion of tns2 and the ptb domain , respectively . both abs detected tns2 expression in the glomeruli of tns2 mice , but tns2 protein expression was lost in nph homozygotes ( tns2 ) . in compound heterozygotes ( tns2 ) , tns2 could be detected only by ab1 but not by ab2 , indicating that the sh2-ptb domain had been deleted . the use of the two antibodies revealed that tns2 protein expression was lost in fvb - tns2 mice , which is in agreement with an earlier report . in contrast , tns2 protein expression and localization in glomeruli was normal in the compound heterozygotes . however , tns2 could be detected by ab1 but not by ab2 , indicating that the sh2-ptb domain had been deleted ; deletion of this domain does not affect tns2 protein stability ( fig . immunohistochemical analysis of tns2 expression in the glomeruli of tns2 , tns2 and tns2 mice . c1 , protein kinase c conserved region 1 domain ; ptpc , protein tyrosine phosphatase catalytic domain ; pten c2 , c2 domain of pten tumor - suppressor protein ; sh2 , src homology 2 domain ; ptb , phospho tyrosine binding . ( b ) immunohistochemical analysis of tns2 expression in the glomeruli of tns2 , tns2 and tns2 mice . both abs detected tns2 expression in the glomeruli of tns2 mice , but tns2 protein expression was lost in nph homozygotes ( tns2 ) . in compound heterozygotes ( tns2 ) , tns2 could be detected only by ab1 but not by ab2 , indicating that the sh2-ptb domain had been deleted . proteinuria was detectable at 4 weeks of age using sds - page , and subsequent cbb staining demonstrated remarkable proteinuria ( fig . 15 ) mice from 4 weeks of age , but not in heterozygote ( tns2 ) mice . histological analysis with pas staining revealed that almost all glomeruli in the compound heterozygote mice showed entire expansion of the mesangial matrix at 12 weeks of age ( fig . representative light microscopy images with pas staining ( upper panel ) of the kidney from tns2 mice ( left ) and tns2 ( right ) mice at 12 weeks of age . tem analysis ( lower panel ) of tns2 mice ( left ) and tns2 ( right ) mice at 12 weeks of age . ultrastructural analysis revealed fused podocyte fps , loss of slit diaphragms and gbm thickening in all glomeruli in the compound heterozygotes at 12 weeks of age ( fig . in addition , homozygous tns2/tns2 mice displayed massive proteinuria similar to that seen in icgn mice ( fig . compound heterozygotes derived from m1 and m2 founders did not show any abnormalities ( data not shown ) . thus , the tns2 mutation was confirmed to be responsible for gs in icgn mice . further , this result suggests that the sh2-ptb domain of tns2 is required for podocyte integrity . 15 ) mice from 4 weeks of age , but not in heterozygote ( tns2 ) mice . representative light microscopy images with pas staining ( upper panel ) of the kidney from tns2 mice ( left ) and tns2 ( right ) mice at 12 weeks of age . tem analysis ( lower panel ) of tns2 mice ( left ) and tns2 ( right ) mice at 12 weeks of age . loss of podocyte tns2 results in increases in the formation of actin stress fibers and cell migration : cell - cell contact and adherence of podocytes to the extracellular matrix of the gbm are crucial for podocyte function . it is well known that cell - to - cell and cell - to - extracellular matrix ( ecm ) adhesions affect morphological changes involved in cell migration . to examine whether tns2 is involved in adhesion and migration in podocytes , mpc5 cells , a conditionally immortalized podocyte cell line , were transfected with sirnas for the coding sequence of tns2 , and two assays were used to determine the effects of tns2 kd on adhesion and migration . reduced levels of tns2 protein ideally should be verified by quantitative western blotting . however , because good tns2 antibodies are not available , we used rt - qpcr to detect knockdown of tns2 mrna . 5.decreased tns2 mrna expression results in increased formation of actin stress fibers . the degree of actin stress fiber formation was classified into three categories ( high to low : a , b and c ) , depending on the thickness and length of the actin stress fibers in the cytoplasm . in tns2 kd podocytes , cells in the stress fiber - rich category , with thick cables , ( type a ) increased , whereas cells lacking stress fibers , without thick cables , ( type c ) decreased , suggesting that tns2 suppression significantly enhanced actin stress fiber formation ( fig . the degree of actin stress fiber formation was classified into three categories ( high to low : a , b and c ) , depending on the thickness and length of the actin stress fibers in the cytoplasm . in tns2 kd podocytes , cells in the stress fiber - rich category , with thick cables , ( type a ) increased , whereas cells lacking stress fibers , without thick cables , ( type c ) decreased , suggesting that tns2 suppression significantly enhanced actin stress fiber formation ( fig . to observe actin reorganization , we created wound gaps in cell monolayers by scratching a straight line with a 200-l tip . tns2 kd produced a tendency toward increased migration in the scratch assay ( data not shown ) . we monitored the actin architecture in tns2 kd podocytes migrating towards the center of the gap by staining with conjugated phalloidin - alexa fluor 594 ( fig . the degree of actin stress fiber formation was classified into three categories ( high to low : a , b and c ) , depending on the thickness and length of the actin stress fibers in the cytoplasm . in tns2-kd podocytes , cells in category a ( rich in stress fibers , with thick cables ) increased , but the number of those in category b was similar to control cells . in contrast , cells in category c ( with few stress fibers ) decreased , suggesting that tns2 suppression significantly enhanced actin stress fiber formation ( fig . next , to quantify cell migration accurately , we performed a transwell migration assay , which is widely used for studying the motility of different types of cells . as expected , tns2 kd in the podocyte cell line significantly enhanced cell migration ( fig . after incubation for 24 hr , migrated cells were fixed and stained with crystal violet and then quantified by counting the number of cells in each well ( n=3 ) . in tns2-kd podocytes , in this study , to test whether tns2 might cause the mutant phenotype , we crossed nph / nph mice with mice that carried a sh2-ptb domain deletion . compound heterozygotes that inherited both tns2 and tns2 displayed marked gs and proteinuria , indicating that these two mutations are allelic , and confirming that tns2 deficiency is responsible for the gs phenotype . further , kd of tns2 expression in the podocyte cell line increased both actin stress fiber formation and migration speed . the currently available evidence suggests that podocytes act as the main component of this barrier , as mutations in a number of podocyte - specific genes have been identified to be responsible for gs [ 23 , 36 ] . thus , podocyte dysfunction is a common determining factor for progression toward many types of kidney diseases . these proteins are involved in actin organization in podocytes and the mutations results in fp effacement . thus , it has become ever clearer that the precise organization and regulation of the actin cytoskeleton in podocytes is essential for the maintenance of normal structure and function and the actin cytoskeleton serves as the common final pathway organizing fp effacement , independent of the cause of podocyte damage [ 12 , 22 ] . the n - terminal region of tns1 interacts with actin at multiple sites , thereby linking the actin cytoskeleton to integrin . both 3 integrin- and 1 integrin - ko mice show podocyte abnormalities and proteinuria similar to that of tns2-deficient mutants . although no tns2 mutation has been found to be associated with human disease , expression of tns2 and tns3 at the mrna and protein levels was found to be largely absent in a panel of diverse human cancer cell lines . the loss of tns3 leads to greater tumor cell motility and consequent metastasis , similar to our in vitro results . it has been reported that the tns2 sh2-ptb domain binds to the dlc1 protein . human dlc1 and tns2 interact and co - localize to punctate structures at focal adhesions , and their interaction is required for tumor suppressive function . in addition , dlc1 kd or ko increases actin stress fiber formation , similar to our result for tns2 kd [ 2 , 37 ] . the mouse podocyte mrna expression database contains mrna expression data from facs - sorted mouse podocytes , as analyzed by rna sequencing . these results suggest that both proteins may play roles in the regulation of actin reorganization in podocytes . our tns2 mice will contribute to determination of which of the tns2-integrin and the tns2-dlc1 signaling axes is essential for the precise organization and regulation of the actin cytoskeleton in podocytes . in conclusion , we show here that tns2 regulates the podocyte cytoskeleton . further analysis of tns2 should provide a better understanding of the molecular mechanisms of podocyte cytoskeleton regulation . moreover , these studies may lead to the development of podocyte - specific drugs for restoration of the actin cytoskeleton in podocytes .

heart disease is the leading cause of death for men and women in the united states , and the predominant etiology is coronary artery disease . new york , ny , usa ) is a highly efficacious lipid - lowering agent that acts as a competitive inhibitor of 3-hydroxy-3-methylglutaryl - coa - reductase.1 primary and secondary prevention studies have shown that the use of 3-hydroxy-3-methylglutaryl - coa - reductase inhibitors ( statins ) in patients with coronary artery disease reduces recurrent events and mortality.2,3 atorvastatin is one of the most studied drugs with regards to clinical data , having been shown to reduce acute coronary heart disease events , coronary revascularization , and rate of stroke . in addition , its efficacy has been confirmed in a variety of populations.68 however , the beneficial effects described above may only be realized if patients take their medicines as prescribed by their health care practitioner ( ie , adhere to treatment ) ; unfortunately , adherence to these medicines is modest at best.9,10 several studies have shown that adherence to statins decreases to 50% by 12 months after the initial prescription,11,12 although some evidence suggests that adherence to atorvastatin may be higher than it is for other statins.13 for medications across a wide range of disease areas 15%20% of new prescriptions are never filled 20%35% of patients who do fill the first prescription fail to fill a second and adherence continues to decline rapidly throughout the first 36 months of therapy14,15 more specifically , statin persistence in older patients 65 years of age , declines over time , with the greatest drop occurring in the first 6 months of treatment.16 multiple barriers to taking medicines have been described by the world health organization and others , the most common being forgetfulness . others include lack of knowledge , fear of adverse events , psychological factors , and cost . intervention strategies that integrate counseling , emotional support , and cost sharing are motivational and have been shown to improve adherence.1723 the objective of this study was to describe and evaluate the effectiveness of an integrated intervention program on patients adherence to statin therapy , using atorvastatin as a specific example . this was a randomized prospective study that involved patients who were prescribed atorvastatin at prairie heart cardiovascular consultants , springfield ill , usa , that performs diagnostic catheterization , angioplasties , implants , and heart surgeries . ten practicing physicians ( all board - certified cardiologists , some of whom are interventionalists , and others noninterventionalists ) participated on the basis of their total clinic volume and clinical research interest . patients who met the study criteria ( described below ) , and were prescribed atorvastatin by a participating physician after study initiation , whether they were considered new or returning practice patients , were eligible to enroll into this trial . eligible patients were randomized using a telephone randomization system to one of two groups : an intervention group and a control group ( with a patient ratio of 3:1 intervention : control ) . all patients randomized to the intervention group were provided adherence counseling from a nurse(via a 510-minute discussion ) , and an adherence tip sheet . patients in the intervention group were also given the opportunity to enroll in the my heartwise program,24 a 12-week guide to managing cholesterol ( included monthly mailing of educational materials ) . the practice physicians also had the discretion to provide eligible patients in the intervention group with a copay relief card ( usable with commercial payers , not medicare ) . the control group received usual care , with no additional adherence counseling or tip sheet . data for this study was sourced from the practice s electronic medical record system and was matched and merged to ims health s ( plymouth meeting , pa , usa ) longitudinal ( lrx ) data . the matching process was based upon patient name , gender , mailing address , and date of birth . this generated a de - identified database that was used for the study s analyses . the ims lrx database covers 65% of all retail prescriptions in the united states and includes mail service and specialty pharmacy provider prescriptions . in this database , the de - identified patient , pharmacy , and prescriber can be tracked , as well as all dispensed prescriptions independent of the method of payment . prescription records are collected directly from pharmacies , which provide encrypted patient identifiers , compliant with health insurance portability and accountability act privacy regulations . the period of analysis for each patient was 180 days from the first fill date of the index atorvastatin prescription ( index date ) to the end of the study . all subjects enrolled met the lipitor physician prescribing information for atorvastatin and were entered into the trial at the physician s discretion . all subjects had to satisfy inclusion criteria to be considered eligible for participation by one of the ten participating physicians of the practice s study team : ( 1 ) be older than 21 years of age and , on the basis of clinical assessment by his or her physician , a candidate for statin therapy ; ( 2 ) have received a first prescription for atorvastatin after study initiation at the practice , including patients who were new to the practice and returning practice patients ( new versus continuing atorvastatin patients were deciphered by requiring claims activity 6 months before and after the index date ) ; and ( 3 ) provide a personally signed and dated informed consent document indicating that the participant ( or a legally acceptable representative ) had been informed of all pertinent aspects of the study . patients were excluded from the study if they were unwilling to participate in the adherence counseling or unwilling to give a written informed consent document . ethics approval for the study was granted by fox commercial institutional review board ( springfield , il , usa ) . a sample size of 500 patients , selected in order to have at least 300 evaluable patients ( 225 intervention patients and 75 control patients ) , was calculated for this study . this sample size provides at least 80% power at a type 1 error rate of 0.05 to detect an effect size ( proportion of days covered [ pdc]intervention pdccontrol)/pdcstandard deviation ) of at least 0.37 . for continuous variables , measures of central tendency ( mean and median ) and measures of dispersion ( standard deviation [ sd ] ) as one of the main objectives of the study was to calculate the adherence to atorvastatin after study enrollment , both the pdc and medication possession ratio ( mpr ) during the 180 days from the patient s index date were used in the analysis . the pdc was calculated as the total number of days with the medication on hand during the 180 days from the index date divided by the total number of days in the study period ( 180 ) . if the patient filled a prescription near the end of the study period that extended beyond the study period , the data for this patient was censored at that point . for example , if a patient filled a 30-day prescription on day 160 , only 20 days supply of that prescription ( ie , up to day 180 ) was used in the numerator to calculate pdc . the mpr was defined as the number of days on medication after study enrollment divided by the number of days between the first fill and the last refill plus the days supply of last refill . the mpr for up to 180 days after the index date was computed : if the last refill in the study period provided supply that lasted beyond 180 days after the index date , the supply of that refill was truncated at day 180 postindex in calculating the mpr . if the patient had evidence of only a single fill , the mpr was set to zero . both pdc and mpr were dichotomized into a categorical adherent / not adherent variable . patients were defined as adherent if their pdc / mpr 80% ; all others were classified as nonadherent . the proportion of patients who met this criterion was reported overall and for the intervention and control groups . exploratory logistic regressions were performed using the adherent or nonadherent categorization as the dependent variable ( adjusted for demographic and baseline characteristics ) , to explore variables that were important in predicting adherence for both intervention and control patients . persistence was measured as the number of days a patient was supplied with medication before experiencing a gap in medication 30 days . persistence was compared between the control and the intervention groups using a time - to - event cox proportional hazards model , adjusted for the same demographic and baseline characteristics used in the logistical regression analyses of pdc and mpr . data for this study was sourced from the practice s electronic medical record system and was matched and merged to ims health s ( plymouth meeting , pa , usa ) longitudinal ( lrx ) data . the matching process was based upon patient name , gender , mailing address , and date of birth . this generated a de - identified database that was used for the study s analyses . the ims lrx database covers 65% of all retail prescriptions in the united states and includes mail service and specialty pharmacy provider prescriptions . in this database , the de - identified patient , pharmacy , and prescriber can be tracked , as well as all dispensed prescriptions independent of the method of payment . prescription records are collected directly from pharmacies , which provide encrypted patient identifiers , compliant with health insurance portability and accountability act privacy regulations . the period of analysis for each patient was 180 days from the first fill date of the index atorvastatin prescription ( index date ) to the end of the study . all subjects enrolled met the lipitor physician prescribing information for atorvastatin and were entered into the trial at the physician s discretion . all subjects had to satisfy inclusion criteria to be considered eligible for participation by one of the ten participating physicians of the practice s study team : ( 1 ) be older than 21 years of age and , on the basis of clinical assessment by his or her physician , a candidate for statin therapy ; ( 2 ) have received a first prescription for atorvastatin after study initiation at the practice , including patients who were new to the practice and returning practice patients ( new versus continuing atorvastatin patients were deciphered by requiring claims activity 6 months before and after the index date ) ; and ( 3 ) provide a personally signed and dated informed consent document indicating that the participant ( or a legally acceptable representative ) had been informed of all pertinent aspects of the study . patients were excluded from the study if they were unwilling to participate in the adherence counseling or unwilling to give a written informed consent document . ethics approval for the study was granted by fox commercial institutional review board ( springfield , il , usa ) . a sample size of 500 patients , selected in order to have at least 300 evaluable patients ( 225 intervention patients and 75 control patients ) , was calculated for this study . this sample size provides at least 80% power at a type 1 error rate of 0.05 to detect an effect size ( proportion of days covered [ pdc]intervention pdccontrol)/pdcstandard deviation ) of at least 0.37 . for continuous variables , measures of central tendency ( mean and median ) and measures of dispersion ( standard deviation [ sd ] ) as one of the main objectives of the study was to calculate the adherence to atorvastatin after study enrollment , both the pdc and medication possession ratio ( mpr ) during the 180 days from the patient s index date were used in the analysis . the pdc was calculated as the total number of days with the medication on hand during the 180 days from the index date divided by the total number of days in the study period ( 180 ) . if the patient filled a prescription near the end of the study period that extended beyond the study period , the data for this patient was censored at that point . for example , if a patient filled a 30-day prescription on day 160 , only 20 days supply of that prescription ( ie , up to day 180 ) was used in the numerator to calculate pdc . the mpr was defined as the number of days on medication after study enrollment divided by the number of days between the first fill and the last refill plus the days supply of last refill . the mpr for up to 180 days after the index date was computed : if the last refill in the study period provided supply that lasted beyond 180 days after the index date , the supply of that refill was truncated at day 180 postindex in calculating the mpr . if the patient had evidence of only a single fill , the mpr was set to zero . both pdc and mpr were dichotomized into a categorical adherent / not adherent variable . patients were defined as adherent if their pdc / mpr 80% ; all others were classified as nonadherent . the proportion of patients who met this criterion was reported overall and for the intervention and control groups . exploratory logistic regressions were performed using the adherent or nonadherent categorization as the dependent variable ( adjusted for demographic and baseline characteristics ) , to explore variables that were important in predicting adherence for both intervention and control patients . persistence was measured as the number of days a patient was supplied with medication before experiencing a gap in medication 30 days . persistence was compared between the control and the intervention groups using a time - to - event cox proportional hazards model , adjusted for the same demographic and baseline characteristics used in the logistical regression analyses of pdc and mpr . the study initially included 500 patients ( 125 control patients and 375 patients who received the adherence intervention ) . after matching with the lrx database , 97 controls and however , only 93 controls and 300 intervention patients actually had any lrx claims available for analysis during the study window . of this group , 57 controls and 180 intervention patients had an atorvastatin prescription after enrollment in the study , which served as the index date . after applying the study requirement of claims activity 6 months before and after the index date , only 53 controls ( seven new users [ first atorvastatin prescription after randomization ] and 46 continuing users [ evidence of atorvastatin prescription within 6 months prior to randomization ] ) and 155 intervention patients ( 14 new users and 141 continuing users ) remained eligible for analysis ( figure 1 ) . table 1 provides the descriptive data around the baseline demographic and clinical characteristics for both the control and intervention cohorts . among the control group , the average age was 67.8 years , compared with 69.5 years for the intervention group . most of the control group ( 58.5% ) was 65 years of age or older ; similarly , 65.2% of the intervention group was older than 65 years of age . the sex distribution was predominantly male for both groups ( 67.9% of the controls and 58.7% of the intervention group ) . most patients ( 67.9% of the controls and 70.3% of the intervention group ) were in , the control population was taking an average of 7.8 unique prescriptions , as compared to 7.1 for the intervention group . along the same lines , the control group filled an average of 32.8 prescriptions ( for any medication ) in the 6 months prior to the index date , whereas the intervention group filled an average of 26.2 prescriptions . a prior cv event was identified in 98.1% of the controls and 85.2% of the intervention group in the preindex period . control patients received more antidiabetic ( control 30.2% ; intervention 28.4% ) and antihypertensive ( control 96.2% ; intervention 94.2% ) medications compared to intervention patients . a history of hypertension was similar across the groups , with 79.2% of the controls having the comorbid condition as compared to 81.9% of the intervention group . adherence to index therapy was measured using both pdc and mpr ( table 2 ) , with results stratified as to whether the patient was considered a new user or a continuing user , on the basis of the presence ( or absence ) of atorvastatin prescriptions in the 6-month preindex period . on the basis of pdc ( 0.80 ) , approximately 72% of all patients were considered adherent ( 76.4% based upon mpr ) , with a mean pdc of 0.82 ( 0.81 based upon mpr ) . the mean pdc for the new - user control and intervention groups were similar , averaging 0.70 ( sd , 0.27 ) , whereas for continuing users , the control group exhibited a pdc of 0.83 ( sd , 0.24 ) and the intervention group 0.84 ( sd , 0.22 ) . the mean mpr for new users within the control group was 0.58 ( sd , 0.44 ) and was 0.62 ( sd , 0.42 ) for new users within the intervention group . three of the seven new users in the control group ( 42.9% ) were considered to have good adherence ( pdc and mpr 0.80 ) while five ( 35.7% when examining pdc ) and six ( 42.9% when examining mpr ) new users in the intervention group were considered to have good adherence . continuing users within the control group had a mean mpr of 0.83 ( sd , 0.29 ) as compared with 0.84 ( sd , 0.28 ) for those in the intervention group . in general , the continuing users in both groups were considered to have good adherence , with 76.1% of the control group ( when using pdc , versus 80.4% when using mpr ) and 75.2% of the intervention group ( when using pdc , versus 80.1% when using mpr ) having pdc > 0.80 . figure 2a shows the overall adherence ( pdc ) over time of both the control and intervention groups , up to 12 months postindex . for the control group , the mean pdc decreased from an average of 0.89 ( 3 months postindex ) to 0.73 ( 12 months postindex ) ; this decline was almost exactly matched in the intervention group , going from 0.91 ( 3 months postindex ) to 0.74 ( 12 months postindex ) . not all study patients had 12 months of follow - up for this analysis , and these were censored at the end of follow - up ( the control group had 33 patients , and 101 remained in the intervention group ) . similarly , the percentage of patients who were considered to have good adherence ( pdc 0.80 ) decreased from 84.9% ( 3 months postindex ) to 60.6% ( 12 months postindex ) in the control group and from 82.6% ( 3 months postindex ) to 58.4% ( 12 months postindex ) in the intervention group ( figure 2b ) . after adjusting for baseline demographic and clinical characteristics , there was no significant difference in pdc with index therapy between the two patient cohorts ( odds ratio = 1.03 for intervention versus control ; p = 0.95 ) ( table 3 ) . patients aged 6574 years ( as compared with patients aged 1854 years ) with a prior cv event or history of hypertension were less likely to be adherent to index therapy over 6 months , whereas males , patients aged 5564 years or 75 + years ( as compared with the patients aged 1854 years ) , and patients with a history of diabetes were more likely to be adherent over the 6-month postindex period . the same logistic regression model with mpr as the dependent variable yielded similar results ( odds ratio = 1.14 for intervention versus control ; p < 0.75 ) . patients aged 6574 years ( as compared with patients aged 1854 years ) and with a history of hypertension were less likely to be adherent to index therapy over 6 months . patients who were male , aged 5564 years or 75 + years ( as compared with patients aged 1854 years ) , with a prior cv event , or with a history of diabetes were more likely to be adherent over the 6-month postindex period . table 2 provides the descriptive data for persistence with the index therapy over the 6-month postindex period . index atorvastatin patients who were deemed continuing users had more persistent days over the 6-month postindex period compared with new users . for continuing users , the control group had mean persistent days of 151.6 ( sd , 50.2 ) compared with 150.9 ( sd , 50.9 ) for the intervention group . the new users had fewer persistent days ( control 111.4 days , sd , 69.6 days ; intervention 112.0 days , sd , 58.8 days ) compared with the continuing users . the cox proportional hazards model of the risk of discontinuation with index therapy ( table 3 ) did not show a significant difference between the intervention and control groups after adjusting for baseline demographic and clinical characteristics ( hazard ratio 0.83 , p < 0.55 ) . patients aged 6574 years ( as compared with patients aged 1854 years ) or with a history of hypertension were at greater risk for discontinuing their index therapy , whereas males and patients aged 5564 years or 75 + years ( as compared with patients aged 1854 years ) , with a prior cv event , or with a history of diabetes were at a lower risk of discontinuing index therapy . table 1 provides the descriptive data around the baseline demographic and clinical characteristics for both the control and intervention cohorts . among the control group , the average age was 67.8 years , compared with 69.5 years for the intervention group . most of the control group ( 58.5% ) was 65 years of age or older ; similarly , 65.2% of the intervention group was older than 65 years of age . the sex distribution was predominantly male for both groups ( 67.9% of the controls and 58.7% of the intervention group ) . most patients ( 67.9% of the controls and 70.3% of the intervention group ) were in , the control population was taking an average of 7.8 unique prescriptions , as compared to 7.1 for the intervention group . along the same lines , the control group filled an average of 32.8 prescriptions ( for any medication ) in the 6 months prior to the index date , whereas the intervention group filled an average of 26.2 prescriptions . a prior cv event was identified in 98.1% of the controls and 85.2% of the intervention group in the preindex period . control patients received more antidiabetic ( control 30.2% ; intervention 28.4% ) and antihypertensive ( control 96.2% ; intervention 94.2% ) medications compared to intervention patients . a history of hypertension was similar across the groups , with 79.2% of the controls having the comorbid condition as compared to 81.9% of the intervention group . adherence to index therapy was measured using both pdc and mpr ( table 2 ) , with results stratified as to whether the patient was considered a new user or a continuing user , on the basis of the presence ( or absence ) of atorvastatin prescriptions in the 6-month preindex period . on the basis of pdc ( 0.80 ) , approximately 72% of all patients were considered adherent ( 76.4% based upon mpr ) , with a mean pdc of 0.82 ( 0.81 based upon mpr ) . the mean pdc for the new - user control and intervention groups were similar , averaging 0.70 ( sd , 0.27 ) , whereas for continuing users , the control group exhibited a pdc of 0.83 ( sd , 0.24 ) and the intervention group 0.84 ( sd , 0.22 ) . the mean mpr for new users within the control group was 0.58 ( sd , 0.44 ) and was 0.62 ( sd , 0.42 ) for new users within the intervention group . three of the seven new users in the control group ( 42.9% ) were considered to have good adherence ( pdc and mpr 0.80 ) while five ( 35.7% when examining pdc ) and six ( 42.9% when examining mpr ) new users in the intervention group were considered to have good adherence . continuing users within the control group had a mean mpr of 0.83 ( sd , 0.29 ) as compared with 0.84 ( sd , 0.28 ) for those in the intervention group . in general , the continuing users in both groups were considered to have good adherence , with 76.1% of the control group ( when using pdc , versus 80.4% when using mpr ) and 75.2% of the intervention group ( when using pdc , versus 80.1% when using mpr ) having pdc > 0.80 . figure 2a shows the overall adherence ( pdc ) over time of both the control and intervention groups , up to 12 months postindex . for the control group , the mean pdc decreased from an average of 0.89 ( 3 months postindex ) to 0.73 ( 12 months postindex ) ; this decline was almost exactly matched in the intervention group , going from 0.91 ( 3 months postindex ) to 0.74 ( 12 months postindex ) . not all study patients had 12 months of follow - up for this analysis , and these were censored at the end of follow - up ( the control group had 33 patients , and 101 remained in the intervention group ) . similarly , the percentage of patients who were considered to have good adherence ( pdc 0.80 ) decreased from 84.9% ( 3 months postindex ) to 60.6% ( 12 months postindex ) in the control group and from 82.6% ( 3 months postindex ) to 58.4% ( 12 months postindex ) in the intervention group ( figure 2b ) . after adjusting for baseline demographic and clinical characteristics , there was no significant difference in pdc with index therapy between the two patient cohorts ( odds ratio = 1.03 for intervention versus control ; p = 0.95 ) ( table 3 ) . patients aged 6574 years ( as compared with patients aged 1854 years ) with a prior cv event or history of hypertension were less likely to be adherent to index therapy over 6 months , whereas males , patients aged 5564 years or 75 + years ( as compared with the patients aged 1854 years ) , and patients with a history of diabetes were more likely to be adherent over the 6-month postindex period . the same logistic regression model with mpr as the dependent variable yielded similar results ( odds ratio = 1.14 for intervention versus control ; p < 0.75 ) . patients aged 6574 years ( as compared with patients aged 1854 years ) and with a history of hypertension were less likely to be adherent to index therapy over 6 months . patients who were male , aged 5564 years or 75 + years ( as compared with patients aged 1854 years ) , with a prior cv event , or with a history of diabetes were more likely to be adherent over the 6-month postindex period . table 2 provides the descriptive data for persistence with the index therapy over the 6-month postindex period . index atorvastatin patients who were deemed continuing users had more persistent days over the 6-month postindex period compared with new users . for continuing users , the control group had mean persistent days of 151.6 ( sd , 50.2 ) compared with 150.9 ( sd , 50.9 ) for the intervention group . the new users had fewer persistent days ( control 111.4 days , sd , 69.6 days ; intervention 112.0 days , sd , 58.8 days ) compared with the continuing users . the cox proportional hazards model of the risk of discontinuation with index therapy ( table 3 ) did not show a significant difference between the intervention and control groups after adjusting for baseline demographic and clinical characteristics ( hazard ratio 0.83 , p < 0.55 ) . patients aged 6574 years ( as compared with patients aged 1854 years ) or with a history of hypertension were at greater risk for discontinuing their index therapy , whereas males and patients aged 5564 years or 75 + years ( as compared with patients aged 1854 years ) , with a prior cv event , or with a history of diabetes were at a lower risk of discontinuing index therapy . this trial was designed to evaluate the effectiveness of an integrated intervention program ( nurse counseling , adherence tip sheet , copay relief card , option to enroll in my heartwise program ) on patients adherence to statin therapy , using atorvastatin as an example . our findings indicate that both the control and intervention groups were quite similar ( no inferential statistics were conducted ) both groups were from a relatively sick population exhibiting polypharmacy behavior ( at least 85% of each group had a prior cv event ) . most patients who had one , two , or three atorvastatin prescription refills during the study period obtained a 90-day supply ( data not shown ) . in general , adherence and persistence for both the control and intervention groups were essentially the same , regardless of whether pdc or mpr was used as the outcome of interest . these unadjusted results were further validated by the findings of the logistic regression and cox proportional hazards models , which did not indicate a significant difference among the cohorts of interest . as the control group s adherence was initially high , there was little room for improvement as a result of the intervention . in addition , the large number of continuing users and the level of treatment patients received at this particular cardiology practice group could explain the high adherence rate observed in this study , which in turn could partially explain the lack of a significant impact due to the intervention . the literature confirms that new users often exhibit lower adherence rates as compared with continuing users.13 in a recent retrospective database analysis of treatment - nave hyperlipidemia patients , 65% of patients were considered nonadherent ( pdc = 0.33).25 the overall cost of medication nonadherence to the health care system is staggering . between one third and two thirds of medication - related hospital admissions are linked to poor adherence.9 total cost estimates for nonadherence amount to at least us$300 billion a year.26,27 some general reasons for nonadherence include the patient being unconvinced of the need for therapy and/or its effectiveness , medication side effects , difficulty with regimen administration , forgetfulness , and out - of - pocket costs . as such , adherence interventions seek to directly address some , if not all , of these barriers . evidence suggests that effective interventions combine education , motivation , support , reminders , and rewards , as a successful program must be delivered by a trusted source , be personalized to the patient s situation , reinforce medical need and expected outcomes , segment and target at - risk populations , and reinforce and reward initiation and maintenance.2830 some interventions directed at patients with hypertension have resulted in an improvement in adherence or clinical outcomes . two such studies of complex intervention programs , involving provision of care at the worksite , special pill containers , counseling , reminders , self - monitoring , support groups , and feedback and reinforcement , reported such positive effects.31,32 another study tested a telephone - linked computer system for monitoring and counseling hypertension patients . when adjusted for age , sex , and baseline adherence , patients using the telephone - linked computer system demonstrated a greater improvement in medication adherence than did those receiving usual care.33 a previous systematic review of adherence interventions concluded that all interventions that were effective for long - term care were complex ( including combinations of more convenient care , information , counseling , reminders , self - monitoring , reinforcement , family therapy , and other forms of additional supervision or attention ) ; even so , the most effective ones did not lead to large improvements in adherence and treatment outcomes.33 finally , the relative cost - effectiveness of adherence interventions should be taken into consideration when designing and implementing adherence - improving programs.34,35 there are several limitations that are worth noting here . the reader should be aware that both the patients and clinicians were not blinded to the study . however , because many of the patients were older than 65 years of age , the impact of discount cards was likely limited , as medicare patients did not qualify to receive them . although inferential statistics were not provided , the demographic profiles of the two cohorts were a bit disparate . the number of new users was small compared with the number of continuing users , an issue that should be taken into consideration when interpreting the findings ( as a general guide , studies with a single intervention group and a control group need to include at least 60 participants per group if they are to have at least 80% power to detect an absolute difference of 25% in the proportion of patients judged to have adequate adherence).33 in addition , one should consider that perhaps 180 days of follow - up was not sufficient to identify a meaningful difference between the two cohorts . regarding the data source , lrx does not track mail order prescriptions ; as such , patients who may have been excluded due to lack of atorvastatin claims may have filled their prescriptions via this channel . in addition , administrative / pharmacy claims do not address whether or not the patient filled the prescription that was written for a particular medication , nor do they reveal the actual underlying barriers to adherence . this study suggests that the impact of an integrated program including nurse counseling , copay relief cards , and a monthly newsletter did not significantly improve atorvastatin adherence relative to usual care in the studied patient population . because adherence and persistence may be affected by numerous factors , including comorbidities and economic conditions , further evaluation of differences in adherence and persistence among the patients may be warranted .

backgroundthis trial evaluated the effectiveness of an integrated intervention program that included a 3-to-5-minute nurse counseling session , copay relief cards , and a monthly newsletter on adherence to atorvastatin treatment.methods and resultsa prospective , integrated ( composed of nurse counseling , adherence tip sheet , copay relief card , opportunity to enroll in 12-week cholesterol management program ) randomized interventional study was designed involving patients > 21 years of age who were prescribed atorvastatin at a large single - specialty cardiovascular physician practice in illinois from march 2010 to may 2011 . data from the practice s electronic medical record were matched / merged to ims health s longitudinal data . a total of 500 patients were enrolled ( 125 in the control arm ; 375 in the intervention arm ) . after data linkage , 53 control patients and 155 intervention patients were included in the analysis.resultsmean age was 67.8 years ( control ) and 69.5 years ( intervention ) ; 67.9% and 58.7% , respectively , were male . the mean 6-month adherence rate was 0.82 in both arms . the mean proportion of days covered for both the new - user control and intervention groups was the same , averaging 0.70 day ( standard deviation [ sd ] , 0.27 day ) ; for continuing users , the proportion of days covered for the control group was 0.83 ( sd , 0.24 ) and for the intervention group was 0.84 ( sd , 0.22 ) . for continuing users , the control group had mean persistent days of 151.6 ( sd , 50.2 ) compared with 150.9 days ( sd , 50.9 ) for the intervention group . new users had fewer persistent days ( control 111.4 days , sd , 69.6 days ; intervention 112.0 days , sd , 58.8 days ) compared with continuing users . the cox proportional hazards model of the risk of discontinuation with index therapy was not significantly different between the intervention and control groups ( hazard ratio 0.83 , p = 0.55).conclusionthe integrated intervention program did not significantly improve atorvastatin adherence relative to usual care in the studied patient population .

Introduction Methods Data source Sample selection Statistical analysis Results Patient demographic and clinical characteristics Adherence Persistence Discussion Conclusion

in addition , its efficacy has been confirmed in a variety of populations.68 however , the beneficial effects described above may only be realized if patients take their medicines as prescribed by their health care practitioner ( ie , adhere to treatment ) ; unfortunately , adherence to these medicines is modest at best.9,10 several studies have shown that adherence to statins decreases to 50% by 12 months after the initial prescription,11,12 although some evidence suggests that adherence to atorvastatin may be higher than it is for other statins.13 for medications across a wide range of disease areas 15%20% of new prescriptions are never filled 20%35% of patients who do fill the first prescription fail to fill a second and adherence continues to decline rapidly throughout the first 36 months of therapy14,15 more specifically , statin persistence in older patients 65 years of age , declines over time , with the greatest drop occurring in the first 6 months of treatment.16 multiple barriers to taking medicines have been described by the world health organization and others , the most common being forgetfulness . intervention strategies that integrate counseling , emotional support , and cost sharing are motivational and have been shown to improve adherence.1723 the objective of this study was to describe and evaluate the effectiveness of an integrated intervention program on patients adherence to statin therapy , using atorvastatin as a specific example . this was a randomized prospective study that involved patients who were prescribed atorvastatin at prairie heart cardiovascular consultants , springfield ill , usa , that performs diagnostic catheterization , angioplasties , implants , and heart surgeries . patients who met the study criteria ( described below ) , and were prescribed atorvastatin by a participating physician after study initiation , whether they were considered new or returning practice patients , were eligible to enroll into this trial . eligible patients were randomized using a telephone randomization system to one of two groups : an intervention group and a control group ( with a patient ratio of 3:1 intervention : control ) . all patients randomized to the intervention group were provided adherence counseling from a nurse(via a 510-minute discussion ) , and an adherence tip sheet . patients in the intervention group were also given the opportunity to enroll in the my heartwise program,24 a 12-week guide to managing cholesterol ( included monthly mailing of educational materials ) . the practice physicians also had the discretion to provide eligible patients in the intervention group with a copay relief card ( usable with commercial payers , not medicare ) . data for this study was sourced from the practice s electronic medical record system and was matched and merged to ims health s ( plymouth meeting , pa , usa ) longitudinal ( lrx ) data . all subjects had to satisfy inclusion criteria to be considered eligible for participation by one of the ten participating physicians of the practice s study team : ( 1 ) be older than 21 years of age and , on the basis of clinical assessment by his or her physician , a candidate for statin therapy ; ( 2 ) have received a first prescription for atorvastatin after study initiation at the practice , including patients who were new to the practice and returning practice patients ( new versus continuing atorvastatin patients were deciphered by requiring claims activity 6 months before and after the index date ) ; and ( 3 ) provide a personally signed and dated informed consent document indicating that the participant ( or a legally acceptable representative ) had been informed of all pertinent aspects of the study . a sample size of 500 patients , selected in order to have at least 300 evaluable patients ( 225 intervention patients and 75 control patients ) , was calculated for this study . for continuous variables , measures of central tendency ( mean and median ) and measures of dispersion ( standard deviation [ sd ] ) as one of the main objectives of the study was to calculate the adherence to atorvastatin after study enrollment , both the pdc and medication possession ratio ( mpr ) during the 180 days from the patient s index date were used in the analysis . the proportion of patients who met this criterion was reported overall and for the intervention and control groups . persistence was compared between the control and the intervention groups using a time - to - event cox proportional hazards model , adjusted for the same demographic and baseline characteristics used in the logistical regression analyses of pdc and mpr . data for this study was sourced from the practice s electronic medical record system and was matched and merged to ims health s ( plymouth meeting , pa , usa ) longitudinal ( lrx ) data . all subjects had to satisfy inclusion criteria to be considered eligible for participation by one of the ten participating physicians of the practice s study team : ( 1 ) be older than 21 years of age and , on the basis of clinical assessment by his or her physician , a candidate for statin therapy ; ( 2 ) have received a first prescription for atorvastatin after study initiation at the practice , including patients who were new to the practice and returning practice patients ( new versus continuing atorvastatin patients were deciphered by requiring claims activity 6 months before and after the index date ) ; and ( 3 ) provide a personally signed and dated informed consent document indicating that the participant ( or a legally acceptable representative ) had been informed of all pertinent aspects of the study . a sample size of 500 patients , selected in order to have at least 300 evaluable patients ( 225 intervention patients and 75 control patients ) , was calculated for this study . for continuous variables , measures of central tendency ( mean and median ) and measures of dispersion ( standard deviation [ sd ] ) as one of the main objectives of the study was to calculate the adherence to atorvastatin after study enrollment , both the pdc and medication possession ratio ( mpr ) during the 180 days from the patient s index date were used in the analysis . the proportion of patients who met this criterion was reported overall and for the intervention and control groups . persistence was compared between the control and the intervention groups using a time - to - event cox proportional hazards model , adjusted for the same demographic and baseline characteristics used in the logistical regression analyses of pdc and mpr . the study initially included 500 patients ( 125 control patients and 375 patients who received the adherence intervention ) . after applying the study requirement of claims activity 6 months before and after the index date , only 53 controls ( seven new users [ first atorvastatin prescription after randomization ] and 46 continuing users [ evidence of atorvastatin prescription within 6 months prior to randomization ] ) and 155 intervention patients ( 14 new users and 141 continuing users ) remained eligible for analysis ( figure 1 ) . table 1 provides the descriptive data around the baseline demographic and clinical characteristics for both the control and intervention cohorts . among the control group , the average age was 67.8 years , compared with 69.5 years for the intervention group . most of the control group ( 58.5% ) was 65 years of age or older ; similarly , 65.2% of the intervention group was older than 65 years of age . the sex distribution was predominantly male for both groups ( 67.9% of the controls and 58.7% of the intervention group ) . most patients ( 67.9% of the controls and 70.3% of the intervention group ) were in , the control population was taking an average of 7.8 unique prescriptions , as compared to 7.1 for the intervention group . along the same lines , the control group filled an average of 32.8 prescriptions ( for any medication ) in the 6 months prior to the index date , whereas the intervention group filled an average of 26.2 prescriptions . control patients received more antidiabetic ( control 30.2% ; intervention 28.4% ) and antihypertensive ( control 96.2% ; intervention 94.2% ) medications compared to intervention patients . adherence to index therapy was measured using both pdc and mpr ( table 2 ) , with results stratified as to whether the patient was considered a new user or a continuing user , on the basis of the presence ( or absence ) of atorvastatin prescriptions in the 6-month preindex period . the mean pdc for the new - user control and intervention groups were similar , averaging 0.70 ( sd , 0.27 ) , whereas for continuing users , the control group exhibited a pdc of 0.83 ( sd , 0.24 ) and the intervention group 0.84 ( sd , 0.22 ) . the mean mpr for new users within the control group was 0.58 ( sd , 0.44 ) and was 0.62 ( sd , 0.42 ) for new users within the intervention group . three of the seven new users in the control group ( 42.9% ) were considered to have good adherence ( pdc and mpr 0.80 ) while five ( 35.7% when examining pdc ) and six ( 42.9% when examining mpr ) new users in the intervention group were considered to have good adherence . continuing users within the control group had a mean mpr of 0.83 ( sd , 0.29 ) as compared with 0.84 ( sd , 0.28 ) for those in the intervention group . in general , the continuing users in both groups were considered to have good adherence , with 76.1% of the control group ( when using pdc , versus 80.4% when using mpr ) and 75.2% of the intervention group ( when using pdc , versus 80.1% when using mpr ) having pdc > 0.80 . figure 2a shows the overall adherence ( pdc ) over time of both the control and intervention groups , up to 12 months postindex . for the control group , the mean pdc decreased from an average of 0.89 ( 3 months postindex ) to 0.73 ( 12 months postindex ) ; this decline was almost exactly matched in the intervention group , going from 0.91 ( 3 months postindex ) to 0.74 ( 12 months postindex ) . not all study patients had 12 months of follow - up for this analysis , and these were censored at the end of follow - up ( the control group had 33 patients , and 101 remained in the intervention group ) . similarly , the percentage of patients who were considered to have good adherence ( pdc 0.80 ) decreased from 84.9% ( 3 months postindex ) to 60.6% ( 12 months postindex ) in the control group and from 82.6% ( 3 months postindex ) to 58.4% ( 12 months postindex ) in the intervention group ( figure 2b ) . after adjusting for baseline demographic and clinical characteristics , there was no significant difference in pdc with index therapy between the two patient cohorts ( odds ratio = 1.03 for intervention versus control ; p = 0.95 ) ( table 3 ) . index atorvastatin patients who were deemed continuing users had more persistent days over the 6-month postindex period compared with new users . for continuing users , the control group had mean persistent days of 151.6 ( sd , 50.2 ) compared with 150.9 ( sd , 50.9 ) for the intervention group . the new users had fewer persistent days ( control 111.4 days , sd , 69.6 days ; intervention 112.0 days , sd , 58.8 days ) compared with the continuing users . the cox proportional hazards model of the risk of discontinuation with index therapy ( table 3 ) did not show a significant difference between the intervention and control groups after adjusting for baseline demographic and clinical characteristics ( hazard ratio 0.83 , p < 0.55 ) . patients aged 6574 years ( as compared with patients aged 1854 years ) or with a history of hypertension were at greater risk for discontinuing their index therapy , whereas males and patients aged 5564 years or 75 + years ( as compared with patients aged 1854 years ) , with a prior cv event , or with a history of diabetes were at a lower risk of discontinuing index therapy . table 1 provides the descriptive data around the baseline demographic and clinical characteristics for both the control and intervention cohorts . among the control group , the average age was 67.8 years , compared with 69.5 years for the intervention group . most of the control group ( 58.5% ) was 65 years of age or older ; similarly , 65.2% of the intervention group was older than 65 years of age . the sex distribution was predominantly male for both groups ( 67.9% of the controls and 58.7% of the intervention group ) . most patients ( 67.9% of the controls and 70.3% of the intervention group ) were in , the control population was taking an average of 7.8 unique prescriptions , as compared to 7.1 for the intervention group . along the same lines , the control group filled an average of 32.8 prescriptions ( for any medication ) in the 6 months prior to the index date , whereas the intervention group filled an average of 26.2 prescriptions . control patients received more antidiabetic ( control 30.2% ; intervention 28.4% ) and antihypertensive ( control 96.2% ; intervention 94.2% ) medications compared to intervention patients . adherence to index therapy was measured using both pdc and mpr ( table 2 ) , with results stratified as to whether the patient was considered a new user or a continuing user , on the basis of the presence ( or absence ) of atorvastatin prescriptions in the 6-month preindex period . the mean pdc for the new - user control and intervention groups were similar , averaging 0.70 ( sd , 0.27 ) , whereas for continuing users , the control group exhibited a pdc of 0.83 ( sd , 0.24 ) and the intervention group 0.84 ( sd , 0.22 ) . the mean mpr for new users within the control group was 0.58 ( sd , 0.44 ) and was 0.62 ( sd , 0.42 ) for new users within the intervention group . three of the seven new users in the control group ( 42.9% ) were considered to have good adherence ( pdc and mpr 0.80 ) while five ( 35.7% when examining pdc ) and six ( 42.9% when examining mpr ) new users in the intervention group were considered to have good adherence . continuing users within the control group had a mean mpr of 0.83 ( sd , 0.29 ) as compared with 0.84 ( sd , 0.28 ) for those in the intervention group . in general , the continuing users in both groups were considered to have good adherence , with 76.1% of the control group ( when using pdc , versus 80.4% when using mpr ) and 75.2% of the intervention group ( when using pdc , versus 80.1% when using mpr ) having pdc > 0.80 . figure 2a shows the overall adherence ( pdc ) over time of both the control and intervention groups , up to 12 months postindex . for the control group , the mean pdc decreased from an average of 0.89 ( 3 months postindex ) to 0.73 ( 12 months postindex ) ; this decline was almost exactly matched in the intervention group , going from 0.91 ( 3 months postindex ) to 0.74 ( 12 months postindex ) . not all study patients had 12 months of follow - up for this analysis , and these were censored at the end of follow - up ( the control group had 33 patients , and 101 remained in the intervention group ) . similarly , the percentage of patients who were considered to have good adherence ( pdc 0.80 ) decreased from 84.9% ( 3 months postindex ) to 60.6% ( 12 months postindex ) in the control group and from 82.6% ( 3 months postindex ) to 58.4% ( 12 months postindex ) in the intervention group ( figure 2b ) . after adjusting for baseline demographic and clinical characteristics , there was no significant difference in pdc with index therapy between the two patient cohorts ( odds ratio = 1.03 for intervention versus control ; p = 0.95 ) ( table 3 ) . index atorvastatin patients who were deemed continuing users had more persistent days over the 6-month postindex period compared with new users . for continuing users , the control group had mean persistent days of 151.6 ( sd , 50.2 ) compared with 150.9 ( sd , 50.9 ) for the intervention group . the new users had fewer persistent days ( control 111.4 days , sd , 69.6 days ; intervention 112.0 days , sd , 58.8 days ) compared with the continuing users . the cox proportional hazards model of the risk of discontinuation with index therapy ( table 3 ) did not show a significant difference between the intervention and control groups after adjusting for baseline demographic and clinical characteristics ( hazard ratio 0.83 , p < 0.55 ) . patients aged 6574 years ( as compared with patients aged 1854 years ) or with a history of hypertension were at greater risk for discontinuing their index therapy , whereas males and patients aged 5564 years or 75 + years ( as compared with patients aged 1854 years ) , with a prior cv event , or with a history of diabetes were at a lower risk of discontinuing index therapy . this trial was designed to evaluate the effectiveness of an integrated intervention program ( nurse counseling , adherence tip sheet , copay relief card , option to enroll in my heartwise program ) on patients adherence to statin therapy , using atorvastatin as an example . our findings indicate that both the control and intervention groups were quite similar ( no inferential statistics were conducted ) both groups were from a relatively sick population exhibiting polypharmacy behavior ( at least 85% of each group had a prior cv event ) . in general , adherence and persistence for both the control and intervention groups were essentially the same , regardless of whether pdc or mpr was used as the outcome of interest . the literature confirms that new users often exhibit lower adherence rates as compared with continuing users.13 in a recent retrospective database analysis of treatment - nave hyperlipidemia patients , 65% of patients were considered nonadherent ( pdc = 0.33).25 the overall cost of medication nonadherence to the health care system is staggering . when adjusted for age , sex , and baseline adherence , patients using the telephone - linked computer system demonstrated a greater improvement in medication adherence than did those receiving usual care.33 a previous systematic review of adherence interventions concluded that all interventions that were effective for long - term care were complex ( including combinations of more convenient care , information , counseling , reminders , self - monitoring , reinforcement , family therapy , and other forms of additional supervision or attention ) ; even so , the most effective ones did not lead to large improvements in adherence and treatment outcomes.33 finally , the relative cost - effectiveness of adherence interventions should be taken into consideration when designing and implementing adherence - improving programs.34,35 there are several limitations that are worth noting here . however , because many of the patients were older than 65 years of age , the impact of discount cards was likely limited , as medicare patients did not qualify to receive them . the number of new users was small compared with the number of continuing users , an issue that should be taken into consideration when interpreting the findings ( as a general guide , studies with a single intervention group and a control group need to include at least 60 participants per group if they are to have at least 80% power to detect an absolute difference of 25% in the proportion of patients judged to have adequate adherence).33 in addition , one should consider that perhaps 180 days of follow - up was not sufficient to identify a meaningful difference between the two cohorts . this study suggests that the impact of an integrated program including nurse counseling , copay relief cards , and a monthly newsletter did not significantly improve atorvastatin adherence relative to usual care in the studied patient population .

whole cell recordings whole cell current recordings of cultured pfc neurons employed standard voltage - clamp techniques as those we described previously ( 21 , 22 ) . the external solution for recording nmdar - mediated current contained ( in mm ) : 127 nacl , 20 cscl , 1 cacl2 , 10 hepes , 5 bacl2 , 12 glucose , 0.001 tetrodotoxin , and glycine 0.02 , ph 7.37.4 , 300305 mosm / liter . the internal solution contained ( in mm ) : 180 n - methyl - d - glucamine , 4 mgcl2 , 40 hepes , 0.5 1,2-bis(2-aminophenoxy)ethane - n , n , n,n-tetraacetic acid , 12 phosphocreatine , 3 na2atp , and 0.5 na2gtp , 0.1 leupeptin , ph 7.2 - 3 , 265270 mosm / liter . recordings were obtained with an axon instruments ( union city , ca ) 200b patch clamp amplifier that was monitored by an ibm pc running pclamp 8 with a digidata 1320 series interface . electrode resistances were normally 24 m in bath solution . following seal rupture , series resistance ( 410 m ) attention was applied to monitor the series resistance , and recordings were stopped when a significant increase ( > 20% ) occurred . the whole cell nmdar - mediated current was evoked by nmda ( 100 m ) application for 2 s every 30 s with neurons held at 60 mv . the array of drug capillaries was positioned a few hundred micrometers from the cell under recording . solution changes were controlled by the sf-77b fast - step solution stimulus delivery device ( warner instruments , hamden , ct ) . data were analyzed with axograph ( axon instruments ) and kaleidagraph ( albeck software ) . anova was performed to compare the differential degrees of current regulation between experimental groups subjected to different drug treatment . , we performed the standard whole cell recording techniques in layer v pfc pyramidal neurons ( 21 , 23 ) . patch pipettes ( 59 m ) were filled with the following internal solution ( in mm ) : 130 cesium methanesulfonate , 10 cscl , 4 nacl , 1 mgcl2 , 10 hepes , 5 egta , 2.2 qx-314 , 12 phosphocreatine , 5 mgatp , 0.5 na2gtp , 0.1 leupeptin , ph 7.27.3 , 265270 mosm / liter . pfc slices ( 300 m ) were perfused at room temperature ( 2224 c ) , with artificial cerebrospinal fluid was bubbled with 95% o2/5% co2 containing 6-cyano-7-nitroquinoxaline-2,3-dione ( 20 m ) and bicuculline ( 10 m ) to block -amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid / kainite receptors and gabaa receptors , respectively . neurons were observed with a 40 water - immersion lens and illuminated with near infrared ir light , and the image was captured with an ir - sensitive charge - coupled device camera . all recordings were performed using a multiclamp 700a amplifier . upon application of negative pressure , the membrane was tightly sealed with resistance ( 210 g ) . with additional suction , evoked currents were generated with a pulse from a stimulation isolation unit controlled by an s48 pulse generator ( astro - med ) . a bipolar stimulating electrode ( fredrick haer company ) was positioned 100m from the neuron upon recording . prior to stimulation , neurons ( clamped at 70 mv ) were depolarized to + 60 mv for 3 s to fully eliminate the voltage - dependent mg block of nmdar . age - matched saline controls were done side - by - side with drug - injected animals on each day of experiments . to minimize variations between slices , the stimulus with the same intensity was delivered by the stimulating electrode placed at the same location . the agents we used include serotonin , 8-hydroxy-2(di - n - propylamino)tetralin ( 8-oh - dpat ) , -me-5ht , ( )-2,5-dimethoxy-4-iodoamphetamine , ketanserin , benzothiazole , colchicine ( sigma ) , dynamin inhibitory peptide ( tocris ) , 4-amino-5-(4-chlorophenyl)-7-(t - butyl)pyrazolo[3,4-d]pyrimidine ( pp2 ) and 4-amino-7-phenylpyrazol[3,4-d]pyrimidine ( pp3 ) ( calbiochem ) . they were made up as concentrated stocks in water or dmso and stored at 20 c . animal treatment young male rats ( 2528 days old ) were administered intraperitoneally with the drugs as described in the text . for stereotaxic injection , rats were anesthetized with pentobarbital sodium ( 50 mg / kg intraperitoneal ) and then mounted into a stereotaxic apparatus ( david kopf instruments ) . fluoxetine ( fluox ) ( 2 l , 0.34 mg / ml , dissolved in saline ) were injected unilaterally into the pfc region using a hamilton syringe ( 22-gauge needle ) at a rate of 0.5 l / min . the coordinates of the lateral pfc used are 1.01.4 mm lateral from midline , 2.2 mm anterior to bregma , and 3.3 mm dorsal to ventral . small interfering rna to knock down the endogenous -arrestin expression , we used the small interfering rna ( sirna ) that specifically targets -arrestin1 or -arrestin2 mrna ( 24 ) . the sirna oligonucleotide sequences were : 5-ggcgagucuacgugacacutt-3 ( for -arrestin1 ) and 5-ggaccggaasguguuugugtt-3 ( for -arrestin2 ) . the sirna ( purchased from ambion , austin , tx ) was co - transfected with enhanced gfp into cultured pfc neurons ( 11 days in vitro ) using the lipofectamine 2000 method . biochemical , immunocytochemical , or electrophysiological experiments were performed in neurons after 23 days of transfection . cultured pfc neurons ( 2 10 cells / cm , 14 days in vitro ) in 3.5-cm dishes were washed twice at 37 c with 1 ml of microtubule stabilizing buffer containing ( 0.1 m mes ( ph 6.75 ) , 1 mm mgso4 , 2 mm egta , 0.1 mm edta , and 4 m glycerol ) . cultures were then incubated at 37 c for 5 min in 600 l of soluble tubulin extraction buffer ( 0.1 m mes ( ph 6.75 ) , 1 mm mgso4 , 2 mm egta , 0.1 mm edta , 4 m glycerol , and 0.1% triton x-100 ) with the addition of protease inhibitor mixture tablets ( roche applied science ) . the soluble extract was centrifuged at 37 c for 2 min , and the supernatant was collected . equal amounts of total protein were analyzed by western blotting using anti--tubulin ( sigma ) . the intensity of tubulin bands was quantitatively analyzed with image ( national institutes of health ) . figure 1.activation of 5-ht2a / c receptors counteracts the 5-ht1a -induced reduction of nmdar - mediated ionic and synaptic currents in pfc pyramidal neurons . a and c , plot of normalized peak nmdar currents ( a ) or nmdar - epsc ( c ) as a function of time and 8-oh - dpat ( 5-ht1a agonist , 20 m ) application in neurons treated with or without -me-5ht ( 5-ht2a / c agonist , 20 m ) . each point ( c ) represents the average peak ( mean s.e . ) of three consecutive nmdar - epsc . b and d , representative current traces taken from the records used to construct a or c ( at time points denoted by # ) . scale bars : 100 pa , 1 s ( b ) ; 100 pa , 100 ms ( d ) . e , plot of peak nmdar - epsc showing the effect of 8-oh - dpat ( 20 m ) in the presence of -me-5ht ( 20 m ) and ketanserin ( 5-ht2a / c antagonist , 20 m ) . f , cumulative data ( mean s.e . ) summarizing the percentage reduction of nmdar - epsc by 8-oh - dpat in the presence or absence of various 5-ht receptor agonists or antagonists . * , p < 0.001 , anova . activation of 5-ht2a / c receptors counteracts the 5-ht1a -induced reduction of nmdar - mediated ionic and synaptic currents in pfc pyramidal neurons . a and c , plot of normalized peak nmdar currents ( a ) or nmdar - epsc ( c ) as a function of time and 8-oh - dpat ( 5-ht1a agonist , 20 m ) application in neurons treated with or without -me-5ht ( 5-ht2a / c agonist , 20 m ) . each point ( c ) represents the average peak ( mean s.e . ) of three consecutive nmdar - epsc . b and d , representative current traces taken from the records used to construct a or c ( at time points denoted by # ) . scale bars : 100 pa , 1 s ( b ) ; 100 pa , 100 ms ( d ) . e , plot of peak nmdar - epsc showing the effect of 8-oh - dpat ( 20 m ) in the presence of -me-5ht ( 20 m ) and ketanserin ( 5-ht2a / c antagonist , 20 m ) . f , cumulative data ( mean s.e . ) summarizing the percentage reduction of nmdar - epsc by 8-oh - dpat in the presence or absence of various 5-ht receptor agonists or antagonists . * , immunocytochemical staining the detection of surface gfp - nr2b ( 25 ) was performed as how we described before ( 21 , 22 ) . briefly , cultured pfc neurons were treated with various agents after transfection , and then fixed in 4% paraformaldehyde for 30 min at room temperature without permeabilization . after incubating in 5% bovine serum albumin to block nonspecific staining , cells were incubated with the anti - gfp antibody ( 1:100 , chemicon , temecula , ca ) for 1 h at room temperature . after three washes in phosphate - buffered saline , cells were incubated with a rhodamine - conjugated secondary antibody ( 1 : 200 , sigma ) for 1 h at room temperature . after washing in phosphate - buffered saline , coverslips were mounted on slides with vectashield mounting media ( vector laboratories , burlingame , ca ) . fluorescence images were detected using a 60 objective with a cooled charge - coupled device camera mounted on a nikon microscope . a 50-m segment of dendrite was selected from the equal distance away from the soma of four to six individual neurons . signal was counted as clusters when its intensity was 2- to 3-fold greater than the overall fluorescence on the dendritic shaft . biochemical measurement of surface receptors after treatment , pfc slices were incubated with artificial cerebrospinal fluid containing 1 mg / ml sulfo - nhs - lc - biotin ( pierce ) for 20 min on ice . the slices were then rinsed three times in tris - buffered saline to quench the biotin reaction , followed by homogenization in 300 l of modified radioimmune precipitation assay buffer ( 1% triton x-100 , 0.1% sds , 0.5% deoxycholic acid , 50 mm napo4 , 150 mm nacl , 2 mm edta , 50 mm naf , 10 mm sodium pyrophosphate , 1 mm sodium orthovanadate , 1 mm phenylmethylsulfonyl fluoride , and 1 mg / ml leupeptin ) . 15 g of homogenates was removed to measure total proteins . for surface protein detection , 150 g of homogenates was incubated with 100 l of 50% neutravidin - agarose ( pierce ) for 2 h at 4 c , and bound proteins were resuspended in 25 l of sds sample buffer and boiled . quantitative western blots were performed on both total and biotinylated ( surface ) proteins using anti - nr1 , anti - nr2b ( upstate biotechnology , lake placid , ny ) , and anti - gabaar 2/3 ( chemicon ) . activation of 5-ht1a and 5-ht2a / c receptors regulates nmdar - mediated ionic and synaptic currents in a counteractive manner we have previously found that activation of 5-ht1a receptors reduces nmdar currents in pfc pyramidal neurons ( 21 ) . to examine the potential interactions between 5-ht1a and 5-ht2a / c receptors on nmdar functions , we tested the impact of 5-ht2 activation on 5-ht1a regulation of nmdar currents by preincubating pfc cultures with the 5-ht2a / c agonist -me-5ht ( 20 m , 1030 min ) . application of nmda ( 100 m ) elicited an inward current that was partially desensitized and was completely eliminated by the nmda receptor antagonist d - aminophosphonovalerate ( 50 m ) . as shown in fig . 1 ( a and b ) , application of the 5-ht1a agonist 8-oh - dpat ( 20 m ) reversibly reduced nmdar currents ( 21.3 0.7% , n = 16 ) . however , in -me-5ht - treated neurons , the reduction of nmdar current by 8-oh - dpat was substantially attenuated ( 6.4 0.7% , n = 8) . -me-5ht alone did not significantly affect nmdar currents ( 1 m : 4.0 0.8% , n = 5 ; 20 m : 5.7 1.4% , n = 7 ) . another 5-ht2a / c agonist doi also had no effect on nmdar currents at low doses ( 0.05 m : 4.2 1.2% , n = 5 ; 0.1 m : 4.0 2.5% , n = 5 ) , which are different from the inhibitory effect of doi shown before ( 26 ) . the discrepancy may be due to different experimental procedures in recording nmda - induced currents . in a previous study ( 26 ) , microdrops of nmda ( 1 mm , every 15 min ) were applied to pfc slices , which did not allow the accurate detection of peak nmda currents because of the slow diffusion of the ligand . moreover , it was not a pure postsynaptic preparation like dissociated neurons , which could have indirect effects due to changes in the circuit . our results suggest that 5-ht2a / c activation alone does not directly affect nmdar currents but opposes 5-ht1a regulation of nmdar currents in pfc pyramidal neurons . figure 2.inhibition of 5-ht2a / c receptors unmasks the 5-ht1a -mediated reduction of nmdar - epsc in response to 5-ht . a , representative nmdar - epsc traces ( average of three trials ) showing the regulation of nmdar - epsc by 5-ht ( 10 , 40 , or 100 m ) in neurons treated with or without the 5-ht2a / c antagonist ketanserin ( ket , 20 m ) . b , dose - response curves summarizing the percentage reduction of nmdar - epsc by different concentrations of 5-ht in neurons treated with or without ketanserin . * , 0.001 , anova . inhibition of 5-ht2a / c receptors unmasks the 5-ht1a -mediated reduction of nmdar - epsc in response to 5-ht . a , representative nmdar - epsc traces ( average of three trials ) showing the regulation of nmdar - epsc by 5-ht ( 10 , 40 , or 100 m ) in neurons treated with or without the 5-ht2a / c antagonist ketanserin ( ket , 20 m ) . b , dose - response curves summarizing the percentage reduction of nmdar - epsc by different concentrations of 5-ht in neurons treated with or without ketanserin . * , we further tested the influence of 5-ht2 activation on 5-ht1a regulation of nmdar - epsc mediated by synaptic nmda receptors in pfc slices . as shown in fig . 1 ( c and d ) , application of 8-oh - dpat potently reduced the amplitude of nmdar - epsc ( 37.1 2.1% ; n = 10 ( fig . 1f ) ) . however , this reduction was significantly attenuated in the presence of -me-5ht ( 10.4 0.7% ; n = 13 ( fig . 1f ) ) . to verify that 5-ht2a / c receptors were mediating this regulatory effect of -me-5ht , we pre - treated pfc slices with the specific 5-ht2a / c antagonist ketanserin ( 20 m ) . as shown in fig . 1e , -me-5ht failed to oppose the effect of 5-ht1a on nmdar - epsc in the presence of ketanserin ( 36.8 7.1% ; n = 5 ( fig . 1f ) ) . in contrast , the reduction of nmdar - epsc by 8-oh - dpat was intact in the presence of the 5-ht4 agonist benzothiazole ( 20 m , 33 2.9% ; n = 5 ( fig . these results indicate that 5-ht2a / c receptor activation selectively counteracts 5-ht1a regulation of nmdar functions in pfc pyramidal neurons . we next examined what would happen when both 5-ht1a and 5-ht2a / c receptors are co - activated by serotonin . if both signals oppose each other , then blocking one receptor activation would unmask the effect of the other . thus , we tested the effect of serotonin on nmdar - epsc in pfc slices treated with or without 5-ht2 antagonists . as shown in fig . 2 ( a and b ) , different concentrations of serotonin reduced the amplitude of nmdar - epsc to different extents ( 10 m:21 2.3% , n = 4 ; 40 m : 28.5 4.9% , n = 4 ; 100 m : 29.4 4.8% , n = 5 ) . however , in the presence of the 5-ht2a / c antagonist ketanserin ( 20 m ) , the reduction of nmdar - epsc by serotonin was greatly augmented ( 10 m : 35.2 2.2% , n = 7 ; 40 m : 43.0 2.0% , n = 6 ; 100 m:47 1.9% , n = 7 ) . it suggests that 5-ht2 receptor activation in response to serotonin masks part of the inhibitory action of 5-ht1a receptors on nmdar channels . to assess whether the serotonergic regulation of nmdars , which we found in vitro , is also occurring in vivo , we tested whether endogenous serotonin , via the activation of 5-ht1a and 5-ht2a / c receptors , could regulate nmdar functions in a similar manner in intact animals . first , we examined nmdar - epsc in pfc slices from animals intraperitoneally injected with 5-ht1a or 5-ht2a / c agonists . as shown in fig . 3a , the amplitude of nmdar - epsc was significantly smaller in animals intraperitoneally injected with the 5-ht1a agonist 8-oh - dpat ( saline : 379.8 8.9 pa , n = 8 ; dpat : 166 11.5 pa , n = 11 ; p < 0.001 , anova ) , whereas it was largely unaltered by injecting the 5-ht2a / c agonist -me-5ht ( 349 12.2 pa , n = 10 ) . however , the effect of 8-oh - dpat injection was blocked by injecting -me-5ht ( dpat + -me-5ht : 335 15.9 pa , n = 12 ) . these results indicate that activation of 5-ht1a receptors in vivo down - regulates nmdar functions , and this effect of 5-ht1a is opposed by 5-ht2a / c receptor activation in vivo . moreover , the amplitude of -amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptor - epsc was unchanged by 8-oh - dpat injection ( saline : 113 11.8 pa , n = 11 ; dpat : 112 9.9 pa , n = 10 ) , suggesting that 5-ht1a activation in pfc is specifically targeting postsynaptic nmdars rather than presynaptic glutamate release . figure 3.the synaptic nmda response is reduced in pfc pyramidal neurons from animals with systemic administration of 5-ht1a agonist or the serotonin re - uptake inhibitor fluoxetine , which is opposed by injecting 5-ht2a / c agonist . a and b , dot plots showing the peak amplitude of nmdar - epsc recorded in pfc pyramidal neurons from animals with a single intraperitoneal injection of different drugs . a , saline , 8-oh - dpat , -me-5ht , or 8-oh - dpat plus -me-5ht ; b , saline , fluoxetine , way-100635 , way-100635 plus fluoxetine , ketanserin , or ketanserin plus fluoxetine . inset : representative nmdar - epsc traces recorded in pfc pyramidal neurons from animals with different drug injection . c , plot of normalized peak nmdar - epsc showing the effect of bath application of 8-oh - dpat ( 40 m ) in pfc pyramidal neurons from animals intraperitoneally injected with fluoxetine ( 20 mg / kg ) or saline . the synaptic nmda response is reduced in pfc pyramidal neurons from animals with systemic administration of 5-ht1a agonist or the serotonin re - uptake inhibitor fluoxetine , which is opposed by injecting 5-ht2a / c agonist . a and b , dot plots showing the peak amplitude of nmdar - epsc recorded in pfc pyramidal neurons from animals with a single intraperitoneal injection of different drugs . a , saline , 8-oh - dpat , -me-5ht , or 8-oh - dpat plus -me-5ht ; b , saline , fluoxetine , way-100635 , way-100635 plus fluoxetine , ketanserin , or ketanserin plus fluoxetine . inset : representative nmdar - epsc traces recorded in pfc pyramidal neurons from animals with different drug injection . c , plot of normalized peak nmdar - epsc showing the effect of bath application of 8-oh - dpat ( 40 m ) in pfc pyramidal neurons from animals intraperitoneally injected with fluoxetine ( 20 mg / kg ) or saline . figure 4.activation of 5-ht2a / c receptors attenuates the 5-ht1a -induced microtubule depolymerization and the effect of microtubule depolymerizer on nmdar - epsc . a , western blot analysis of free tubulin in lysates of cultured pfc neurons treated with or without 8-oh - dpat ( 40m , 30 min ) in the absence or presence of -me-5ht ( 20 m , added 10 min before 8-oh - dpat treatment ) . free tubulin level was normalized to control ( ) , based on the intensity of the tubulin band from western blot analyses . c , plot of normalized peak nmdar - epsc showing the effect of microtubule depolymerizer colchicine ( 30 m ) in the absence or presence of -me-5ht ( 20 m ) . d , cumulative data ( mean s.e . ) illustrating the percent reduction of nmdar - epsc by colchicine in neurons with or without the exposure to -me-5ht . * , p < 0.05 , anova . activation of 5-ht2a / c receptors attenuates the 5-ht1a -induced microtubule depolymerization and the effect of microtubule depolymerizer on nmdar - epsc . a , western blot analysis of free tubulin in lysates of cultured pfc neurons treated with or without 8-oh - dpat ( 40m , 30 min ) in the absence or presence of -me-5ht ( 20 m , added 10 min before 8-oh - dpat treatment ) . free tubulin level was normalized to control ( ) , based on the intensity of the tubulin band from western blot analyses . c , plot of normalized peak nmdar - epsc showing the effect of microtubule depolymerizer colchicine ( 30 m ) in the absence or presence of -me-5ht ( 20 m ) . d , cumulative data ( mean s.e . ) illustrating the percent reduction of nmdar - epsc by colchicine in neurons with or without the exposure to -me-5ht . * , p < 0.05 , anova . next , we examined nmdar - epsc in pfc slices from animals intraperitoneally injected with the serotonin re - uptake inhibitor fluoxetine ( 20 mg / kg ) to elevate endogenous 5-ht levels at synapses . as shown in fig . 3b , the amplitude of nmdar - epsc in fluoxetine - injected animals was significantly smaller , compared with saline controls ( saline : 403.5 15.8 pa , n = 11 ; fluox : 195.1 12.4 pa , n = 14 ; p < 0.001 , anova ) . this effect of fluoxetine was blocked by injecting the 5-ht1a antagonist way-100635 ( way : 367 12.9 pa , n = 12 ; way + fluox : 390 15.8 pa , n = 12 ) , suggesting the mediation by 5-ht1a receptors . moreover , the reducing effect of fluoxetine on nmdar - epsc was potentiated by injecting the 5-ht2a / c antagonist ketanserin ( ket : 392 12.3 pa , n = 11 ; ket + fluox : 59.6 5.2 pa , n = 12 ; p < 0.001 , anova ) , revealing the counteracting effect of 5-ht2a / c on 5-ht1a regulation of synaptic nmda responses . moreover , the effect of bath application of 8-oh - dpat ( 40 m ) on nmdar - epsc was occluded in animals injected with fluoxetine , as compared with saline controls ( fig . 3c , saline : 40.2 1.9% , n = 5 , fluox : 10.5 2.5% , n = 6 ) , confirming the common mechanism underlying the in vivo fluoxetine effect and in vitro 8-oh - dpat effect on nmdars . in contrast to the strong effect in pfc pyramidal neurons , fluoxetine injection failed to affect nmdar - epsc in striatal medium spiny neurons ( saline : 375 15.9 pa , n = 8 ; fluox : 351 13.9 pa , n = 10 ) , suggesting the specificity of serotonergic regulation of nmdars in pfc . figure 5.activation of 5-ht2a / c receptors induces the phosphorylation and activation of erk in neuronal dendrites in cultured pfc neurons . a and b , immunocytochemical images of cultured pfc neurons stained with phospho - erk ( a , green ) or erk ( b , green ) plus the nucleus marker topro3 ( red ) in the absence ( vehicle ) or presence of -me-5ht ( 20 m , 3 min ) or glutamate ( 100 m , 3 min ) . activation of 5-ht2a / c receptors induces the phosphorylation and activation of erk in neuronal dendrites in cultured pfc neurons . a and b , immunocytochemical images of cultured pfc neurons stained with phospho - erk ( a , green ) or erk ( b , green ) plus the nucleus marker topro3 ( red ) in the absence ( vehicle ) or presence of -me-5ht ( 20 m , 3 min ) or glutamate ( 100 m , 3 min ) . figure 6.the counteractive effect of 5-ht2a / c on 5-ht1a regulation of nmdar function is dependent on the -arrestin / erk pathway . a , top : western blots of -arrestin 1/2 in pfc cultures transfected without or with sirna against -arrestin 1/2 or a scrambled sirna . b , top : western blots of phospho - erk in the absence or presence of -me-5ht ( 20 m , 3 min ) in pfc cultures transfected with or without -arrestin 1/2 sirna . bottom : quantification of phospho - erk under different conditions . c , plot of peak nmdar currents as a function of time and 8-oh - dpat ( 20m ) application in the presence of-me-5ht ( 20 m ) in gfp - positive neurons transfected with or without sirna against -arrestin 1/2 . d , cumulative data ( mean s.e . ) summarizing the percentage reduction of nmdar currents by 8-oh - dpat in the absence or presence of-me-5ht under different conditions . * , p < 0.001 , anova . the counteractive effect of 5-ht2a / c on 5-ht1a regulation of nmdar function is dependent on the -arrestin / erk pathway . a , top : western blots of -arrestin 1/2 in pfc cultures transfected without or with sirna against -arrestin 1/2 or a scrambled sirna . b , top : western blots of phospho - erk in the absence or presence of -me-5ht ( 20 m , 3 min ) in pfc cultures transfected with or without -arrestin 1/2 sirna . bottom : quantification of phospho - erk under different conditions . c , plot of peak nmdar currents as a function of time and 8-oh - dpat ( 20m ) application in the presence of-me-5ht ( 20 m ) in gfp - positive neurons transfected with or without sirna against -arrestin 1/2 . d , cumulative data ( mean s.e . ) summarizing the percentage reduction of nmdar currents by 8-oh - dpat in the absence or presence of-me-5ht under different conditions . * , . to further test the effect of 5-ht on nmdars in pfc networks in vivo without affecting other circuits , we also performed stereotaxic injection of fluoxetine to pfc to elevate the local 5-ht levels . we found that the amplitude of nmdar - epsc in pfc pyramidal neurons localized at the proximity of injection sites was significantly smaller , compared with saline controls ( saline : 401 10.1 pa , n = 11 ; fluox : 162 13.2 pa , n = 11 , p < 0.001 , anova ) . the opposing regulation of nmda receptors by 5-ht1a and 5-ht2 receptors depends on microtubule stability next , we investigated the potential mechanism by which 5-ht2 receptors counteract the effect of 5-ht1a on nmdar currents in pfc neurons . previously , we have found that 5-ht1a disrupts nmda receptor trafficking by destabilizing microtubule integrity ( 21 ) . thus , we examined whether microtubule dynamics is the convergent target of 5-ht1a- and 5-ht2a - mediated signaling . to test this , we compared the level of free ( depolymerized ) tubulin in pfc cultures subjected to 8-oh - dpat treatment in the absence or presence of -me5ht . 4 ( a and b ) , application of 8-oh - dpat ( 40 m , 20 min ) caused a potent increase in free tubulin ( 1.7 0.3-fold increase , n = 3 , p < 0.001 , anova ) , however , this effect was significantly blocked by -me-5ht ( 20 m , 0.6 0.2-fold increase , n = 3 ) , indicating that 5-ht2 activation could increase microtubule stability and prevent 5-ht1a - induced microtubule depolymerization . we then tested whether the counteractive effect of 5-ht2 on 5-ht1a regulation of nmdar currents is due to the opposing regulation of microtubule dynamics by 5-ht1a and 5-ht2 receptors . 4 ( c and d ) , bath application of the microtubule depolymerizer , colchicine ( 30 m ) , gradually reduced nmdar - epsc in pfc slices ( 38.0 2.9% , n = 5 , also see ref . 22 ) , mimicking and occluding the 5-ht1a effect ( 21 ) . however , this inhibitory effect of colchicine was largely attenuated in the presence of -me-5ht ( 20 m , 11.2 13.4% , n = 6 ) . these results suggest that activation of 5-ht1a and 5-ht2 receptors could regulate nmdar currents in a counteractive manner by converging on microtubule dynamics . activation of 5-ht2 receptors induces erk activation in neuronal processes how could 5-ht2 activation increase microtubule stability ? evidence has shown that activation of erk stabilizes microtubule integrity ( 27 , 28 ) . moreover , our previous findings have suggested that 5-ht1a reduces erk activity , which in turn reduces map2 phosphorylation and microtubule stability ( 21 ) . thus , we speculate that 5-ht2 receptors might activate erk , leading to increased phosphorylation of map2 and its association with microtubules . to test this , we measured the activation of erk1/2 in response to 5-ht2a / c agonists with an antibody that recognizes activated ekr1/2 , which are doubly phosphorylated at thr-202/tyr-204 in the activation loop of the kinases ( 29 ) . as a positive control , we also treated neurons with glutamate , which was reported to activate erk ( 30 ) . 5a , application of -me-5ht ( 20 m , 3 min ) or glutamate ( 100 m , 3 min ) increased erk phosphorylation , as compared with vehicle - treated neurons . unlike glutamate treatment , the -me-5ht - activated erk did not co - localize with topro3 ( a nucleus marker ) staining , suggesting that 5-ht2-activated erk is mainly targeted to the cytoplasm rather than nucleus . among all experimental groups , these data suggest that 5-ht2 receptors induce erk activation , which may oppose the down - regulation of erk by 5-ht1a receptors . figure 7.the counteractive effect of 5-ht2a / c on 5-ht1a regulation of nmdar function involves clathrin / dynamin - mediated endocytosis . a , western blots of phospho - erk and total erk in pfc cultures treated with or without -me-5ht ( 20 m , 3 min ) in the absence or presence of the cell - permeable ( myristoylated ) or non - permeable dynamin inhibitory peptide ( both 50 m ) , or a cell - permeable scrambled control peptide ( 50 m ) . this scrambled peptide was fused with the protein transduction domain of the human immunodeficiency virus tat protein ( ygrkkrrqrrr , 53 ) , which rendered it cell - permeable . c , plot of normalized peak nmdar - epsc showing the effect of 8-oh - dpat ( 20 m ) in the presence of -me-5ht ( 20 m ) in cells dialyzed with the dynamin inhibitory peptide ( 50 m ) or a scrambled control peptide ( 50 m ) . inset , representative traces of nmdar - epsc taken at time points denoted by # . d , cumulative data ( mean s.e . ) summarizing the percentage reduction of nmdar - epsc by 8-oh - dpat in the absence or presence of -me-5ht in neurons injected with or without the dynamin inhibitory peptide . * , the counteractive effect of 5-ht2a / c on 5-ht1a regulation of nmdar function involves clathrin / dynamin - mediated endocytosis . a , western blots of phospho - erk and total erk in pfc cultures treated with or without -me-5ht ( 20 m , 3 min ) in the absence or presence of the cell - permeable ( myristoylated ) or non - permeable dynamin inhibitory peptide ( both 50 m ) , or a cell - permeable scrambled control peptide ( 50 m ) . this scrambled peptide was fused with the protein transduction domain of the human immunodeficiency virus tat protein ( ygrkkrrqrrr , 53 ) , which rendered it cell - permeable . c , plot of normalized peak nmdar - epsc showing the effect of 8-oh - dpat ( 20 m ) in the presence of -me-5ht ( 20 m ) in cells dialyzed with the dynamin inhibitory peptide ( 50 m ) or a scrambled control peptide ( 50 m ) . inset , representative traces of nmdar - epsc taken at time points denoted by # . d , cumulative data ( mean s.e . ) summarizing the percentage reduction of nmdar - epsc by 8-oh - dpat in the absence or presence of -me-5ht in neurons injected with or without the dynamin inhibitory peptide . * , the -arrestin - mediated pathway is involved in the counteractive effects of 5-ht2 receptors on 5-ht1a regulation of nmdar currents next , we sought to identify the signaling mechanism underlying 5-ht2 activation of erk . recently , it has been shown that some g proteins form a signaling complex with the multifunctional adaptor and transducer molecule , -arrestins 1/2 , which recruits and activates components of the mitogen - activated protein kinase cascades ( 31 , 32 ) . to examine the possibility that 5-ht2 receptors activate erk through the -arrestin pathway , we transfected pfc cultures with sirna against -arrestin1 or -2 to knock down their expression . 6a , the protein level of -arrestin1 or -2 was selectively reduced by -arrestin1 sirna or -arrestin2 sirna , respectively , but not by a scrambled sirna . the -me-5ht - induced activation of erk1/2 was markedly blocked by knockdown of -arrestin1 or -2 ( fig . -arrestin is highly expressed in postsynaptic synapses of glutamatergic neurons ( 33 ) , where both 5-ht receptors and nmda receptors are abundant . to test whether 5-ht2 activation of erk is required for the counteracting effect of 5-ht2 on 5-ht1a modulation of nmdar channels , we examined the effect of 8-oh - dpat on nmdar currents in the presence of -me-5ht in pfc cultures transfected with sirna against -arrestin1 or -2 . no significant differences in nmdar current densities were found in neurons with different transfections ( gfp : 20.1 1.3 pa / picofarad , n = 11 ; -arrestin1 sirna : 20 1.2 pa / picofarad , n = 7 ; -arrestin2 sirna : 19.5 0.8 pa / picofarad , n = 11 ) . in addition , the effect of 8-oh - dpat on nmdar currents was the same in all experimental groups . however , -me-5ht failed to counteract the 8-oh - dpat reduction of nmdar currents in gfp - positive neurons transfected with -arrestin1 or -arrestin2 sirna ( fig . 6d : -arrestin2 sirna : 20.0 1.1% , n = 11 ) , as compared with neurons transfected with gfp alone ( 7.4 1.2% ; taken together , these data suggest that -arrestins are involved in the counteractive effect of 5-ht2 on 5-ht1a regulation of nmdar currents in pfc neurons . because dynamin - dependent endocytosis of g protein - coupled receptor is required for g protein - coupled receptor/-arrestin - induced erk signaling ( 34 ) , we further examined the role of dynamin in the counteractive effect of 5-ht2 on 5-ht1a regulation of nmdar currents . pfc cultures were treated with a dynamin inhibitory peptide , which competes for binding to amphiphysin and hence inhibits the clathrin / dynamin - dependent endocytosis ( 35 ) . as shown in fig . 7 ( a and b ) , treatment of pfc cultures with the membrane - permeable dynamin inhibitory peptide ( 50 m ) markedly blocked the -me-5ht - induced erk activation , whereas the membrane - impermeable dynamin inhibitory peptide or a cell - permeable scrambled control peptide was ineffective . furthermore , in neurons injected with the dynamin inhibitory peptide ( 50 m ) , -me-5ht failed to counteract the 5-ht1a reduction of nmdar - epsc ( 35.8 6.3% , n = 5 ( fig . 7 , c and d ) ) . together , these data suggest that dynamin - based 5-ht2 receptor endocytosis is involved in 5-ht2 activation of erk and 5-ht2 opposing of 5-ht1a regulation of nmdar currents . figure 8.inhibition of src prevents 5-ht2a / c from opposing 5-ht1a regulation of nmdar currents . a , western blots of phospho - erk and total erk in pfc cultures treated with or without -me-5ht ( 20 m , 3 min ) in the absence or presence of the src kinase inhibitor pp2 or its inactive homolog pp3 ( both 10 m , added 30 min before -me-5ht treatment ) . c , plot of normalized peak nmdar - epsc showing the effect of 8-oh - dpat ( 20 m ) in the presence of -me-5ht ( 20 m ) in cells injected with pp2 ( 20 m ) or pp3 ( 20 m ) . inset , representative traces of nmdar - epsc ( average of three trials ) taken at time points denoted by # . d , cumulative data ( mean s.e . ) showing the percent reduction of nmdar - epsc by 8-oh - dpat in the absence or presence of -me-5ht under different conditions . * , 0.001 , anova . inhibition of src prevents 5-ht2a / c from opposing 5-ht1a regulation of nmdar currents . a , western blots of phospho - erk and total erk in pfc cultures treated with or without -me-5ht ( 20 m , 3 min ) in the absence or presence of the src kinase inhibitor pp2 or its inactive homolog pp3 ( both 10 m , added 30 min before -me-5ht treatment ) . c , plot of normalized peak nmdar - epsc showing the effect of 8-oh - dpat ( 20 m ) in the presence of -me-5ht ( 20 m ) in cells injected with pp2 ( 20 m ) or pp3 ( 20 m ) . inset , representative traces of nmdar - epsc ( average of three trials ) taken at time points denoted by # . d , cumulative data ( mean s.e . ) showing the percent reduction of nmdar - epsc by 8-oh - dpat in the absence or presence of -me-5ht under different conditions . * , previous studies have suggested that src - mediated tyrosine phosphorylation of dynamin is involved in g protein - coupled receptor - induced erk signaling ( 31 , 36 ) . to test whether src kinase activity is required for 5-ht2 activation of erk and the counteractive effect of 5-ht2 on 5-ht1a regulation of nmdar currents , we measured the effect of -me-5ht in pfc neurons treated with the src kinase inhibitor , pp2 . as shown in fig . 8 ( a and b ) , application of pp2 ( 20 m ) , but not the inactive analog pp3 ( 20 m ) , significantly blocked the -me-5ht - induced erk phosphorylation . the total erk level was not affected in cells subjected to different treatments . moreover , in the presence of pp2 , but not pp3 , -me-5ht lost the ability to oppose 8-oh - dpat reduction of nmdar - epsc ( fig . 8c : pp2 : 32.7 3.7% , n = 7 ; fig . 8d : pp3 : 8.8 3.3% , n = 4 ) . taken together , these results suggest that src kinase activation is involved in 5-ht2 signaling . activation of 5-ht2 receptors opposes 5-ht1a reduction of surface nr2b subunits in a -arrestin - dependent manner if 5-ht1a and 5-ht2a / c regulate microtubule dynamics in a counteractive manner , it is possible that they may alter nmdar trafficking on microtubules in an opposite way . to test this , we performed immunocytochemical experiments in cultured pfc neurons transfected with gfp - tagged nr2b subunits ( the gfp tag is positioned at the extracellular n terminus of nr2b ) . surface nr2b receptors were detected with the anti - gfp primary antibody , followed by rhodamine - conjugated secondary antibody in non - permeabilized conditions . consistent with our previous findings ( 21 ) , application of 8-oh - dpat ( 40 m , 5 min ) , caused a marked reduction in the number and size of surface nr2b clusters ( fig . 9 , a and b ) , as compared with control neurons ( cluster density : 43.6 1.7 clusters/50 m in controls versus 22.7 0.8 clusters/50 m in dpat - treated cells ; cluster size : 0.33 0.02 m in controls versus 0.18 0.02 m in dpat - treated cells ( fig . treatment with -me-5ht ( 40 m , 30 min ) significantly prevented the 8-oh - dpat - induced reduction of surface nr2b subunits ( cluster density : 40.0 2.7 clusters/50 m ; cluster size : 0.3 0.03 m ( fig . the -me-5ht treatment itself did not affect the distribution of surface nr2b clusters ( data not shown ) . the fluorescence intensity of surface nr2b clusters ( average gray value per pixel ) was not significantly changed in neurons subject to various treatments ( fig . next , we examined the signaling molecules involved in 5-ht2 blockade of 5-ht1a reduction of surface nr2b clusters . to test this , we co - transfected pfc cultures with -arrestin2 sirna and gfp - tagged nr2b subunits . as shown in fig . 9 ( d and e ) , transfection of -arrestin2 alone did not affect the distribution of surface nr2b clusters ( cluster density : 38.1 3.1 clusters/50 m ; cluster size : 0.29 0.01 m ( fig . 9 g ) ) or the reducing effect of 8-oh - dpat on nr2b clusters ( cluster density : 22.8 0.8 clusters/50 m ; cluster size : 0.16 0.03 m ( fig . 9 g ) ) . however , knockdown of -arrestin2 prevented -me-5ht from blocking the 8-oh - dpat - induced reduction of surface nr2b clusters on dendrites ( cluster density : 23.4 1.6 clusters/50 m ; cluster size : 0.17 0.02 m ( fig . these results suggest that activation of 5-ht2 receptors opposes 5-ht1a reduction of the surface nr2b level through a -arrestin - dependent mechanism . finally , we tested whether the change on nmdar - epsc amplitudes in pfc neurons by acute fluoxetine treatment of intact animals can be accounted for by the altered number of nmda receptors on the cell membrane . surface biotinylation experiments ( 23 ) were performed to measure levels of surface nr1 and nr2b in pfc slices from animals intraperitoneally injected with fluoxetine ( 20 mg / kg ) . surface proteins were labeled with sulfo - nhs - lc - biotin , and then biotinylated surface proteins were separated from non - labeled intracellular proteins by reaction with neutravidin beads . surface and total proteins were subjected to electrophoresis and probed with an antibody against the nr1 or nr2b subunit . as shown in fig . 10 ( a and b ) , the surface levels of nr1 and nr2b in pfc slices were significantly lower in animals exposed to fluoxetine ( nr1 : 70 3% of control ; nr2b : 60 6% of control ; n = 4 ) . consistent with electrophysiological results , the fluoxetine - induced reduction of surface nmdars was more prominent in animals co - injected with the 5-ht2a / c antagonist ketanserin ( nr1 : 29 3% of control ; nr2b : 30 1% of control ; n = 4 ) and was largely blocked in animals co - injected with the 5-ht1a antagonist way-100635 ( nr1 : 82 5% of control ; nr2b : 84 1% of control ; n = 4 ) . the surface gabaar 2 subunit level , as well as the total nr1 or nr2b level , in pfc slices was unchanged by any of these drug administrations . taken together , these results suggest that endogenous serotonin regulates pfc nmdar surface expression in vivo through 5-ht1a and 5-ht2a / c receptors in a counteractive manner . figure 9.activation of 5-ht2a / c receptors opposes the 5-ht1a -induced decrease of surface nr2b clusters on neuronal dendrites . a f , immunocytochemical images of surface gfp - nr2b clusters in pfc cultures transfected with or without-arrestin2 sirna . cultures were untreated ( control ) or treated with 8-oh - dpat ( 40 m , 5 min ) in the absence or presence of -me-5ht ( 20 m , added 10 min before 8-oh - dpat treatment ) . enlarged versions of the boxed regions of dendrites are shown beneath each of the images . g , quantitative analysis of surface gfp - nr2b clusters ( cluster density , size , and intensity ) along dendrites under different treatments . * , p < 0.01 , anova . activation of 5-ht2a / c receptors opposes the 5-ht1a -induced decrease of surface nr2b clusters on neuronal dendrites . a f , immunocytochemical images of surface gfp - nr2b clusters in pfc cultures transfected with or without-arrestin2 sirna . cultures were untreated ( control ) or treated with 8-oh - dpat ( 40 m , 5 min ) in the absence or presence of -me-5ht ( 20 m , added 10 min before 8-oh - dpat treatment ) . enlarged versions of the boxed regions of dendrites are shown beneath each of the images . g , quantitative analysis of surface gfp - nr2b clusters ( cluster density , size , and intensity ) along dendrites under different treatments . * , p < 0.01 , anova . nr1 and nr2 subunits have to be co - assembled before leaving the endoplasmic reticulum and being transported along microtubules in dendrites to synapses . our previous study ( 21 ) shows that 5-ht1a receptors primarily target nr2b subunit - containing nmda receptors , consistent with nr2b being the cargo of the microtubule motor kif17 . with the application of fluoxetine plus ketanserin , the surface levels of nr1 and nr2b were reduced to a similar degree ( surface nr2a was also reduced , data not shown ) . it suggests that this reduction likely results from endogenous nr1/nr2b heteromers and nr1/nr2a / nr2b triheteromers . it is well known that many antidepressants and antipsychotics exert their actions by inhibiting serotonin reuptake and thus enhance serotonergic transmission ( 37 , 38 ) . because serotonin has multiple receptor subtypes , systemic elevation of serotonin can induce diverse physiological effects in neurons ( 2 ) . the molecular mechanisms for serotonin to regulate cellular targets through different subtypes of receptors remain to be identified . we have previously found that 5-ht2 and 5-ht4 receptors are linked to regulate gabaa receptors ( 9 , 39 ) , whereas 5-ht1a receptors are linked to regulate nmda receptors in pfc neurons ( 21 ) . the specific coupling of different 5-ht receptors to distinct ion channels allows serotonin to regulate multiple targets in a precise but flexible manner . here , we provide evidence showing that the 5-ht1a regulation of nmdars can be modified by 5-ht2a / c receptor activation , which provides a mechanism to fine - tune the effect of serotonin on nmdar - mediated synaptic transmission and plasticity . under normal physiological conditions , which of the one or more 5-ht receptors in pfc pyramidal neurons that are activated by serotonin is determined by the serotonergic projection from dorsal raphe to cellular compartments rich in different subtypes of receptors ( 40 ) . agonist binding studies indicate that 5-ht1 and 5-ht2 receptors have different affinities ( nanomolar versus low micromolar ) to 5-ht , suggesting that 5-ht1 receptor activation may play a dominant role in response to serotonin . microdialysis studies have shown that synaptic concentration of serotonin can reach up to 6 mm ( 41 ) , suggesting that sometimes all 5-ht receptors could be fully activated at synapses . both 5-ht1a and 5-ht2a receptors are expressed at dendritic shafts and spines of pfc pyramidal neurons ( 42 , 43 ) , where nmda receptors are abundant , prompting us to speculate that both receptors may interact with nmda receptors in synergistic or opposite ways . our studies in pfc pyramidal neurons from both acute slices and intact animals treated with various 5-ht - related drugs indicate that blocking 5-ht2a / c receptors unmasks the ability of 5-ht1a receptors to reduce nmdar currents , suggesting that 5-ht1a and 5-ht2a / c receptors converge on nmdar channels in a counteractive manner . similar to serotonin , it has been shown that , in response to dopamine , d1 and d2 receptors that are linked to distinct signaling cascades regulate cortical gabaergic inhibition in an opposing manner ( 44 ) . figure 10.the surface nmdar level is reduced in pfc from fluoxetine - injected animals . a , western blot analysis showing the surface and total nr1 , nr2b , and gabaar 2 subunits in lysates of pfc slices taken from animals receiving a single intraperitoneal injection of saline , fluoxetine , ketanserin , ketanserin plus fluoxetine , way-100635 , or way-100635 plus fluoxetine ( all drugs are 20 mg / kg ) . b , quantification of surface nmdar subunits in pfc slices from animals treated with different drugs . * , p < 0.01 ; * * , p < 0.001 ; anova . the surface nmdar level is reduced in pfc from fluoxetine - injected animals . a , western blot analysis showing the surface and total nr1 , nr2b , and gabaar 2 subunits in lysates of pfc slices taken from animals receiving a single intraperitoneal injection of saline , fluoxetine , ketanserin , ketanserin plus fluoxetine , way-100635 , or way-100635 plus fluoxetine ( all drugs are 20 mg / kg ) . b , quantification of surface nmdar subunits in pfc slices from animals treated with different drugs . * , p < 0.01 ; * * , p < 0.001 ; anova . our previous study has shown that activation of 5-ht1a receptors suppresses nmdar currents by reducing microtubule stability and the ensuing nmdar trafficking along dendritic microtubules ( 21 ) . in this study , we found that activation of 5-ht2a / c receptor opposes the ability of 5-ht1a to induce microtubule depolymerization , suggesting that microtubule dynamics and the microtubule - based nmdar transport are regulated by 5-ht1a and 5-ht2a / c receptors in a counteractive fashion . the stability of microtubules is regulated by different microtubule - associated proteins ( maps ) in distinct neuronal compartments . the phosphorylation state of map2 , a dendrite - specific map , determines the ability of map2 to associate and stabilize dendritic microtubules ( 45 ) . our previous study suggests that 5-ht1a activation suppresses erk activity , which leads to the decreased map2 phosphorylation , map2-microtubule interaction , and microtubule stability ( 21 ) . it is possible that 5-ht1a and 5-ht2a / c oppositely regulates erk activity , thereby controlling microtubule stability in a counteractive manner . consistently , our biochemical and immunocytochemical data show that 5-ht2a / c activation potently enhances erk activity in the dendrites of pfc cultures . phospholipase c and inositol 1,4,5-trisphosphate , two downstream molecules of classic 5-ht2 signaling , are not involved in this regulation ( data not shown ) . recent studies have suggested that the scaffolding protein -arrestin , which binds to the third intracellular loop of certain g protein - coupled receptors , induces erk activation ( 31 , 32 ) . 5-ht2a receptors are found to bind purified -arrestin ( 46 ) . however , whether 5-ht2a activates mitogen - activated protein kinase via -arrestin pathway is essentially unknown . our data show that knockdown of -arrestin1/2 not only blocks the 5-ht2a / c - induced erk activation but also eliminates the counteractive effect of 5-ht2a / c on 5-ht1a reduction of nmdar currents . in addition , we found that the 5-ht2a / c action is dependent on src activation and clarthin / dynamin - mediated endocytosis of the receptor . taken together , 5-ht2a , via the -arrestin / src / dynamin cascade , induces erk activation to oppose the effect of 5-ht1a on nmdar functions . several mechanisms have been proposed for the regulation of nmdar functions , including altering the phosphorylation state and biophysical properties of the channel ( 47 , 48 ) and changing nmdar trafficking and channel numbers at the membrane ( 49 , 50 ) . our previous finding suggests that 5-ht1a decreases surface nr2b clusters in a microtubule - dependent manner ( 21 ) , consistent with the role of cytoskeleton - based transport on nmdar insertion to the plasma membrane ( 51 , 52 ) . our immunocytochemical data show that the 5-ht1a reduction of surface nr2b clusters is attenuated by pretreatment with a 5-ht2a / c agonist , confirming that 5-ht1a and 5-ht2a / c oppositely regulate nmdar trafficking . moreover , 5-ht2a / c opposes the 5-ht1a reduction of surface nr2b clusters in a -arrestin - dependent manner , consistent with the -arrestin dependence of the 5-ht2a / c action on 5-ht1a regulation of nmdar currents . surface biotinylation experiments using pfc from intact animals subject to acute fluoxetine treatment have also confirmed the results found in cultured neurons . based on the experimental data , we speculate that , in response to serotonin , both 5-ht1a and 5-ht2a / c receptors localized in pfc pyramidal neurons are activated , which converge to regulate nmdar trafficking and function in an opposite manner , by coupling to distinct signaling cascades and differentially affecting microtubule stability . this study may provide significant insights into the complex regulation of nmdar - mediated synaptic transmission and plasticity by different serotonin receptors in the pfc network .

abnormal serotonin - glutamate interaction in prefrontal cortex ( pfc ) is implicated in the pathophysiology of many mental disorders , including schizophrenia and depression . however , the mechanisms by which this interaction occurs remain unclear . our previous study has shown that activation of 5-ht1a receptors inhibits n - methyl - d - aspartate ( nmda ) receptor ( nmdar ) currents in pfc pyramidal neurons by disrupting microtubule - based transport of nmdars . here we found that activation of 5-ht2a / c receptors significantly attenuated the effect of 5-ht1a on nmdar currents and microtubule depolymerization . the counteractive effect of 5-ht2a / c on 5-ht1a regulation of synaptic nmdar response was also observed in pfc pyramidal neurons from intact animals treated with various 5-ht - related drugs . moreover , 5-ht2a / c stimulation triggered the activation of extracellular signal - regulated kinase ( erk ) in dendritic processes . inhibition of the -arrestin / src / dynamin signaling blocked 5-ht2a / c activation of erk and the counteractive effect of 5-ht2a / c on 5-ht1a regulation of nmdar currents . immunocytochemical studies showed that 5-ht2a / c treatment blocked the inhibitory effect of 5-ht1a on surface nr2b clusters on dendrites , which was prevented by cellular knockdown of -arrestins . taken together , our study suggests that serotonin , via 5-ht1a and 5-ht2a / c receptor activation , regulates nmdar functions in pfc neurons in a counteractive manner . 5-ht2a / c , by activating erk via the -arrestin - dependent pathway , opposes the 5-ht1a disruption of microtubule stability and nmdar transport . these findings provide a framework for understanding the complex interactions between serotonin and nmdars in pfc , which could be important for cognitive and emotional control in which both systems are highly involved .

EXPERIMENTAL PROCEDURES RESULTS DISCUSSION

the internal solution contained ( in mm ) : 180 n - methyl - d - glucamine , 4 mgcl2 , 40 hepes , 0.5 1,2-bis(2-aminophenoxy)ethane - n , n , n,n-tetraacetic acid , 12 phosphocreatine , 3 na2atp , and 0.5 na2gtp , 0.1 leupeptin , ph 7.2 - 3 , 265270 mosm / liter . figure 1.activation of 5-ht2a / c receptors counteracts the 5-ht1a -induced reduction of nmdar - mediated ionic and synaptic currents in pfc pyramidal neurons . e , plot of peak nmdar - epsc showing the effect of 8-oh - dpat ( 20 m ) in the presence of -me-5ht ( 20 m ) and ketanserin ( 5-ht2a / c antagonist , 20 m ) . activation of 5-ht2a / c receptors counteracts the 5-ht1a -induced reduction of nmdar - mediated ionic and synaptic currents in pfc pyramidal neurons . e , plot of peak nmdar - epsc showing the effect of 8-oh - dpat ( 20 m ) in the presence of -me-5ht ( 20 m ) and ketanserin ( 5-ht2a / c antagonist , 20 m ) . activation of 5-ht1a and 5-ht2a / c receptors regulates nmdar - mediated ionic and synaptic currents in a counteractive manner we have previously found that activation of 5-ht1a receptors reduces nmdar currents in pfc pyramidal neurons ( 21 ) . to examine the potential interactions between 5-ht1a and 5-ht2a / c receptors on nmdar functions , we tested the impact of 5-ht2 activation on 5-ht1a regulation of nmdar currents by preincubating pfc cultures with the 5-ht2a / c agonist -me-5ht ( 20 m , 1030 min ) . another 5-ht2a / c agonist doi also had no effect on nmdar currents at low doses ( 0.05 m : 4.2 1.2% , n = 5 ; 0.1 m : 4.0 2.5% , n = 5 ) , which are different from the inhibitory effect of doi shown before ( 26 ) . our results suggest that 5-ht2a / c activation alone does not directly affect nmdar currents but opposes 5-ht1a regulation of nmdar currents in pfc pyramidal neurons . figure 2.inhibition of 5-ht2a / c receptors unmasks the 5-ht1a -mediated reduction of nmdar - epsc in response to 5-ht . a , representative nmdar - epsc traces ( average of three trials ) showing the regulation of nmdar - epsc by 5-ht ( 10 , 40 , or 100 m ) in neurons treated with or without the 5-ht2a / c antagonist ketanserin ( ket , 20 m ) . inhibition of 5-ht2a / c receptors unmasks the 5-ht1a -mediated reduction of nmdar - epsc in response to 5-ht . a , representative nmdar - epsc traces ( average of three trials ) showing the regulation of nmdar - epsc by 5-ht ( 10 , 40 , or 100 m ) in neurons treated with or without the 5-ht2a / c antagonist ketanserin ( ket , 20 m ) . * , we further tested the influence of 5-ht2 activation on 5-ht1a regulation of nmdar - epsc mediated by synaptic nmda receptors in pfc slices . 1e , -me-5ht failed to oppose the effect of 5-ht1a on nmdar - epsc in the presence of ketanserin ( 36.8 7.1% ; n = 5 ( fig . these results indicate that 5-ht2a / c receptor activation selectively counteracts 5-ht1a regulation of nmdar functions in pfc pyramidal neurons . however , in the presence of the 5-ht2a / c antagonist ketanserin ( 20 m ) , the reduction of nmdar - epsc by serotonin was greatly augmented ( 10 m : 35.2 2.2% , n = 7 ; 40 m : 43.0 2.0% , n = 6 ; 100 m:47 1.9% , n = 7 ) . it suggests that 5-ht2 receptor activation in response to serotonin masks part of the inhibitory action of 5-ht1a receptors on nmdar channels . to assess whether the serotonergic regulation of nmdars , which we found in vitro , is also occurring in vivo , we tested whether endogenous serotonin , via the activation of 5-ht1a and 5-ht2a / c receptors , could regulate nmdar functions in a similar manner in intact animals . these results indicate that activation of 5-ht1a receptors in vivo down - regulates nmdar functions , and this effect of 5-ht1a is opposed by 5-ht2a / c receptor activation in vivo . figure 3.the synaptic nmda response is reduced in pfc pyramidal neurons from animals with systemic administration of 5-ht1a agonist or the serotonin re - uptake inhibitor fluoxetine , which is opposed by injecting 5-ht2a / c agonist . c , plot of normalized peak nmdar - epsc showing the effect of bath application of 8-oh - dpat ( 40 m ) in pfc pyramidal neurons from animals intraperitoneally injected with fluoxetine ( 20 mg / kg ) or saline . the synaptic nmda response is reduced in pfc pyramidal neurons from animals with systemic administration of 5-ht1a agonist or the serotonin re - uptake inhibitor fluoxetine , which is opposed by injecting 5-ht2a / c agonist . c , plot of normalized peak nmdar - epsc showing the effect of bath application of 8-oh - dpat ( 40 m ) in pfc pyramidal neurons from animals intraperitoneally injected with fluoxetine ( 20 mg / kg ) or saline . figure 4.activation of 5-ht2a / c receptors attenuates the 5-ht1a -induced microtubule depolymerization and the effect of microtubule depolymerizer on nmdar - epsc . c , plot of normalized peak nmdar - epsc showing the effect of microtubule depolymerizer colchicine ( 30 m ) in the absence or presence of -me-5ht ( 20 m ) . activation of 5-ht2a / c receptors attenuates the 5-ht1a -induced microtubule depolymerization and the effect of microtubule depolymerizer on nmdar - epsc . c , plot of normalized peak nmdar - epsc showing the effect of microtubule depolymerizer colchicine ( 30 m ) in the absence or presence of -me-5ht ( 20 m ) . moreover , the reducing effect of fluoxetine on nmdar - epsc was potentiated by injecting the 5-ht2a / c antagonist ketanserin ( ket : 392 12.3 pa , n = 11 ; ket + fluox : 59.6 5.2 pa , n = 12 ; p < 0.001 , anova ) , revealing the counteracting effect of 5-ht2a / c on 5-ht1a regulation of synaptic nmda responses . in contrast to the strong effect in pfc pyramidal neurons , fluoxetine injection failed to affect nmdar - epsc in striatal medium spiny neurons ( saline : 375 15.9 pa , n = 8 ; fluox : 351 13.9 pa , n = 10 ) , suggesting the specificity of serotonergic regulation of nmdars in pfc . figure 5.activation of 5-ht2a / c receptors induces the phosphorylation and activation of erk in neuronal dendrites in cultured pfc neurons . activation of 5-ht2a / c receptors induces the phosphorylation and activation of erk in neuronal dendrites in cultured pfc neurons . figure 6.the counteractive effect of 5-ht2a / c on 5-ht1a regulation of nmdar function is dependent on the -arrestin / erk pathway . the counteractive effect of 5-ht2a / c on 5-ht1a regulation of nmdar function is dependent on the -arrestin / erk pathway . we found that the amplitude of nmdar - epsc in pfc pyramidal neurons localized at the proximity of injection sites was significantly smaller , compared with saline controls ( saline : 401 10.1 pa , n = 11 ; fluox : 162 13.2 pa , n = 11 , p < 0.001 , anova ) . the opposing regulation of nmda receptors by 5-ht1a and 5-ht2 receptors depends on microtubule stability next , we investigated the potential mechanism by which 5-ht2 receptors counteract the effect of 5-ht1a on nmdar currents in pfc neurons . we then tested whether the counteractive effect of 5-ht2 on 5-ht1a regulation of nmdar currents is due to the opposing regulation of microtubule dynamics by 5-ht1a and 5-ht2 receptors . these results suggest that activation of 5-ht1a and 5-ht2 receptors could regulate nmdar currents in a counteractive manner by converging on microtubule dynamics . moreover , our previous findings have suggested that 5-ht1a reduces erk activity , which in turn reduces map2 phosphorylation and microtubule stability ( 21 ) . to test this , we measured the activation of erk1/2 in response to 5-ht2a / c agonists with an antibody that recognizes activated ekr1/2 , which are doubly phosphorylated at thr-202/tyr-204 in the activation loop of the kinases ( 29 ) . figure 7.the counteractive effect of 5-ht2a / c on 5-ht1a regulation of nmdar function involves clathrin / dynamin - mediated endocytosis . a , western blots of phospho - erk and total erk in pfc cultures treated with or without -me-5ht ( 20 m , 3 min ) in the absence or presence of the cell - permeable ( myristoylated ) or non - permeable dynamin inhibitory peptide ( both 50 m ) , or a cell - permeable scrambled control peptide ( 50 m ) . * , the counteractive effect of 5-ht2a / c on 5-ht1a regulation of nmdar function involves clathrin / dynamin - mediated endocytosis . a , western blots of phospho - erk and total erk in pfc cultures treated with or without -me-5ht ( 20 m , 3 min ) in the absence or presence of the cell - permeable ( myristoylated ) or non - permeable dynamin inhibitory peptide ( both 50 m ) , or a cell - permeable scrambled control peptide ( 50 m ) . * , the -arrestin - mediated pathway is involved in the counteractive effects of 5-ht2 receptors on 5-ht1a regulation of nmdar currents next , we sought to identify the signaling mechanism underlying 5-ht2 activation of erk . to test whether 5-ht2 activation of erk is required for the counteracting effect of 5-ht2 on 5-ht1a modulation of nmdar channels , we examined the effect of 8-oh - dpat on nmdar currents in the presence of -me-5ht in pfc cultures transfected with sirna against -arrestin1 or -2 . 6d : -arrestin2 sirna : 20.0 1.1% , n = 11 ) , as compared with neurons transfected with gfp alone ( 7.4 1.2% ; taken together , these data suggest that -arrestins are involved in the counteractive effect of 5-ht2 on 5-ht1a regulation of nmdar currents in pfc neurons . because dynamin - dependent endocytosis of g protein - coupled receptor is required for g protein - coupled receptor/-arrestin - induced erk signaling ( 34 ) , we further examined the role of dynamin in the counteractive effect of 5-ht2 on 5-ht1a regulation of nmdar currents . together , these data suggest that dynamin - based 5-ht2 receptor endocytosis is involved in 5-ht2 activation of erk and 5-ht2 opposing of 5-ht1a regulation of nmdar currents . figure 8.inhibition of src prevents 5-ht2a / c from opposing 5-ht1a regulation of nmdar currents . a , western blots of phospho - erk and total erk in pfc cultures treated with or without -me-5ht ( 20 m , 3 min ) in the absence or presence of the src kinase inhibitor pp2 or its inactive homolog pp3 ( both 10 m , added 30 min before -me-5ht treatment ) . inhibition of src prevents 5-ht2a / c from opposing 5-ht1a regulation of nmdar currents . a , western blots of phospho - erk and total erk in pfc cultures treated with or without -me-5ht ( 20 m , 3 min ) in the absence or presence of the src kinase inhibitor pp2 or its inactive homolog pp3 ( both 10 m , added 30 min before -me-5ht treatment ) . to test whether src kinase activity is required for 5-ht2 activation of erk and the counteractive effect of 5-ht2 on 5-ht1a regulation of nmdar currents , we measured the effect of -me-5ht in pfc neurons treated with the src kinase inhibitor , pp2 . activation of 5-ht2 receptors opposes 5-ht1a reduction of surface nr2b subunits in a -arrestin - dependent manner if 5-ht1a and 5-ht2a / c regulate microtubule dynamics in a counteractive manner , it is possible that they may alter nmdar trafficking on microtubules in an opposite way . however , knockdown of -arrestin2 prevented -me-5ht from blocking the 8-oh - dpat - induced reduction of surface nr2b clusters on dendrites ( cluster density : 23.4 1.6 clusters/50 m ; cluster size : 0.17 0.02 m ( fig . these results suggest that activation of 5-ht2 receptors opposes 5-ht1a reduction of the surface nr2b level through a -arrestin - dependent mechanism . taken together , these results suggest that endogenous serotonin regulates pfc nmdar surface expression in vivo through 5-ht1a and 5-ht2a / c receptors in a counteractive manner . figure 9.activation of 5-ht2a / c receptors opposes the 5-ht1a -induced decrease of surface nr2b clusters on neuronal dendrites . activation of 5-ht2a / c receptors opposes the 5-ht1a -induced decrease of surface nr2b clusters on neuronal dendrites . here , we provide evidence showing that the 5-ht1a regulation of nmdars can be modified by 5-ht2a / c receptor activation , which provides a mechanism to fine - tune the effect of serotonin on nmdar - mediated synaptic transmission and plasticity . our studies in pfc pyramidal neurons from both acute slices and intact animals treated with various 5-ht - related drugs indicate that blocking 5-ht2a / c receptors unmasks the ability of 5-ht1a receptors to reduce nmdar currents , suggesting that 5-ht1a and 5-ht2a / c receptors converge on nmdar channels in a counteractive manner . our previous study has shown that activation of 5-ht1a receptors suppresses nmdar currents by reducing microtubule stability and the ensuing nmdar trafficking along dendritic microtubules ( 21 ) . in this study , we found that activation of 5-ht2a / c receptor opposes the ability of 5-ht1a to induce microtubule depolymerization , suggesting that microtubule dynamics and the microtubule - based nmdar transport are regulated by 5-ht1a and 5-ht2a / c receptors in a counteractive fashion . our previous study suggests that 5-ht1a activation suppresses erk activity , which leads to the decreased map2 phosphorylation , map2-microtubule interaction , and microtubule stability ( 21 ) . it is possible that 5-ht1a and 5-ht2a / c oppositely regulates erk activity , thereby controlling microtubule stability in a counteractive manner . consistently , our biochemical and immunocytochemical data show that 5-ht2a / c activation potently enhances erk activity in the dendrites of pfc cultures . our data show that knockdown of -arrestin1/2 not only blocks the 5-ht2a / c - induced erk activation but also eliminates the counteractive effect of 5-ht2a / c on 5-ht1a reduction of nmdar currents . in addition , we found that the 5-ht2a / c action is dependent on src activation and clarthin / dynamin - mediated endocytosis of the receptor . taken together , 5-ht2a , via the -arrestin / src / dynamin cascade , induces erk activation to oppose the effect of 5-ht1a on nmdar functions . our previous finding suggests that 5-ht1a decreases surface nr2b clusters in a microtubule - dependent manner ( 21 ) , consistent with the role of cytoskeleton - based transport on nmdar insertion to the plasma membrane ( 51 , 52 ) . our immunocytochemical data show that the 5-ht1a reduction of surface nr2b clusters is attenuated by pretreatment with a 5-ht2a / c agonist , confirming that 5-ht1a and 5-ht2a / c oppositely regulate nmdar trafficking . moreover , 5-ht2a / c opposes the 5-ht1a reduction of surface nr2b clusters in a -arrestin - dependent manner , consistent with the -arrestin dependence of the 5-ht2a / c action on 5-ht1a regulation of nmdar currents . based on the experimental data , we speculate that , in response to serotonin , both 5-ht1a and 5-ht2a / c receptors localized in pfc pyramidal neurons are activated , which converge to regulate nmdar trafficking and function in an opposite manner , by coupling to distinct signaling cascades and differentially affecting microtubule stability .

even though chronic obstructive pulmonary disease ( copd ) is one of the leading chronic disorders in malaysia , many cases are not diagnosed at an early stage . this is mainly due to patient ignorance , because often the symptoms ( ie , dyspnea and chronic cough ) are considered to be part of normal aging , and thus , medical attention is not sought.1 coughing and the production of sputum are accepted by most smokers as a natural side effect of smoking instead of a possible sign of the initial development of a lung disorder . the prevalence of moderate - to - severe copd in those patients 30 years and above in malaysia was projected to be 4.7% in 2003 , which translates to 448,000 cases , but this figure is now expected to be larger due to population aging.2 many prevalence studies have shown that the percentage of potentially undiagnosed airflow obstruction in both western35 and asian countries is approximately 3%15%.6 for example , in spain , 14.3% of men and 3.9% of women aged 4079 years have been reported to have some form of obstructed airflow,4 while the prevalence of copd in individuals aged between 40 and 80 years is 10.2% and increases with age , smoking consumption , and lower educational levels.7 in a general population - based sample in the uk , 9.9% of patients were observed to have airflow obstruction , with 52% of cases being undiagnosed.3 in the us , meanwhile , the percentages of white men and women aged 45 years and above having obstructive airflow were 14.2% and 9.9% , respectively , as noted in the national health and nutrition examination survey.5 finally , a study in korea observed undiagnosed airflow obstruction in 12.4% of men and 3.5% of women.6 despite the existence of evidence - based validated spirometry as a diagnostic method , many patients with copd continue to be undiagnosed or misdiagnosed.8 most undiagnosed copd patients are found to have been exposed to cigarettes for a period of time in their life.912 buffels et al13 showed that the screening of smokers and ex - smokers revealed a prevalence of copd of 7.4%18% in individuals aged 3570 years . in those who smoked , there was a 2.3-fold excess in its prevalence when three or more symptoms ( chronic cough , wheezing , and shortness of breath ) were present , compared to when these symptoms were absent.6 likewise , a study by coultas et al5 revealed that subjects with undiagnosed airflow obstruction had a higher prevalence of smoking ( 82.3% ) than those subjects with no airflow obstruction ( 54.2%).5 for current smokers , early detection with spirometry and intervention has been recommended by the us preventive services task force for the prevention of copd.14 it should be noted , however , that although screening with spirometry is a good way to diagnose and classify copd according to air - flow limitation,8 the malaysian clinical practice guidelines ( cpg ) for copd15 do not recommend the indiscriminate use of spirometry for the purpose of screening potential patients for copd , since it is labor intensive and time consuming to screen every patient at risk of developing copd with spirometry , which makes such a broad screening program not cost effective . the us preventive services task force also discourages the use of spirometry for asymptomatic screening for copd.14 similarly , the clinical practice guideline update from the american college of physicians , american college of chest physicians , american thoracic society , and european respiratory society each state that there is no proven benefit of using spirometry to screen adults who have no smoking history and no respiratory symptoms.16 early diagnosis may not reverse the disease but does at least offer the opportunity to improve a patient s quality of life by reducing the symptoms and preventing progression of the disease . once it reaches its more advanced stages , there is an increased risk of developing other comorbidities with an associated reduction in the quality of life of the patient , and an increase in the costs of treatment . there is , therefore , a need for a simple method to help identify persons who might have the early stages of copd . in places where spirometry is not available such as in remote areas , alternative options to the doctor visit include copd questionnaires followed by the use of handheld spirometric devices ( hsds ) as a guide in choosing the correct treatment for the patient.1719 according to the global initiative for chronic obstructive lung disease ( gold ) , there are four stages of copd , based on the air flow limitation at forced expiratory volume in 1 second ( fev1 ) on the spirometry test . simple validated questionnaires , such as the copd population screener ( copd - ps ) questionnaire,20 diagnostic score for copd ( ds - copd),21 and the international primary care airways guidelines ( ipag ) questionnaire12,15 have been developed , and hsds ( vitalograph copd-622 and the piko-612,23 ) have been shown to provide a precise screening tool for people at risk of developing copd . a recent multicenter , cluster - randomized study ( search1 ) involving 8,770 volunteers has shown that dual - combination assessment using questionnaire screening and hsd offered better copd detection than the use of either method in isolation.19 based on the above result , the hypothesis of this research was that the combination of the copd - ps questionnaire and the vitalograph copd-6 represented an accurate tool to detect patients at risk of copd ( ie , those with a score of 5 for the questionnaire and a ratio of fev1/forced expiratory volume in 6 seconds ( fev6 ) of < 0.75 ) . the rationale for choosing what we have termed the dual vitalqplus tool rather than the combination of ipag with piko-6 is the easy availability of copd-6 in governmental health care facilities in malaysia . other than the choice of an hsd , the five - question copd - ps does not need any body weight , height , and body mass index calculations , in contrast to the eight - question ipag . due to the chronic progressive nature of the disease , the copd - ps questionnaire surveys a time frame focusing on 4 weeks of shortness of breath and 12 months of breathing problems.20 we proposed , therefore , to preselect patients using the copd - ps questionnaire based on age , smoking history , and symptoms , and then , for patients with a score of 5 , to undertake further tests with the vitalograph copd-6 . this dual - method assessment , which we have termed as vitalq - plus , may increase the likelihood of identifying potentially undiagnosed copd patients for further evaluation with diagnostic spirometry . it is worth noting that fev6 has been proven to be easily reproducible and can be used as surrogate parameter for forced vital capacity ( fvc).2427 a meta - analysis by jing et al summarized from eleven trials , concluded that fev1/fev6 is a sensitive and specific test and can be used as a valid alternative for fev1/fvc ( fev1/fvc < 0.70 as copd ) in the diagnosis of airway obstruction.25 estimates from this meta - analysis for the effectiveness of fev1/fev6 in the diagnosis of airway obstruction show a sensitivity of 89% and a specificity of 98% . fev1/fev6 had a sensitivity of 86.09% and a positive predictive value of 100% in the detection of airway obstruction when fev1/fvc is taken as the gold standard.28 the current study aimed to investigate and pilot the feasibility and application of dual - combination assessment vitalqplus as a screening tool for the early detection of copd , especially in primary care settings . this was a cross - sectional study to perform screening of potentially undiagnosed copd patients using a validated five - item questionnaire ( copd - ps ) and hsd ( copd-6 device ; model 4000 , vitalograph , inc , ennis , ireland ) on patients who were 35 years old and above , were smokers or ex - smokers , and were without any previous medical diagnosis of respiratory disease . patients were excluded if they were pregnant or if they had been prescribed an inhaler ( such as a bronchodilator and/or glucocorticoid ) . smoking status was measured according to the world health organization ( who ) smoking and tobacco use policy . a smoker was defined as someone who smokes any tobacco product , either daily or occasionally.22 hsds use the ratio of fev1/fev6 instead of the diagnostic ratio fev1/fvc in detecting the airflow limitation of patient.22 studies by rosa et al24 and represas represas et al29 have reported that the best sensitivity for the fev1/fev6 ratio for copd-6 is > 0.70 . additionally , the fev1/fev6 ratio is normally higher than the fev1/fvc ratio because fev6 only records the first 6 seconds of expiratory volume , while fvc accounts for the whole expiratory volume . the lungs of a healthy person can generally empty more than 80% of their volume in 6 seconds or less . due to this reason , we decided to choose fev1/fev6 < 0.75 as the cutoff point to detect copd . prior to the present study , we performed a preliminary study , which showed that fev1 and fev1/fev6 values measured by the copd-6 correlated well with those measured by a standard spirometer ( r=0.98 and r=0.99 , respectively ; n=19 ) . after answering the copd - ps , patients who were smokers and above the age of 35 years were asked to perform the forced expiratory maneuver using the hsd . the hsd had an accuracy of at least 3% of reading or 0.05 l with flows between 0 and 14 l / s.30 a new calibration was performed on each day of data collection . a measurement was deemed to be satisfactory when a beep sound was heard , indicating that expiration of at least 6 seconds had been achieved . symbol on the hsd screen was considered void , because this symbol indicated incorrect technique such as coughing , air leakage between the lips and the mouthpiece , and/or interruptions in blowing . potentially undiagnosed copd patients were also categorized according to the gold guidelines based on their fev1 percentage , so as to estimate patient staging in copd . a systematic sampling method was employed across three geographical regions in malaysia , including the northern ( kedah , perlis , and penang ) and central regions ( perak , selangor , negeri sembilan , and kuala lumpur ) of west malaysia ( peninsular malaysia ) as well as east malaysia ( sabah and sarawak ) . firstly , a list of governmental health care clinics was compiled by selecting at least three clinics from each state . the selected clinics were : 1 ) luyang district of sabah , 2 ) kampar district of perak , and 3 ) gelugor district of penang . all statistical analyses were carried out using spss software for windows version 16.0 ( spss inc . , the numerical data were expressed as mean standard deviation ( sd ) for parametric data , and median ( interquartile range ) for non - parametric data . the data were then further analyzed based on smoking status ( active smoker versus ex - smoker ) , location ( sabah versus perak and penang ) , and copd status ( potentially undiagnosed versus healthy / non - copd ) . pearson correlation was also used to analyze the relationship between age and fev6 and the fev1/fev6 ratio . descriptive analysis was performed for all potential covariates , outcome measures , and results . one - way analysis of variance ( anova ) was used to measure the difference in characteristics between the patients from three health centers . the kruskal this study was registered in the national malaysia research registry ( registration number nmrr-13 - 1195 - 17901 ) , and ethics approval was granted by the medical research ethics committee ( mrec ) of the ministry of health , malaysia . a total of 88 patients were recruited from the three different governmental primary care health centers situated in sabah , east malaysia ( n=66 ) ; in the state of perak , central region of west malaysia ( n=12 ) ; and in penang island , northern region of west malaysia ( n=10 ) . five patients were excluded because they were not able to perform the vitalograph or because they had a previous medical history of respiratory disease ( figure 1 ) . the characteristics of the patients in the three governmental primary care health centers are shown in table 1 . no statistically significant differences were observed between the three centers in the copd - ps scores , but there was a statistically significant difference in terms of fev1/fev6 ratio ( mean and median ) between the samples from sabah , perak , and penang ( p<0.05 ) . it must be noted that only 24.1% ( 20/83 ) patients were recruited from perak and penang compared to 75.9% ( 63/83 ) from sabah , mainly due to a lack of patients that fulfilled the inclusion criteria . at the copd questionnaire screening stage , 34.9% of the patients had a score of 5 , indicating that they had a potential chance of having copd . conversely , more than half of the patients had a normal score of < 5 . there was a moderate , negative correlation between the two variables ( r=0.368 , p<0.05 ) , with lower fev1 associated with older age . furthermore , it was found that the association with age is intermediate and negative in respect to fev6 ( r=0.324 , p<0.05 ) , and low in respect to fev1/fev6 ( r=0.163 , p0.05 ) . a summary of the results comparison is presented in table 2 . for fev1 and fev1/fev6 values , the group with fev1/fev6 < 0.75 was significantly smaller than the group with fev1/fev6 0.75 . median fev1 was 2.4 l ( range , 0.93.29 ) in the fev1/fev6 0.75 group , while the median fev1 for fev1/fev6 < 0.75 was 1.8 l ( range , 0.71.47 ) . the 63 patients in sabah had significantly lower average fev1 , compared to the 20 patients in perak and penang . this may be due to the higher numbers of elderly patients and active smokers in sabah compared to both perak and penang . an attempt was made to assess the impact of cigarette smoking by comparing active smokers with ex - smokers in terms of their risk of having undiagnosed copd , but in each case , the outcomes were not statistically significant ( table 2 ) . results of the comparison between healthy / non - copd subjects and those subjects with potentially undiagnosed copd are presented in table 3 . subjects that had positive outcomes on both tests ( ie , a copd - ps score 5 and a vitalograph score < 0.75 ) were classified as potentially experiencing undiagnosed copd , while subjects that had a negative outcome on both tests ( copd - ps score < 5 ; vitalograph score 0.75 ) were categorized as healthy / non - copd subjects . it is noteworthy that there were 28 subjects who were not classified in either category because they had either a copd - ps score < 5 and a vitalograph score < 0.75 ( n=6 ) , or a copd - ps score 5 and a vitalograph score 0.75 ( n=22 ) . the average age of the subjects in the potentially undiagnosed copd category was almost 20 years older than the subjects in the healthy group . other than significantly older age in the potentially undiagnosed copd group , fev1 , fev6 , and fev1/fev6 were also significantly lower than in the healthy / non - copd group . the percentage of active smokers was 85.7% in the potentially undiagnosed copd group , 16.9% more than in the healthy / non - copd group . in our current study , we found out that 15.7% ( n=83 ) of patients screened using copd-6 had fev1/fev6 < 0.75 , which was interpreted as suggesting a high potential for a positive diagnosis with copd . apart from copd-6 measurement , the copd - ps symptoms - based questionnaire found that 35.9% of patients screened had a score > 5 , indicating a need for further evaluation to confirm a copd diagnosis . when the copd - ps assessment was combined with copd-6 , the results showed that 8.4% of patients had both a copd - ps score of 5 and a copd-6 measurement of fev1/fev6 < 0.75 . our current study showed that symptomatic patients with a score of 5 had a 2.2 times higher chance of recording fev1/fev6 < 0.75 compared to patients without symptoms ( 24.1% versus 11.1% , respectively ) , which is consistent with the study by shin et al.6 preselection with a screening questionnaire may , therefore , reduce costs by allowing a more targeted choice of patients to undergo spirometry testing . in a study by salameh et al,21 us$4,150 was saved by combining spirometry and ds - copd questionnaires compared to the systematic use of spirometry alone in 100 patients . generally , spirometry testing in malaysia costs above rm1,000 ( equivalent to us$271)31 in the private sector and is free of charge in the government sector . due to differences between the apparatus used , the cost of the device to perform a vitalograph ( approximately rm600 ) is less than 1/10 the cost of using a spirometer ( approximately rm7,000 for a spirometer , which is equivalent to us$2,000).31 this means that the vitalograph is sufficiently affordable for general practitioners or community pharmacies to make this device available to detect potential copd patients.32 because spirometers are not controlled under medical services regulations , there are no restrictions on who can perform this testing . lung foundation australia makes screening with handheld devices available as a program in community pharmacies in australia.33 if this program were implemented in community pharmacies in malaysia , the screening charges to perform vitalograph testing should be approximately rm6.00 per person ( equivalent to us$1.70 per person).6 it must be noted that patient recruitment in the perak and penang states of malaysia was challenging . the average ratio of hospitals per district was 3.2:2.2:1.1 ( penang , perak , and sabah , respectively).34 the greater accessibility to health care services evident in perak and penang probably meant that the copd - prone patients in these regions had already been screened and had already received treatment for copd ( thus being excluded from selection for this study ) . based on our subgroup analysis in sabah , the high percentage of potentially undiagnosed copd in this area is worrying . apart from the lack of access to health care facilities , it is suspected that lower penetration of mass media coverage contributes to a lack of awareness regarding lung health and the harm posed by cigarette smoking in rural parts of sabah . the prevalence of smuggled , nontaxed cigarettes into major cities in sabah from the surrounding countries might also be a contributing factor.35,36 many trials have advocated different types of screening questionnaires12,20,3740 to select the most probable copd subjects , followed by spirometry to confirm the air obstruction diagnosis . a recent trial has also reported using a similar two - step approach with an ipag questionnaire and piko-6,12 instead of the copd - ps and copd-6 used in our present study . in the former study , a positive ipag questionnaire for possible copd ( 17 points ) was obtained in 594 ( 55.1% ) subjects,12 while our copd - ps questionnaire yielded a result of 34.9% ( scores 5 ) . with piko-6 ( a similar device to copd-6 ) , 139 ( 12.9% ) subjects fulfilled the criteria for possible copd ( fev1/fev6 < 0.7),12 while our present study revealed that 15.7% ( 13/83 ) of patients screened by copd-6 had fev1/fev6 < 0.75 . when combined assessment results were considered , our current study yielded 8.4% ( table 3 ) , and sichletidis et al s study12 yielded 10.4% , which could be due to the difference in mean age between sichletidis et al s cohort ( 65.311.4 years of age ) and our cohort ( 53.31.1 years of age ) . in spirometer - defined copd screening in smokers aged 40 years and without prior respiratory diagnosis , undiagnosed copd is 18.9% ( n=818).41 our current study yielded 15.7% ( 13/83 ) of potentially undiagnosed copd patients with the aid of copd-6 , 3.2% lower than reported by tinkelman et al.41 this could be due to the younger age cutoff in our present study : a 35-year - old cutoff rather than the 40-year - old cutoff in the tinkelman trial.41 previous studies have shown that up to 30% of smokers experienced undiagnosed copd,41 while undiagnosed copd in the overall population is between 10% and 20% . in a case - finding study,42 patients aged 4075 years , who were smokers with no previous diagnosis of pulmonary disease , but with acute respiratory infection , yielded a 27% incidence of undiagnosed copd . this percentage is almost twice as high as in our current study ( 27% versus 15.7% , respectively ) . another study of undiagnosed copd by vandevoorde et al found a 29.5% incidence ( n=146 ) in active smokers aged 4070 years who smoked at least 15 packs / year.26 a cross - sectional study in male smokers aged 4065 years yielded undiagnosed airway obstruction in approximately 29.9% of cases.9 this study used a questionnaire followed by spirometry for subjects without previous diagnosis of copd . in england , spirometry - defined , undiagnosed copd was found in 10.8% ( n=8,215 ) in subjects aged 35 years,11 while in spain , undiagnosed copd is approximately 7.04% ( n=4,035 ) in the age range of 4069 years.7 however , the studies conducted in england and spain recruited from the general population , while in our previous study , only smoking patients were recruited.43 in addition , lkke et al followed a general population sample for 25 years and also concluded that the absolute risk of developing copd among continuous smokers was at least 25%.44 a local pilot cross - sectional study conducted in malaysia by ching et al17 using a similar handheld spirometer managed to detect 10.6% copd patients . compared to our current study , their study did not exclude any copd patient or patients on inhalers . nevertheless , ching et al s findings17 are congruent with our study in showing that copd - positive patients are older on average than non - copd patients . the chances of participants in the older group being identified as a potential patient with copd are higher when compared to the younger group of patients , as shown in our current study . age is an unmodified factor in smokers for developing copd , because the older the age of a smoker , the longer they have been exposed to cigarettes . findings from a korean trial of copd screening suggest that advanced age increases the number of undiagnosed airway obstructions.6 the authors found that the association between potentially undiagnosed airflow obstruction and age was particularly strong , with prevalence increasing from 4.6% in those aged 4049 years to 40% in those aged 6069 years ( approximately ten times the prevalence was shown in the older age group ) . our result yielded a median age of 61 years for fev1/fev6 < 0.75 and a copd - ps score 5 group , which was significantly older than those with fev1/fev6 0.75 and a copd - ps score < 5 . this age difference between the groups is significant , which means older age groups have a higher risk of recording fev1/fev6 below 0.75 . even though smoking and age are generally associated with an increased prevalence of undiagnosed airway obstruction , for smokers , the presence of respiratory complaints may be interpreted as a minor consequence of smoking cigarettes and not as a sign of a more serious airway obstruction . this may cause unnecessary delay in seeking professional advice for the worsening symptoms of airway obstruction . with vitalqplus , combining both the screening questionnaire ( copd - ps ) and the copd-6 , there is an easy opportunity to make patients more aware of their lung symptoms and to encourage the provision of early treatment . our findings show that patients with more symptoms ( scores of 5 ) yield twice the percentage of outcomes of fev1/fev6 < 0.75 compared to patients with fewer copd symptoms ( scores < 5 ) . the chance of identifying a potentially undiagnosed case of copd is 24.1% if a patient scores 5 . this high incidence of copd in symptomatic patients indicates the need for further evaluation with spirometry to confirm the presence of airway obstruction . in a trial by ferguson et al for example , four out of five patients with copd were current or former smokers;45 some groups have advocated mass screening of asymptomatic smokers by using office spirometry.5 table 4 shows the participants copd gold staging based on fev1 percentage . the majority of our patients fell into stage ii gold classification : 61.5% were in stage ii , 23.1% in stage i , and 15.4% in stage iii . tinkelman et al found different results when screening for copd with spirometry.41 his gold classification was mild ( stage i ) , moderate ( stage ii ) , and severe copd ( stage iii ) , which yielded 57.4% , 36.8% , and 5.8% of patients with undiagnosed copd in those stages , respectively . our current study , however , showed more potentially undiagnosed copd patients at stage ii , while tinkelman et al s study yielded more patients in stage i.41 the study undertaken by sandelowsky et al42 meanwhile , showed a similar result to our present findings , with 45% of patients in stage i , 53% in stage ii , 3% in stage iii , and 0% in stage iv . this result is slightly different in our study because we focused on patients who were smokers , while other studies were performed on the overall population . the generalizability of the present findings requires careful consideration , because the sample was not randomly chosen . the current study adopted a cross - sectional methodology , and consequently , caution should be used in determining causality among variables.46 in addition to this limitation , the small number of participants , especially from perak and penang , might limit the generalizability of the findings . even though our findings suggested a quantitative difference between sabah and peninsular malaysia , more research this dual - combination assessment , vitalqplus , could potentially be used by clinicians to identify individuals at risk of copd and in selecting specific patients for spirometry measurement . with this approach , considerable time and costs could be saved in respect to the early diagnosis of copd , especially in high risk categories of patients such as smokers .

backgroundthis study utilized a validated combination of a copd population screener ( copd - ps ) questionnaire and a handheld spirometric device as a screening tool for patients at high risk of copd , such as smokers . the study aimed to investigate and pilot the feasibility and application of this combined assessment , which we termed the vitalqplus , as a screening tool for the early detection of copd , especially in primary care settings.methodsthis was a cross - sectional study screening potentially undiagnosed copd patients using a validated five - item copd - ps questionnaire together with a handheld spirometric device . patients were recruited from selected malaysian government primary care health centers.resultsof the total of 83 final participants , only 24.1% ( 20/83 ) were recruited from perak and penang ( peninsular malaysia ) compared to 75.9% ( 63/83 ) from sabah ( borneo region ) . our dual assessment approach identified 8.4% of the surveyed patients as having potentially undiagnosed copd . when only the vitalograph copd-6 screening tool was used , 15.8% of patients were detected with a forced expiratory volume in 1 second / forced expiratory volume in 6 seconds ( fev1/fev6 ) ratio at < 0.75 , while 35.9% of patients were detected with the copd - ps questionnaire . these findings suggested that this dual assessment approach has a greater chance of identifying potentially undiagnosed copd patients compared to the vitalograph copd-6 or copd - ps questionnaire when used alone . our findings show that patients with more symptoms ( scores of 5 ) yielded twice the percentage of outcomes of fev1/fev6 < 0.75 compared to patients with fewer copd symptoms ( scores < 5).conclusionwith the availability of a simple screening questionnaire and the copd-6 , there is an opportunity easily to make patients more aware of their lung symptoms and to encourage the provision of early treatment . the proposed dual assessment approach , which we termed the vitalqplus , may play a profound role in the early diagnosis of copd , which is crucial in improving the clinical management of the disease .

Introduction Methods Results Discussion Study limitations Conclusion

this is mainly due to patient ignorance , because often the symptoms ( ie , dyspnea and chronic cough ) are considered to be part of normal aging , and thus , medical attention is not sought.1 coughing and the production of sputum are accepted by most smokers as a natural side effect of smoking instead of a possible sign of the initial development of a lung disorder . the prevalence of moderate - to - severe copd in those patients 30 years and above in malaysia was projected to be 4.7% in 2003 , which translates to 448,000 cases , but this figure is now expected to be larger due to population aging.2 many prevalence studies have shown that the percentage of potentially undiagnosed airflow obstruction in both western35 and asian countries is approximately 3%15%.6 for example , in spain , 14.3% of men and 3.9% of women aged 4079 years have been reported to have some form of obstructed airflow,4 while the prevalence of copd in individuals aged between 40 and 80 years is 10.2% and increases with age , smoking consumption , and lower educational levels.7 in a general population - based sample in the uk , 9.9% of patients were observed to have airflow obstruction , with 52% of cases being undiagnosed.3 in the us , meanwhile , the percentages of white men and women aged 45 years and above having obstructive airflow were 14.2% and 9.9% , respectively , as noted in the national health and nutrition examination survey.5 finally , a study in korea observed undiagnosed airflow obstruction in 12.4% of men and 3.5% of women.6 despite the existence of evidence - based validated spirometry as a diagnostic method , many patients with copd continue to be undiagnosed or misdiagnosed.8 most undiagnosed copd patients are found to have been exposed to cigarettes for a period of time in their life.912 buffels et al13 showed that the screening of smokers and ex - smokers revealed a prevalence of copd of 7.4%18% in individuals aged 3570 years . in those who smoked , there was a 2.3-fold excess in its prevalence when three or more symptoms ( chronic cough , wheezing , and shortness of breath ) were present , compared to when these symptoms were absent.6 likewise , a study by coultas et al5 revealed that subjects with undiagnosed airflow obstruction had a higher prevalence of smoking ( 82.3% ) than those subjects with no airflow obstruction ( 54.2%).5 for current smokers , early detection with spirometry and intervention has been recommended by the us preventive services task force for the prevention of copd.14 it should be noted , however , that although screening with spirometry is a good way to diagnose and classify copd according to air - flow limitation,8 the malaysian clinical practice guidelines ( cpg ) for copd15 do not recommend the indiscriminate use of spirometry for the purpose of screening potential patients for copd , since it is labor intensive and time consuming to screen every patient at risk of developing copd with spirometry , which makes such a broad screening program not cost effective . the us preventive services task force also discourages the use of spirometry for asymptomatic screening for copd.14 similarly , the clinical practice guideline update from the american college of physicians , american college of chest physicians , american thoracic society , and european respiratory society each state that there is no proven benefit of using spirometry to screen adults who have no smoking history and no respiratory symptoms.16 early diagnosis may not reverse the disease but does at least offer the opportunity to improve a patient s quality of life by reducing the symptoms and preventing progression of the disease . once it reaches its more advanced stages , there is an increased risk of developing other comorbidities with an associated reduction in the quality of life of the patient , and an increase in the costs of treatment . there is , therefore , a need for a simple method to help identify persons who might have the early stages of copd . in places where spirometry is not available such as in remote areas , alternative options to the doctor visit include copd questionnaires followed by the use of handheld spirometric devices ( hsds ) as a guide in choosing the correct treatment for the patient.1719 according to the global initiative for chronic obstructive lung disease ( gold ) , there are four stages of copd , based on the air flow limitation at forced expiratory volume in 1 second ( fev1 ) on the spirometry test . simple validated questionnaires , such as the copd population screener ( copd - ps ) questionnaire,20 diagnostic score for copd ( ds - copd),21 and the international primary care airways guidelines ( ipag ) questionnaire12,15 have been developed , and hsds ( vitalograph copd-622 and the piko-612,23 ) have been shown to provide a precise screening tool for people at risk of developing copd . a recent multicenter , cluster - randomized study ( search1 ) involving 8,770 volunteers has shown that dual - combination assessment using questionnaire screening and hsd offered better copd detection than the use of either method in isolation.19 based on the above result , the hypothesis of this research was that the combination of the copd - ps questionnaire and the vitalograph copd-6 represented an accurate tool to detect patients at risk of copd ( ie , those with a score of 5 for the questionnaire and a ratio of fev1/forced expiratory volume in 6 seconds ( fev6 ) of < 0.75 ) . other than the choice of an hsd , the five - question copd - ps does not need any body weight , height , and body mass index calculations , in contrast to the eight - question ipag . due to the chronic progressive nature of the disease , the copd - ps questionnaire surveys a time frame focusing on 4 weeks of shortness of breath and 12 months of breathing problems.20 we proposed , therefore , to preselect patients using the copd - ps questionnaire based on age , smoking history , and symptoms , and then , for patients with a score of 5 , to undertake further tests with the vitalograph copd-6 . this dual - method assessment , which we have termed as vitalq - plus , may increase the likelihood of identifying potentially undiagnosed copd patients for further evaluation with diagnostic spirometry . it is worth noting that fev6 has been proven to be easily reproducible and can be used as surrogate parameter for forced vital capacity ( fvc).2427 a meta - analysis by jing et al summarized from eleven trials , concluded that fev1/fev6 is a sensitive and specific test and can be used as a valid alternative for fev1/fvc ( fev1/fvc < 0.70 as copd ) in the diagnosis of airway obstruction.25 estimates from this meta - analysis for the effectiveness of fev1/fev6 in the diagnosis of airway obstruction show a sensitivity of 89% and a specificity of 98% . fev1/fev6 had a sensitivity of 86.09% and a positive predictive value of 100% in the detection of airway obstruction when fev1/fvc is taken as the gold standard.28 the current study aimed to investigate and pilot the feasibility and application of dual - combination assessment vitalqplus as a screening tool for the early detection of copd , especially in primary care settings . this was a cross - sectional study to perform screening of potentially undiagnosed copd patients using a validated five - item questionnaire ( copd - ps ) and hsd ( copd-6 device ; model 4000 , vitalograph , inc , ennis , ireland ) on patients who were 35 years old and above , were smokers or ex - smokers , and were without any previous medical diagnosis of respiratory disease . additionally , the fev1/fev6 ratio is normally higher than the fev1/fvc ratio because fev6 only records the first 6 seconds of expiratory volume , while fvc accounts for the whole expiratory volume . the lungs of a healthy person can generally empty more than 80% of their volume in 6 seconds or less . potentially undiagnosed copd patients were also categorized according to the gold guidelines based on their fev1 percentage , so as to estimate patient staging in copd . a systematic sampling method was employed across three geographical regions in malaysia , including the northern ( kedah , perlis , and penang ) and central regions ( perak , selangor , negeri sembilan , and kuala lumpur ) of west malaysia ( peninsular malaysia ) as well as east malaysia ( sabah and sarawak ) . a total of 88 patients were recruited from the three different governmental primary care health centers situated in sabah , east malaysia ( n=66 ) ; in the state of perak , central region of west malaysia ( n=12 ) ; and in penang island , northern region of west malaysia ( n=10 ) . the characteristics of the patients in the three governmental primary care health centers are shown in table 1 . no statistically significant differences were observed between the three centers in the copd - ps scores , but there was a statistically significant difference in terms of fev1/fev6 ratio ( mean and median ) between the samples from sabah , perak , and penang ( p<0.05 ) . it must be noted that only 24.1% ( 20/83 ) patients were recruited from perak and penang compared to 75.9% ( 63/83 ) from sabah , mainly due to a lack of patients that fulfilled the inclusion criteria . at the copd questionnaire screening stage , 34.9% of the patients had a score of 5 , indicating that they had a potential chance of having copd . median fev1 was 2.4 l ( range , 0.93.29 ) in the fev1/fev6 0.75 group , while the median fev1 for fev1/fev6 < 0.75 was 1.8 l ( range , 0.71.47 ) . this may be due to the higher numbers of elderly patients and active smokers in sabah compared to both perak and penang . subjects that had positive outcomes on both tests ( ie , a copd - ps score 5 and a vitalograph score < 0.75 ) were classified as potentially experiencing undiagnosed copd , while subjects that had a negative outcome on both tests ( copd - ps score < 5 ; vitalograph score 0.75 ) were categorized as healthy / non - copd subjects . it is noteworthy that there were 28 subjects who were not classified in either category because they had either a copd - ps score < 5 and a vitalograph score < 0.75 ( n=6 ) , or a copd - ps score 5 and a vitalograph score 0.75 ( n=22 ) . the average age of the subjects in the potentially undiagnosed copd category was almost 20 years older than the subjects in the healthy group . the percentage of active smokers was 85.7% in the potentially undiagnosed copd group , 16.9% more than in the healthy / non - copd group . in our current study , we found out that 15.7% ( n=83 ) of patients screened using copd-6 had fev1/fev6 < 0.75 , which was interpreted as suggesting a high potential for a positive diagnosis with copd . apart from copd-6 measurement , the copd - ps symptoms - based questionnaire found that 35.9% of patients screened had a score > 5 , indicating a need for further evaluation to confirm a copd diagnosis . when the copd - ps assessment was combined with copd-6 , the results showed that 8.4% of patients had both a copd - ps score of 5 and a copd-6 measurement of fev1/fev6 < 0.75 . our current study showed that symptomatic patients with a score of 5 had a 2.2 times higher chance of recording fev1/fev6 < 0.75 compared to patients without symptoms ( 24.1% versus 11.1% , respectively ) , which is consistent with the study by shin et al.6 preselection with a screening questionnaire may , therefore , reduce costs by allowing a more targeted choice of patients to undergo spirometry testing . due to differences between the apparatus used , the cost of the device to perform a vitalograph ( approximately rm600 ) is less than 1/10 the cost of using a spirometer ( approximately rm7,000 for a spirometer , which is equivalent to us$2,000).31 this means that the vitalograph is sufficiently affordable for general practitioners or community pharmacies to make this device available to detect potential copd patients.32 because spirometers are not controlled under medical services regulations , there are no restrictions on who can perform this testing . lung foundation australia makes screening with handheld devices available as a program in community pharmacies in australia.33 if this program were implemented in community pharmacies in malaysia , the screening charges to perform vitalograph testing should be approximately rm6.00 per person ( equivalent to us$1.70 per person).6 it must be noted that patient recruitment in the perak and penang states of malaysia was challenging . the average ratio of hospitals per district was 3.2:2.2:1.1 ( penang , perak , and sabah , respectively).34 the greater accessibility to health care services evident in perak and penang probably meant that the copd - prone patients in these regions had already been screened and had already received treatment for copd ( thus being excluded from selection for this study ) . a recent trial has also reported using a similar two - step approach with an ipag questionnaire and piko-6,12 instead of the copd - ps and copd-6 used in our present study . in the former study , a positive ipag questionnaire for possible copd ( 17 points ) was obtained in 594 ( 55.1% ) subjects,12 while our copd - ps questionnaire yielded a result of 34.9% ( scores 5 ) . with piko-6 ( a similar device to copd-6 ) , 139 ( 12.9% ) subjects fulfilled the criteria for possible copd ( fev1/fev6 < 0.7),12 while our present study revealed that 15.7% ( 13/83 ) of patients screened by copd-6 had fev1/fev6 < 0.75 . when combined assessment results were considered , our current study yielded 8.4% ( table 3 ) , and sichletidis et al s study12 yielded 10.4% , which could be due to the difference in mean age between sichletidis et al s cohort ( 65.311.4 years of age ) and our cohort ( 53.31.1 years of age ) . in spirometer - defined copd screening in smokers aged 40 years and without prior respiratory diagnosis , undiagnosed copd is 18.9% ( n=818).41 our current study yielded 15.7% ( 13/83 ) of potentially undiagnosed copd patients with the aid of copd-6 , 3.2% lower than reported by tinkelman et al.41 this could be due to the younger age cutoff in our present study : a 35-year - old cutoff rather than the 40-year - old cutoff in the tinkelman trial.41 previous studies have shown that up to 30% of smokers experienced undiagnosed copd,41 while undiagnosed copd in the overall population is between 10% and 20% . another study of undiagnosed copd by vandevoorde et al found a 29.5% incidence ( n=146 ) in active smokers aged 4070 years who smoked at least 15 packs / year.26 a cross - sectional study in male smokers aged 4065 years yielded undiagnosed airway obstruction in approximately 29.9% of cases.9 this study used a questionnaire followed by spirometry for subjects without previous diagnosis of copd . in england , spirometry - defined , undiagnosed copd was found in 10.8% ( n=8,215 ) in subjects aged 35 years,11 while in spain , undiagnosed copd is approximately 7.04% ( n=4,035 ) in the age range of 4069 years.7 however , the studies conducted in england and spain recruited from the general population , while in our previous study , only smoking patients were recruited.43 in addition , lkke et al followed a general population sample for 25 years and also concluded that the absolute risk of developing copd among continuous smokers was at least 25%.44 a local pilot cross - sectional study conducted in malaysia by ching et al17 using a similar handheld spirometer managed to detect 10.6% copd patients . the chances of participants in the older group being identified as a potential patient with copd are higher when compared to the younger group of patients , as shown in our current study . our result yielded a median age of 61 years for fev1/fev6 < 0.75 and a copd - ps score 5 group , which was significantly older than those with fev1/fev6 0.75 and a copd - ps score < 5 . with vitalqplus , combining both the screening questionnaire ( copd - ps ) and the copd-6 , there is an easy opportunity to make patients more aware of their lung symptoms and to encourage the provision of early treatment . our findings show that patients with more symptoms ( scores of 5 ) yield twice the percentage of outcomes of fev1/fev6 < 0.75 compared to patients with fewer copd symptoms ( scores < 5 ) . the chance of identifying a potentially undiagnosed case of copd is 24.1% if a patient scores 5 . tinkelman et al found different results when screening for copd with spirometry.41 his gold classification was mild ( stage i ) , moderate ( stage ii ) , and severe copd ( stage iii ) , which yielded 57.4% , 36.8% , and 5.8% of patients with undiagnosed copd in those stages , respectively . our current study , however , showed more potentially undiagnosed copd patients at stage ii , while tinkelman et al s study yielded more patients in stage i.41 the study undertaken by sandelowsky et al42 meanwhile , showed a similar result to our present findings , with 45% of patients in stage i , 53% in stage ii , 3% in stage iii , and 0% in stage iv . the current study adopted a cross - sectional methodology , and consequently , caution should be used in determining causality among variables.46 in addition to this limitation , the small number of participants , especially from perak and penang , might limit the generalizability of the findings . even though our findings suggested a quantitative difference between sabah and peninsular malaysia , more research this dual - combination assessment , vitalqplus , could potentially be used by clinicians to identify individuals at risk of copd and in selecting specific patients for spirometry measurement . with this approach , considerable time and costs could be saved in respect to the early diagnosis of copd , especially in high risk categories of patients such as smokers .

the trypanosomatidae comprise a large group of parasitic protozoa , some of which cause important diseases in humans . the organisms that have been most extensively studied are trypanosoma brucei , the causative agent of african sleeping sickness , t. cruzi , responsible for chagas disease in south america , and leishmania spp . , which causes leishmaniasis in asia , south america , and mediterranean countries . in order to complete their life cycle , these parasites have to adapt and develop in an insect vector and in a vertebrate host . these single - celled organisms have developed several strategies to modify their surrounding environment , modulate host immune responses , or interfere with the host 's anti - microbial activity . materials secreted by the parasite are involved in these processes helping the parasite survive in an environment more favorable for its own development [ 25 ] . in addition , previous studies indicate that trypanosomatid secreted factors elicit strong immunity and protection against infection in mice and dogs [ 6 , 7 ] . thus secreted factors could be a source of antigens for vaccine development , as demonstrated in the pathogen mycobacterium tuberculosis . nevertheless , all trypanosomatid secreted factors involved in virulence and/or representing putative vaccine targets are not currently known . the availability of three draft trypanosomatid genome sequences provides valuable data for protein - mining using bioinformatic tools , especially for the localization or prediction of function for hypothetical proteins . given that a significant number of trypanosomatid protein - coding genes are annotated as hypothetical , additional studies are needed to ascertain their function . in the present study , we designed an experimental genome - based approach to identify potentially secreted proteins that are conserved among the three main trypanosomatid pathogens and involved in the classical secretory pathway . we hypothesized that a phylogenetic conservation among leishmania , t. cruzi , and t. brucei would indicate evolutionary selection for this family of proteins and suggest an important role for such secreted proteins in the biology of these parasites . our results demonstrate that the bioinformatic analysis , combined with the functional tests , provides a useful and reliable method for the identification of novel secreted proteins , representing potential virulence factors in trypanosomatids . the results are also discussed in relation to the relative importance of the classical and nonclassical secretory pathways for the release of proteins into the extracellular environment . release v 5.0 of the t. cruzi genome was extracted from the integrated t. cruzi genome resource tcruzidb ( http://tcruzidb.org/tcruzidb/ ) . proteins belonging to large families of surface molecules , which include trans - sialidases , mucins , gp63s , and mucin - associated surface proteins , were also discarded . finally orfs encoding proteins bearing a molecular weight ( mw ) above 90 kda the software signalp 3.0 ( http://www.cbs.dtu.dk/services/signalp/ ) was used to predict the presence of a signal peptide and a cleavage site in amino acid sequences . protein sequences having a signal peptide probability greater than 0.8 associated with a cleavage site probability greater than 0.7 were analyzed for the presence of orthologs in the related trypanosoma brucei and leishmania major parasite genomes predicted by jaccard cog clustering in gene db ( http://www.genedb.org/ ) . most of these orthologs were confirmed in tritrypdb ( http://tritrypdb.org/tritrypdb/ ) using orthomcl and genomic context analysis ( gene synteny ) . the tmhmm 2.0 ( http://www.cbs.dtu.dk/services/tmhmm/ ) and the das - tmfilter ( http://mendel.imp.ac.at/das/source.html ) servers were used for the prediction of transmembrane helices in protein sequences . the t. cruzi tcy7 ( or y cl7 ) clone derived from the y strain was used throughout this study . epimastigotes were grown in liver infusion tryptose ( lit ) medium supplemented with 10% fcs at 28c in standard conditions and harvested during the logarithmic growth phase . metacyclic trypomastigotes , obtained from the differentiation of late stationary phase epimastigotes , were used to initiate infection of mouse fibroblasts ( l929 ) . the wild - type ( wt ) promastigote clone from l. infantum ( mhom / ma/67/itmap-263 ) was maintained at 26c by weekly subpassages in sdm 79 medium supplemented with 10% heat - inactivated fcs , 100 u / ml penicillin , and 100 g / ml streptomycin . total rna was extracted from epimastigotes , amastigotes , and trypomastigotes with the rneasy kit ( qiagen , hilden , germany ) according to the manufacturers ' instructions , and treated with dnase i ( dna - free kit , ambion inc . reverse transcription was performed for 1 g of total rna using random hexamers and superscript ii reverse transcriptase ( invitrogen , carlsbad , ca ) according to the manufacturers ' instructions . the cdna ( 4 l of 1/10 dilutions ) from each stage was amplified by pcr in a 20 l reaction volume using 10 l of 2x pcr master mix 1x ( promega , madison , wisconsin ) and 0.5 m gene - specific forward and internal reverse primers ( listed in table 1 ) using the following cycling conditions : 94c for 3 minutes followed by 30 cycles of 94c for 30 seconds , 55c to 58c ( according to the primer pair ) for 30 seconds , 72c for 45 seconds , and a final elongation at 72c for 5 minutes . the encoding genes selected by in silico analysis were cloned into the ptex expression vector , carrying the neomycin resistance gene ( neo ) . full length orfs were amplified from genomic dna with specific reverse and forward primers , including different restriction sites and a 6-histidine - tag in the c - terminal region ( listed in tables 1 and 3 ) . pcr reactions were carried out in 20 l using 0.5 m of each primer , 0.2 mm dntp , 0.4 u of phusion high - fidelity polymerase ( finnzymes , espoo , finland ) , and the following cycling conditions : 98c for 30 seconds followed by 25 cycles of 98c for 10 seconds , 64c to 68c for 15 seconds , 72c for 25 to 60 seconds ( according to gene size ) , and a 72c elongation for 5 minutes . digested and purified fragments were inserted into the dephosphorylated ptex vector digested with the corresponding restriction enzymes . cloned sequences were confirmed by restriction digestion and sequencing . large - scale preparations of the different constructs were performed using the plasmid midi kit ( promega ) . briefly , promastigotes were washed twice with hepes - nacl buffer saline ( 21 mm hepes , 5 mm kcl , 0.7 mm nah2po4 , 137 mm nacl ) , resuspended at 10 cells / ml in hepes - nacl electroporation buffer ( ph 7.2 ) supplemented with 6 mm glucose and cooled on ice for 10 minutes . cells ( 10 ) were combined with 15 g of vector , left on ice for 10 additional minutes , and electroporated using an easyject plus ( eurogentec , seraing , belgium ) apparatus set at 450 v and 450 f , for one pulse . the cells were left on ice for a further 10 minutes and transferred to 4 ml of growth medium . the antibiotic g-418 ( 20 g / ml ) was added 24 hours later , and parasites were subcultured at a dilution of 1/10 in 5 ml sdm in the presence of 20 g / ml g418 . parasites were grown in the presence of gradually increasing concentrations of g418 and were routinely maintained in sdm containing 150 g / ml of g418 . pcr amplifications were carried out to check for the presence of the neo gene and the corresponding gene in transfected parasites . a fragment of 800 bp corresponding to the neo gene was amplified with specific forward and reverse primers ( f5 atgattgaacaagatggattgcacgcagg 3 , r5 tcagaagaactcgtcaagaa 3 ) . full length orfs of the specific genes were amplified with primers listed in table 1 . pcr reactions were carried out in a 20 l reaction volume using 10 l of master mix 1x ( promega , madison , wisconsin ) and 0.5 m neo and gene - specific forward and reverse primers using the following cycling conditions : 94c for 3 minutes followed by 30 cycles of 94c for 30 seconds , 55c to 58c ( according to the primer pair ) for 30 seconds , 72c for 45 seconds to 2 minutes ( according to gene size ) , and a final elongation at 72c for 5 minutes . to analyze the presence of secreted proteins in the supernatant , 1 10 l. infantum promastigotes from log - phase culture were collected by centrifugation , washed twice in hepes - nacl buffer , resuspended in 40 ml of hepes - nacl ( ph 7.2 ) , 11 mm glucose , 200 g / ml g-418 , and incubated for 6 hours at 27c . parasite viability was then assessed as previously described and harvested by centrifugation at 2,100 g for 10 minutes at 4c . the parasite pellet was stored at 80c for subsequent sds - page analysis and the supernatant was filtered through a low retention 0.45 m pvdf filter membrane ( millipore , boston , massachusetts ) . after addition of protease inhibitor cocktail ( sigma - aldrich ) the filtrate was concentrated up to 80-fold using an ultra - centrifugal filter device , according to manufacturers ' instructions ( amicon bioseparations , milliporecorp ) . cell pellets of wild - type and episomally transfected l. infantum promastigotes were resuspended in ripa buffer ( 25 mm tris - hcl ph 7.6 , 150 mm nacl , 1% np-40 , 1% sodium deoxycholate and 0.1% sds ) , incubated on ice for 30 minutes and sonicated three times for 20 seconds . the soluble phase was recovered by centrifugation at 10,000 g for 30 minutes ( 4c ) and the protein concentration was determined using a bradford protein assay ( bio - rad laboratories , hercules , california ) . proteins from parasite lysates ( 35 g ) or from 80 concentrated cell - free supernatants ( 2 g ) were separated on an nupage bis - tris gel ( 4%12% ) in mops - sds running buffer ( invitrogen ) under reducing conditions ( 50 mm dtt ) and transferred to a pvdf membrane ( hybond - p , amersham ) . the membrane was rinsed twice in tbs and incubated for 1 hour in the anti - his hrp conjugate blocking buffer ( qiagen ) . the membrane was then incubated in 1/3000 anti - his hrp conjugate antibody ( qiagen ) for 1 hour and washed seven times for 5 minutes in tbs - t buffer ( tbs-0.5 % tween 20 ) . signals were detected by chemiluminescence emission using the ecl plus western blotting detection system and ecl hyperfilms ( ge healthcare , uk ) . a homologous episomal expression system was devised to further examine the infection in vitro of two secreted proteins from l. infantum . the vector psp-hygluc carrying the firefly - luciferase gene was used to cotransfect l. infantum promastigotes overexpressing the secreted proteins linj19.0410 ( ortholog of tc00.1047053505789.10 ) or linj36.5780 ( ortholog of . recombinant parasites were selected for their growth in increasing concentrations of hygromycin ( up to 300 g / ml ) over a period of several weeks . promastigotes transfected with the ptex vector alone and the psp-hygluc were used as controls for infection experiments . the survival of transfected parasites was evaluated within human leukemia monocyte cells ( thp-1 cells ) . thp-1 cells were cultured in rpmi 1640 medium supplemented with 10% fcs , 2 mm glutamine , 100 iu of penicillin / ml , and 100 g of streptomycin / ml . thp-1 cells in the log - phase of growth were differentiated into macrophages by incubation for 2 days in a medium containing 20 ng / ml of phorbol myristate acetate ( sigma - aldrich ) . thp-1 cells treated with pma were washed with prewarmed medium and then infected with stationary - phase promastigotes of transfected - leishmania in a 24-well plate at a parasite / macrophage ratio of 10 : 1 for 4 hours at 37c with 5% co2 . after different incubation periods ( 24 hours to 96 hours ) luciferase activity was determined using the steady glo reagent ( promega , madison , wi ) , according to the manufacturers ' instructions . after 5 minutes , the plate was read using a multilabel counter victormodel 1420 ( perkin elmer ) . results are expressed as the mean of rlu ( relative luminescence units ) activity of three independent experiments , each performed in triplicate . the preliminary analysis of the 19613 t. cruzi putative proteins from the cl - brener genome was performed to discard potential uncompleted sequences . a total of 1796 sequences were removed manually since they did not bear an initial methionine amino acid . house - keeping genes and sequences belonging to large gene families , like the trans - sialidases , mucins , mucin - associated surface proteins ( masps ) , were discarded given that the main goal was to identify novel secreted proteins . finally , sequences encoding proteins with a molecular weight above 90 kda were eliminated , in order to facilitate subsequent gene cloning . the remaining coding sequences were screened for the presence of both the signal peptide and the peptidase cleavage site with a probability of 0.8 and 0.7 , respectively . a total of 216 sequences were obtained by using these criteria . among them , 91 ( 42% ) were annotated as the final criterion for selected proteins likely to be secreted by the classical eukaryotic pathway was the presence of the signal peptide and the signal peptidase site in orthologs of the related parasites : leishmania major , l. infantum , and t. brucei . among the 91 sequences , only 45 showed orthologs with the signal peptide criteria . the 13 proteins bearing the highest probability for the presence of the signal peptide were selected ( table 2 ) for confirmation of extracellular localization . among the 13 selected genes , tc00.1047053505789.10 and tc00.1047053509835.30 are homologous genes and members of a multigene family ( table 2 ) . these genes show about 40% identity at the protein level and possess the same predicted orthologs in the genedb server . nevertheless in the tritrypdb resource this could be explained by the use of different algorithms ( jaccard cog clustering or orthomcl ) for the prediction of orthologous groups . the beta - tubulin t. cruzi gene ( geneid tc00.1047053506563 ) was added to our sample as a potential negative control for protein secretion . among the 13 selected genes , these genes were included for the functional test because previous studies identified extracellular proteins with putative transmembrane domains as constituents of the secretome of different pathogens , including l. donovani [ 19 , 20 ] . furthermore , in the protozoa toxoplasma gondii , the dense granule protein gra5 is a transmembrane protein that bears a signal peptide and is secreted as a soluble protein into the vacuolar space , before being inserted into the parasitophorous vacuole membrane . based on these studies , we included genes with potential transmembrane domains to test whether the presence of these domains represents a useful criterion for identifying extracellular secreted proteins . reverse transcription - pcr ( rt - pcr ) was performed for the 13 in silico selected genes in the different developmental stages of t. cruzi in order to verify transcription . the beta - tubulin t. cruzi gene ( geneid tc00.1047053506563 ) , constitutively expressed in all the three stages of t. cruzi , was used as a positive control for rna quality . rt - pcr was positive for all genes in the infective trypomastigote and amastigote forms . two genes ( gene i d tc00.1047053511901.30 and tc00.1047053509999.10 ) were negative for the amplification of cdna in the noninfective epimastigote form ( figure 1 ) suggesting a possible stage - specific expression of these genes . nevertheless , since trypanosomatid gene expression is almost exclusively regulated posttranscriptionally , further studies at the protein level have to confirm these observations . a functional test was set up to confirm the presence of selected proteins in the extracellular environment by detection of target proteins in cell - free supernatants . the 13 selected encoding genes of t. cruzi and the gene encoding the beta - tubulin ( negative control ) were amplified from genomic dna . a sequence encoding a 6xhis - tag was added at the c - terminal end of each encoded gene , to allow subsequent detection of the protein in total parasite protein extracts or in concentrated cell - free supernatants ( ccfss ) . amplified pcr products were cloned into the ptex shuttle vector widely used for expression in trypanosomatids . transformation and selection of t. cruzi is not as easy to perform as for leishmania , mainly due to longer periods required for selecting drug - resistant parasites . since we aimed to develop a fast and reliable approach to identify trypanosomatid conserved secreted proteins , we used the related leishmania parasite as the recipient organism for the experimental validation of our selected proteins . thus , l. infantum promastigotes were separately transformed with ptex carrying one of the 14 selected t. cruzi genes ( including the beta - tubulin gene ) , and the recombinant parasites were selected for resistance to geneticin g418 . each parasite population was checked for the presence of both the neo gene and the selected gene whose secretion was to be analyzed . a specific 800 bp fragment , indicative of the presence of the neo gene , was detected in the transfected promastigotes and not in wild - type parasites ( figure 2(a ) ) . moreover , the presence of each candidate gene in recombinant parasites was confirmed using specific primers designed from t. cruzi gene sequences ( figure 2(b ) ) . pcrs performed on wild type leishmania were negative , demonstrating that the amplification was genus specific although the genes are conserved in both trypanosomatids ( data not shown ) . the expression of these genes was screened using an antibody directed against the his - tag carried by the recombinant proteins ( figure 3(a ) ) . western blot analysis demonstrated that ( i ) it was possible to easily and specifically detect the 6xhis tag protein in the extract derived from recombinant parasites , ( ii ) recombinant leishmania expressed a relatively high level of t. cruzi protein , and ( iii ) the molecular weight of the detected tagged protein corresponded to the expected mw ( see table 1 ) . subsequently , an approach was set up to detect recombinant proteins in cell - free supernatants . in order to limit potential contamination by proteins derived from dying organisms , incubation in serum - free mediums was restricted to 6 hours , and the viability of parasite populations was checked before and after this incubation period . parasites and cell - free supernatants were collected if the viability of the cell population was above 98% . western blot analysis of the concentrated cell - free supernatants revealed that among the 14 proteins , only 3 were actively secreted ( tc00.1047053506155.99 , tc00.1047053505789.10 , and tc00.1047053509999.10 ) ( figure 3(b ) ) . these proteins represent genuine secreted material , since ( i ) the overexpression of the beta - tubulin gene does not induce translocation of the beta - tubulin protein into the extracellular space ( difference between lys and ccfs in figure 3(b ) ) , and ( ii ) the detection of the tagged protein in the cell - free supernatant is not related to the level of its expression by leishmania ( low abundance of tc00.1047053506155.99 in figure 3(b ) ) . as expected , a slight molecular weight difference was observed between the tagged protein detected into the whole soluble extract and that detected in the cell - free supernatant that could be explained by the loss of the signal peptide ( figure 3(b ) ) . as anticipated , no cross - reactive band was detected in wild - type parasites ( figure 3(b ) ) . to confirm that the secretion observed was not related to the heterologous expression system , two leishmania genes ( gene i d linj19.0410 and linj36.5780 ) corresponding to the orthologs of tc00.1047053505789.10 and tc00.1047053506155.99 genes were selected to validate our approach . by using the same protocol as above , leishmania expressing the 6xhis tagged proteins as expected , the presence of the tagged protein in the extracellular medium was only detected in the episomally transfected parasites ( figure 4 ) . together , these results indicate that this approach allows the identification of new and genuinely extracellular proteins involved in the endoplasmic reticulum / golgi - dependent secretory pathway . we attempted to determine whether the expression of leishmania - secreted proteins could interfere with the capacity of recombinant parasites to replicate within human macrophages in vitro . both confirmed secreted proteins ( linj19.0410 and linj36.5780 ) from l. infantum were tested by using the luciferase reporter system in transfected parasites overexpressing these proteins . the number of promastigotes cells and luciferase activity were linearly correlated for the different recombinant parasites before macrophage infection ( data not shown ) . results of in vitro infection showed that overexpression of secreted protein linj19.0410 ( ortholog of tc00.1047053505789.10 ) increases the capacity of leishmania to survive in thp-1 differentiated macrophages as early as 24 hours postinfection ( figure 5 ) . furthermore , a statistically significant increase in luciferase activity of recombinant parasites expressing linj19.0410 was maintained throughout the experiments ( p < .05 ) . this effect was not observed in parasites overexpressing linj36.5780 ( ortholog of tc00.1047053506155.99 ) where infectivity levels were similar to the control parasites transfected with the ptex vector alone and the psp-hygluc ( figure 5 ) . in trypanosomatids the secretion process is not fully understood and various pathways including classical and nonclassical mechanisms may contribute to the formation of the extracellular proteome . thus the individual identification of secreted materials would enhance efforts towards understanding mechanisms of protein secretion in these medically important parasites . in an attempt to provide a new approach to analyse the large number of hypothetical conserved proteins in trypanosomatids , we developed an experimental approach to identify hypothetical extracellular proteins likely to be involved in the classical pathway . we combined a web - based bioinformatics approach that used the signal p 3.0 program , one of the most accurate predictors for the presence of a signal peptide sequence , with a functional test that takes advantage of the relative ease to genetically transform leishmania . we assumed that a phylogenetic conservation among leishmania , t. cruzi , and t. brucei would point to evolutionary selection for this family of proteins and indicate an important role for these proteins in the biology of these parasites . using these criteria we selected a pool of 13 trypanosomatid - conserved hypothetical proteins from the t. cruzi database for which secretion was tested . identification of secreted proteins has been hampered in trypanosomatids due to the difficulty in distinguishing genuine secretions from molecules released by lysed , dead , or dying organisms . additionally , the in vitro growth of trypanosomatid developmental stages that occur in mammals is impossible or laborious . characterization by screening cdna libraries with sera raised against culture medium supernatants has been performed for the identification of extracellular proteins [ 22 , 23 ] . however , using this approach proteins with a low abundance or that are poorly immunogenic are likely to be missed . a more exhaustive approach relying on a highly sensitive methodology , like the quantitative mass spectrometry , was recently used to analyze the protein content of the whole conditioned medium from stationary - phase l. donovani promastigotes . nevertheless , proteins produced at low abundance and that are mainly exported to the extracellular compartment are likely to be missed , since the method relies on the comparison of conditioned medium versus cell - associated proteins . indeed proteins that are well known to be extracellular , such as chitinase and sacp ( secreted acid phosphatase ) , were not identified with the silac - based approach . the method we designed allowed us to identify three new trypanosomatid conserved proteins , likely to be secreted through the classical secretory pathway . we were confident that the proteins detected in the cell - free supernatant were genuinely secreted since ( i ) we were not able to detect the beta - tubulin - tagged protein in the cell - free supernatant after overexpression of the gene , ( ii ) the 6-hour incubation time avoided excessive cell death and contamination by proteins released from dead parasites , ( iii ) in the extracellular environment we detected orthologous leishmania proteins ( linj19.0410 , linj36.5780 ) suggesting that secretion in this protein family is likely to be evolutionary conserved , and ( iv ) we detected no relation between the amount of secreted protein and its intracellular expression in the transfected parasites , demonstrating that the translocation of the his - tagged protein into the cell supernatant is not related to the methodology we used . a recent study involving conditioned medium derived from stationary - phase leishmania promastigotes demonstrated that the extracellular proteome was mainly composed of proteins derived from different microvesicles . the parasite growth phase studied by these authors contains the infectious metacyclic parasites and also a high percentage of dying parasites in apoptosis . consequently , the analysis of extracellular material revealed that the main contributors to the extracellular proteome are vesicles likely to be this work leads to the general conclusion that 98% of the proteins of the leishmania secretome lacked a targeting signal , indicating that nonclassical secretion pathways are likely to be the dominant way by which leishmania export proteins . the proteomic characterization of the released / secreted proteins of l. braziliensis promastigotes showed that about 5% of the identified proteins possess a putative n - signal peptide , indicating that protein export may depend on unconventional pathways as suggested in l. donovani . however , some evidence strongly supports the notion that the classical secretory pathway is operational in trypanosomatids and contributes to the composition of the parasite 's secretome . for example , the screening of an l. major cdna library with antiserum raised against culture supernatant from stationary - phase promastigotes led to the detection of 8 proteins bearing a potential signal peptide among the 33 genes identified . moreover some of the well - known leishmania proteins found in the extracellular environment have a signal peptide for secretion , such as gp63 or chitinase [ 3 , 27 ] . in the current work we deliberately decided to experimentally validate the secretion of our candidate proteins during the exponential growth phase of leishmania in order to avoid contamination of the cell - free supernatant by apoptotic vesicles or exosomes . in these conditions we did not detect the secretion of tubulin , even in an overexpression model of leishmania transfectants , while tubulin was identified with a significant score in the leishmania secretome studied by the proteomic analysis performed during the stationary - phase . therefore , our results suggest that tubulin might be associated with exosomes and/or apoptotic vesicles generated by promastigotes in the stationary - phase of growth . we suggest that the composition of the parasite extracellular proteome is variable and depends both on the parasite stage under consideration and on the relative contribution of the various pathways operating in protein secretion . further studies are required to highlight the importance of both classical and nonclassical secretory pathways in different developmental stages of trypanosomatids . since we selected and tested secretion for several genes in the trypanosomatid genomes , our methodology provides a potential tool for genomewide screening to identify extracellular proteins . furthermore , our methodology may complement other strategies , such as the silac approach , for the identification of proteins missed when using proteomic - based approaches . regarding proteins not detected in the extracellular environment , it is important to point out that proteins bearing a signal peptide are not only targets for secretion but also for transfer to specific organelles , for example , lysosomes or the cell surface . thus , these proteins could be retained in specific organelles within the parasite or attach to the cell surface via a gpi - anchor . in this light , analysis of the 13 proteins with gpi - som suggests that 2 out of 13 proteins ( tc00.1047053506417.30 and tc00.1047053506467.29 ) have a predicted glycosyl - inositol phosphate ( gpi ) anchor signature sequences . hence , the presence of the gpi anchored domain may explain the absence of these proteins in the culture supernatant of recombinant parasites . an unexpected finding in our results was the prediction of transmembrane helices in two of our secreted proteins ( tc00.1047053505789.10 and tc00.1047053509999.10 ) . we included proteins with transmembrane domains to test whether their presence in protein sequences is a suitable criterion for the prediction of extracellular secreted proteins . previous studies suggested that the presence of transmembrane helices in protein sequences is not a suitable criterion for discarding potentially secreted proteins [ 19 , 20 ] . in this regard our results provide further evidence that empirical studies are required to verify bioinformatic predictions since two of the extracellular proteins bear transmemebrane domains . however , another speculative explanation for the presence of transmembrane domains is the potential insertion of secreted materials into membranes after secretion , as demonstrated for the pathogen toxoplasma gondii [ 29 , 30 ] . lipocalins are a widely distributed group of mostly extracellular proteins , several of which have been implicated in the regulation of host immune responses , such as , alpha-1-microglobulin and alpha-1-acid glycoprotein . despite a well - conserved tertiary structure of lipocalins , this may explain the absence of a predicted lipocalin signature in the leishmania spp . or t. brucei orthologs . remarkably , the protein encoded by tc00.1047053509835.30 ( homolog of tc00.1047053505789.10 ) lacks a lipocalin signature . although these genes belong to a multigene family ( see table 2 ) and possess about 40% identity at the protein level , our findings suggest a different cellular localization and/or function for the corresponding proteins . a clear assignment of the protein tc00.1047053505789.10 to the lipocalin family would only be possible after nmr or x - ray crystallography structure analysis . further studies are needed to ascertain if this large and diverse group of proteins is present in trypanosomatids and plays a role in the transmission process . a glucosidase ii beta subunit - like protein domain has also been detected in the protein tc00.1047053506155.99 . although mostly localized to the er , glucosidase ii was found in endocytic structures beneath the plasma membrane and has been associated with the protein - tyrosine phosphatase cd45 . there is also evidence that in some cell types glucosidase ii beta is capable of being trafficked to the cell surface . having demonstrated that our methodology is reliable for the identification of extracellular secreted proteins in trypanosomatids , we tested the hypothesis that recombinant leishmania parasites carrying extra copies of leishmania secreted proteins may interfere with their survival or infectivity towards human monocyte - derived macrophages in vitro . results of these assays showed a significant survival advantage to leishmania parasites overexpressing the gene linj19.0410 ( ortholog of tc00.1047053505789.10 ) suggesting that this protein is involved in a process increasing survival and/or replication of the parasite inside its target cell . given that leishmania and t. cruzi do not target the same host cells and follow different cell invasion processes , further experiments in the t. cruzi homologous system are needed to address whether the identified secreted protein tc00.1047053505789.10 from t. cruzi is also involved in host cell invasion and/or replication . current in vitro and in vivo studies are in progress to characterize this protein which represents a potential conserved virulence factor in trypanosomatids . in conclusion , our results show that the bioinformatics method combined with the functional tests , provides a fast and reliable method for the identification of novel extracellular secreted proteins involved in the classical secretory pathway and represents potential virulence factors in trypanosomatids .

extracellular factors produced by leishmania spp . , trypanosoma cruzi , and trypanosoma brucei are important in the host - parasite relationship . here , we describe a genome - based approach to identify putative extracellular proteins conserved among trypanosomatids that are likely involved in the classical secretory pathway . potentially secreted proteins were identified by bioinformatic analysis of the t. cruzi genome . a subset of thirteen genes encoding unknown proteins with orthologs containing a signal peptide sequence in l. infantum , l. major , and t. brucei were transfected into l. infantum . tagged proteins detected in the extracellular medium confirmed computer predictions in about 25% of the hits . secretion was confirmed for two l. infantum orthologs proteins using the same experimental system . infectivity studies of transgenic leishmania parasites suggest that one of the secreted proteins increases parasite replication inside macrophages . this methodology can identify conserved secreted proteins involved in the classical secretory pathway , and they may represent potential virulence factors in trypanosomatids .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Conclusions

the organisms that have been most extensively studied are trypanosoma brucei , the causative agent of african sleeping sickness , t. cruzi , responsible for chagas disease in south america , and leishmania spp . materials secreted by the parasite are involved in these processes helping the parasite survive in an environment more favorable for its own development [ 25 ] . nevertheless , all trypanosomatid secreted factors involved in virulence and/or representing putative vaccine targets are not currently known . in the present study , we designed an experimental genome - based approach to identify potentially secreted proteins that are conserved among the three main trypanosomatid pathogens and involved in the classical secretory pathway . we hypothesized that a phylogenetic conservation among leishmania , t. cruzi , and t. brucei would indicate evolutionary selection for this family of proteins and suggest an important role for such secreted proteins in the biology of these parasites . our results demonstrate that the bioinformatic analysis , combined with the functional tests , provides a useful and reliable method for the identification of novel secreted proteins , representing potential virulence factors in trypanosomatids . the results are also discussed in relation to the relative importance of the classical and nonclassical secretory pathways for the release of proteins into the extracellular environment . release v 5.0 of the t. cruzi genome was extracted from the integrated t. cruzi genome resource tcruzidb ( http://tcruzidb.org/tcruzidb/ ) . proteins belonging to large families of surface molecules , which include trans - sialidases , mucins , gp63s , and mucin - associated surface proteins , were also discarded . finally orfs encoding proteins bearing a molecular weight ( mw ) above 90 kda the software signalp 3.0 ( http://www.cbs.dtu.dk/services/signalp/ ) was used to predict the presence of a signal peptide and a cleavage site in amino acid sequences . protein sequences having a signal peptide probability greater than 0.8 associated with a cleavage site probability greater than 0.7 were analyzed for the presence of orthologs in the related trypanosoma brucei and leishmania major parasite genomes predicted by jaccard cog clustering in gene db ( http://www.genedb.org/ ) . the t. cruzi tcy7 ( or y cl7 ) clone derived from the y strain was used throughout this study . the wild - type ( wt ) promastigote clone from l. infantum ( mhom / ma/67/itmap-263 ) was maintained at 26c by weekly subpassages in sdm 79 medium supplemented with 10% heat - inactivated fcs , 100 u / ml penicillin , and 100 g / ml streptomycin . the cdna ( 4 l of 1/10 dilutions ) from each stage was amplified by pcr in a 20 l reaction volume using 10 l of 2x pcr master mix 1x ( promega , madison , wisconsin ) and 0.5 m gene - specific forward and internal reverse primers ( listed in table 1 ) using the following cycling conditions : 94c for 3 minutes followed by 30 cycles of 94c for 30 seconds , 55c to 58c ( according to the primer pair ) for 30 seconds , 72c for 45 seconds , and a final elongation at 72c for 5 minutes . large - scale preparations of the different constructs were performed using the plasmid midi kit ( promega ) . the antibiotic g-418 ( 20 g / ml ) was added 24 hours later , and parasites were subcultured at a dilution of 1/10 in 5 ml sdm in the presence of 20 g / ml g418 . pcr reactions were carried out in a 20 l reaction volume using 10 l of master mix 1x ( promega , madison , wisconsin ) and 0.5 m neo and gene - specific forward and reverse primers using the following cycling conditions : 94c for 3 minutes followed by 30 cycles of 94c for 30 seconds , 55c to 58c ( according to the primer pair ) for 30 seconds , 72c for 45 seconds to 2 minutes ( according to gene size ) , and a final elongation at 72c for 5 minutes . to analyze the presence of secreted proteins in the supernatant , 1 10 l. infantum promastigotes from log - phase culture were collected by centrifugation , washed twice in hepes - nacl buffer , resuspended in 40 ml of hepes - nacl ( ph 7.2 ) , 11 mm glucose , 200 g / ml g-418 , and incubated for 6 hours at 27c . a homologous episomal expression system was devised to further examine the infection in vitro of two secreted proteins from l. infantum . the vector psp-hygluc carrying the firefly - luciferase gene was used to cotransfect l. infantum promastigotes overexpressing the secreted proteins linj19.0410 ( ortholog of tc00.1047053505789.10 ) or linj36.5780 ( ortholog of . thp-1 cells were cultured in rpmi 1640 medium supplemented with 10% fcs , 2 mm glutamine , 100 iu of penicillin / ml , and 100 g of streptomycin / ml . the preliminary analysis of the 19613 t. cruzi putative proteins from the cl - brener genome was performed to discard potential uncompleted sequences . house - keeping genes and sequences belonging to large gene families , like the trans - sialidases , mucins , mucin - associated surface proteins ( masps ) , were discarded given that the main goal was to identify novel secreted proteins . the remaining coding sequences were screened for the presence of both the signal peptide and the peptidase cleavage site with a probability of 0.8 and 0.7 , respectively . among them , 91 ( 42% ) were annotated as the final criterion for selected proteins likely to be secreted by the classical eukaryotic pathway was the presence of the signal peptide and the signal peptidase site in orthologs of the related parasites : leishmania major , l. infantum , and t. brucei . the 13 proteins bearing the highest probability for the presence of the signal peptide were selected ( table 2 ) for confirmation of extracellular localization . these genes show about 40% identity at the protein level and possess the same predicted orthologs in the genedb server . among the 13 selected genes , these genes were included for the functional test because previous studies identified extracellular proteins with putative transmembrane domains as constituents of the secretome of different pathogens , including l. donovani [ 19 , 20 ] . furthermore , in the protozoa toxoplasma gondii , the dense granule protein gra5 is a transmembrane protein that bears a signal peptide and is secreted as a soluble protein into the vacuolar space , before being inserted into the parasitophorous vacuole membrane . based on these studies , we included genes with potential transmembrane domains to test whether the presence of these domains represents a useful criterion for identifying extracellular secreted proteins . reverse transcription - pcr ( rt - pcr ) was performed for the 13 in silico selected genes in the different developmental stages of t. cruzi in order to verify transcription . the beta - tubulin t. cruzi gene ( geneid tc00.1047053506563 ) , constitutively expressed in all the three stages of t. cruzi , was used as a positive control for rna quality . two genes ( gene i d tc00.1047053511901.30 and tc00.1047053509999.10 ) were negative for the amplification of cdna in the noninfective epimastigote form ( figure 1 ) suggesting a possible stage - specific expression of these genes . a functional test was set up to confirm the presence of selected proteins in the extracellular environment by detection of target proteins in cell - free supernatants . since we aimed to develop a fast and reliable approach to identify trypanosomatid conserved secreted proteins , we used the related leishmania parasite as the recipient organism for the experimental validation of our selected proteins . thus , l. infantum promastigotes were separately transformed with ptex carrying one of the 14 selected t. cruzi genes ( including the beta - tubulin gene ) , and the recombinant parasites were selected for resistance to geneticin g418 . a specific 800 bp fragment , indicative of the presence of the neo gene , was detected in the transfected promastigotes and not in wild - type parasites ( figure 2(a ) ) . moreover , the presence of each candidate gene in recombinant parasites was confirmed using specific primers designed from t. cruzi gene sequences ( figure 2(b ) ) . western blot analysis demonstrated that ( i ) it was possible to easily and specifically detect the 6xhis tag protein in the extract derived from recombinant parasites , ( ii ) recombinant leishmania expressed a relatively high level of t. cruzi protein , and ( iii ) the molecular weight of the detected tagged protein corresponded to the expected mw ( see table 1 ) . western blot analysis of the concentrated cell - free supernatants revealed that among the 14 proteins , only 3 were actively secreted ( tc00.1047053506155.99 , tc00.1047053505789.10 , and tc00.1047053509999.10 ) ( figure 3(b ) ) . these proteins represent genuine secreted material , since ( i ) the overexpression of the beta - tubulin gene does not induce translocation of the beta - tubulin protein into the extracellular space ( difference between lys and ccfs in figure 3(b ) ) , and ( ii ) the detection of the tagged protein in the cell - free supernatant is not related to the level of its expression by leishmania ( low abundance of tc00.1047053506155.99 in figure 3(b ) ) . as expected , a slight molecular weight difference was observed between the tagged protein detected into the whole soluble extract and that detected in the cell - free supernatant that could be explained by the loss of the signal peptide ( figure 3(b ) ) . by using the same protocol as above , leishmania expressing the 6xhis tagged proteins as expected , the presence of the tagged protein in the extracellular medium was only detected in the episomally transfected parasites ( figure 4 ) . together , these results indicate that this approach allows the identification of new and genuinely extracellular proteins involved in the endoplasmic reticulum / golgi - dependent secretory pathway . both confirmed secreted proteins ( linj19.0410 and linj36.5780 ) from l. infantum were tested by using the luciferase reporter system in transfected parasites overexpressing these proteins . in trypanosomatids the secretion process is not fully understood and various pathways including classical and nonclassical mechanisms may contribute to the formation of the extracellular proteome . in an attempt to provide a new approach to analyse the large number of hypothetical conserved proteins in trypanosomatids , we developed an experimental approach to identify hypothetical extracellular proteins likely to be involved in the classical pathway . we combined a web - based bioinformatics approach that used the signal p 3.0 program , one of the most accurate predictors for the presence of a signal peptide sequence , with a functional test that takes advantage of the relative ease to genetically transform leishmania . we assumed that a phylogenetic conservation among leishmania , t. cruzi , and t. brucei would point to evolutionary selection for this family of proteins and indicate an important role for these proteins in the biology of these parasites . using these criteria we selected a pool of 13 trypanosomatid - conserved hypothetical proteins from the t. cruzi database for which secretion was tested . identification of secreted proteins has been hampered in trypanosomatids due to the difficulty in distinguishing genuine secretions from molecules released by lysed , dead , or dying organisms . however , using this approach proteins with a low abundance or that are poorly immunogenic are likely to be missed . nevertheless , proteins produced at low abundance and that are mainly exported to the extracellular compartment are likely to be missed , since the method relies on the comparison of conditioned medium versus cell - associated proteins . indeed proteins that are well known to be extracellular , such as chitinase and sacp ( secreted acid phosphatase ) , were not identified with the silac - based approach . the method we designed allowed us to identify three new trypanosomatid conserved proteins , likely to be secreted through the classical secretory pathway . we were confident that the proteins detected in the cell - free supernatant were genuinely secreted since ( i ) we were not able to detect the beta - tubulin - tagged protein in the cell - free supernatant after overexpression of the gene , ( ii ) the 6-hour incubation time avoided excessive cell death and contamination by proteins released from dead parasites , ( iii ) in the extracellular environment we detected orthologous leishmania proteins ( linj19.0410 , linj36.5780 ) suggesting that secretion in this protein family is likely to be evolutionary conserved , and ( iv ) we detected no relation between the amount of secreted protein and its intracellular expression in the transfected parasites , demonstrating that the translocation of the his - tagged protein into the cell supernatant is not related to the methodology we used . consequently , the analysis of extracellular material revealed that the main contributors to the extracellular proteome are vesicles likely to be this work leads to the general conclusion that 98% of the proteins of the leishmania secretome lacked a targeting signal , indicating that nonclassical secretion pathways are likely to be the dominant way by which leishmania export proteins . the proteomic characterization of the released / secreted proteins of l. braziliensis promastigotes showed that about 5% of the identified proteins possess a putative n - signal peptide , indicating that protein export may depend on unconventional pathways as suggested in l. donovani . however , some evidence strongly supports the notion that the classical secretory pathway is operational in trypanosomatids and contributes to the composition of the parasite 's secretome . for example , the screening of an l. major cdna library with antiserum raised against culture supernatant from stationary - phase promastigotes led to the detection of 8 proteins bearing a potential signal peptide among the 33 genes identified . moreover some of the well - known leishmania proteins found in the extracellular environment have a signal peptide for secretion , such as gp63 or chitinase [ 3 , 27 ] . in the current work we deliberately decided to experimentally validate the secretion of our candidate proteins during the exponential growth phase of leishmania in order to avoid contamination of the cell - free supernatant by apoptotic vesicles or exosomes . therefore , our results suggest that tubulin might be associated with exosomes and/or apoptotic vesicles generated by promastigotes in the stationary - phase of growth . we suggest that the composition of the parasite extracellular proteome is variable and depends both on the parasite stage under consideration and on the relative contribution of the various pathways operating in protein secretion . since we selected and tested secretion for several genes in the trypanosomatid genomes , our methodology provides a potential tool for genomewide screening to identify extracellular proteins . regarding proteins not detected in the extracellular environment , it is important to point out that proteins bearing a signal peptide are not only targets for secretion but also for transfer to specific organelles , for example , lysosomes or the cell surface . in this light , analysis of the 13 proteins with gpi - som suggests that 2 out of 13 proteins ( tc00.1047053506417.30 and tc00.1047053506467.29 ) have a predicted glycosyl - inositol phosphate ( gpi ) anchor signature sequences . hence , the presence of the gpi anchored domain may explain the absence of these proteins in the culture supernatant of recombinant parasites . an unexpected finding in our results was the prediction of transmembrane helices in two of our secreted proteins ( tc00.1047053505789.10 and tc00.1047053509999.10 ) . we included proteins with transmembrane domains to test whether their presence in protein sequences is a suitable criterion for the prediction of extracellular secreted proteins . previous studies suggested that the presence of transmembrane helices in protein sequences is not a suitable criterion for discarding potentially secreted proteins [ 19 , 20 ] . in this regard our results provide further evidence that empirical studies are required to verify bioinformatic predictions since two of the extracellular proteins bear transmemebrane domains . lipocalins are a widely distributed group of mostly extracellular proteins , several of which have been implicated in the regulation of host immune responses , such as , alpha-1-microglobulin and alpha-1-acid glycoprotein . despite a well - conserved tertiary structure of lipocalins , this may explain the absence of a predicted lipocalin signature in the leishmania spp . further studies are needed to ascertain if this large and diverse group of proteins is present in trypanosomatids and plays a role in the transmission process . a glucosidase ii beta subunit - like protein domain has also been detected in the protein tc00.1047053506155.99 . having demonstrated that our methodology is reliable for the identification of extracellular secreted proteins in trypanosomatids , we tested the hypothesis that recombinant leishmania parasites carrying extra copies of leishmania secreted proteins may interfere with their survival or infectivity towards human monocyte - derived macrophages in vitro . results of these assays showed a significant survival advantage to leishmania parasites overexpressing the gene linj19.0410 ( ortholog of tc00.1047053505789.10 ) suggesting that this protein is involved in a process increasing survival and/or replication of the parasite inside its target cell . given that leishmania and t. cruzi do not target the same host cells and follow different cell invasion processes , further experiments in the t. cruzi homologous system are needed to address whether the identified secreted protein tc00.1047053505789.10 from t. cruzi is also involved in host cell invasion and/or replication . in conclusion , our results show that the bioinformatics method combined with the functional tests , provides a fast and reliable method for the identification of novel extracellular secreted proteins involved in the classical secretory pathway and represents potential virulence factors in trypanosomatids .

all study participants had normal glucose tolerance in the oral glucose tolerance test according to world health organization criteria . the exclusion criteria were morbid obesity ( bmi > 40 kg / m ) , extreme physical activity , cardiovascular diseases , hypertension , infectious diseases , or other serious medical problems . all study participants were nonsmokers and not taking anti - inflammatory drugs or drugs affecting carbohydrate and lipid metabolism . they followed the weight - maintaining diet for at least the previous 3 months . before entering the study , all participants underwent a 1-day study protocol that included a brain structural mri and h - mrs at basal state and then the 240-min euglycemic - hyperinsulinemic clamp study , combined with the second brain structural mri and h - mrs after the clamp , in the conditions of euglycemic hyperinsulinemia . the study protocol was approved by the local ethics committee of the medical university of biaystok , and all subjects gave their informed written consent before participation . the bmi was calculated as weight in kilograms divided by the square of height in meters . the waist circumference was measured at the smallest circumference between the rib cage and the iliac crest , with the subject in the standing position . percentage of body fat was assessed by bioelectric impedance analysis using the tanita tbf-511 body fat analyzer ( tanita corp . , insulin sensitivity was evaluated by the euglycemic - hyperinsulinemic clamp technique according to defronzo et al . two venous catheters were inserted into antecubital veins , one for the infusion of the insulin and glucose and the other in the contralateral hand for the blood sampling , with that hand heated to about 60c ( 30 ) . insulin ( actrapid hm ; novo nordisk , copenhagen , denmark ) was given as a primed continuous intravenous infusion for 240 min at 40 mu m min , resulting in constant hyperinsulinemia of about 80 iu / ml . arterialized blood glucose was obtained every 5 min , and 20% dextrose ( 1.11 mol / l ) infusion was adjusted to maintain a glucose level at 5 mmol / l . the rate of whole - body glucose uptake ( m value ) was calculated as the mean glucose infusion rate during the last 40 min of the clamp , corrected for the glucose space and normalized for fat - free mass ( ffm ) . then , participants were divided into subgroups of high insulin sensitivity ( high is ) ( above median from the clamp study , 8.86 mg / kg ffm / min , n = 8 ; m = 11.54 2.71 mg / kg ffm / min ) and low insulin sensitivity ( low is ) ( below median , n = 8 ; m = 7.19 1.87 mg / kg ffm / min ) . after the 4-h clamp , the second structural mri and h - mrs were performed . during the second brain structural mri and h - mrs the glucose infusion rate was continued as during the end of the 4-h period . to maintain euglycemia , the measurement of blood glucose was performed every 10 min , and 20% dextrose infusion was appropriately adjusted . steady - state insulin concentration did not differ during the clamp and the second brain structural mri and h - mrs . brain structural mri imaging and h - mrs examinations were performed on a 1.5 t scanner ( picker eclipse ; picker international inc . , highlands heights , oh ) with the use of a standard circularly polarized head coil . h - mrs examinations were performed with a single voxel point - resolved single voxel localized spectroscopy ( press ) sequence with repetition time ( tr ) = 1,500 ms , echo time ( te ) = 35 ms , number of excitations = 192 , and 2-khz bandwidth . voxels of 2 2 2 cm were positioned in the following regions of interest : left frontal lobe , left temporal lobe , and left thalamus . the signal over the voxel of interest was shimmed to within a line width of 37 hz , and then an unsuppressed signal of water was acquired with eight excitations . subsequently , the multiply optimized insensitive suppression train ( moist ) method was used for suppressing the signal from water . exponential to gaussian transformation was applied as a time - domain apodizing filter . then data were fourier transformed and phase corrected . after application of a legendre polynomial function to approximate the baseline , an automated curve fitting was performed using an iterative , nonlinear least squares fitting procedure by the means levenberg - marquardt algorithm . line shapes of the simulated peaks used in the fitting process were fixed with 85% gaussian and 15% lorentzian fractions . metabolites to cr ratios were analyzed , and the ratios of metabolites to nonsuppressed water signal were calculated according to the following formula : metabolite area 1,000/nonsuppressed water area . the following peaks were assessed : naa at 2.01 ppm and cr at 3.03 ppm , cho at 3.22 ppm , glx complex in the area from 2.11 to 2.45 ppm , and myo - inositol at 3.56 ppm ( fig . h - mrs spectrum obtained from left frontal lobe of the subject from the examined group . thick gray line represents the raw spectrum ; thin black line is the best - fitted spectrum . plasma glucose was measured immediately by the enzymatic method using a glucose analyzer ( ysi 2300 stat plus ; ysi life sciences , yellow springs , oh ) for the oral glucose tolerance test and clamp studies . serum total cholesterol , hdl cholesterol , and triglycerides were assessed by an enzymatic method using commercial kits produced by analco - gbg ( warsaw , poland ) . the concentration of ldl cholesterol was calculated from the friedewald formula : ldl cholesterol = total cholesterol triglycerides/5 hdl cholesterol ( mg / dl ) ( 31 ) . before estimation of the serum concentrations of insulin , the samples were kept frozen at 80c . the serum insulin was measured with immunoradiometric assay ( biosource europe , nivelles , belgium ) . the sensitivity of the assay was 1 iu / ml , and the intra - assay and interassay coefficients of variation were below 2.2 and 6.5% , respectively . the statistics were performed with the statistica 8.0 program ( statsoft , krakow , poland ) . the variables , which did not have a normal distribution , were log transformed before analyses . for the purpose of the data presentation , these variables were antilog transformed again to absolute values in the results . the differences between the groups were estimated with anova . repeated - measures anova with planned comparisons was used to assess the difference between the brain metabolites on h - mrs at baseline and in response to hyperinsulinemia . the relationship between the variables was studied with the pearson product - moment correlation analysis . all study participants had normal glucose tolerance in the oral glucose tolerance test according to world health organization criteria . the exclusion criteria were morbid obesity ( bmi > 40 kg / m ) , extreme physical activity , cardiovascular diseases , hypertension , infectious diseases , or other serious medical problems . all study participants were nonsmokers and not taking anti - inflammatory drugs or drugs affecting carbohydrate and lipid metabolism . they followed the weight - maintaining diet for at least the previous 3 months . before entering the study , all participants underwent a 1-day study protocol that included a brain structural mri and h - mrs at basal state and then the 240-min euglycemic - hyperinsulinemic clamp study , combined with the second brain structural mri and h - mrs after the clamp , in the conditions of euglycemic hyperinsulinemia . the study protocol was approved by the local ethics committee of the medical university of biaystok , and all subjects gave their informed written consent before participation . the bmi was calculated as weight in kilograms divided by the square of height in meters . the waist circumference was measured at the smallest circumference between the rib cage and the iliac crest , with the subject in the standing position . percentage of body fat was assessed by bioelectric impedance analysis using the tanita tbf-511 body fat analyzer ( tanita corp . , insulin sensitivity was evaluated by the euglycemic - hyperinsulinemic clamp technique according to defronzo et al . two venous catheters were inserted into antecubital veins , one for the infusion of the insulin and glucose and the other in the contralateral hand for the blood sampling , with that hand heated to about 60c ( 30 ) . insulin ( actrapid hm ; novo nordisk , copenhagen , denmark ) was given as a primed continuous intravenous infusion for 240 min at 40 mu m min , resulting in constant hyperinsulinemia of about 80 iu / ml . arterialized blood glucose was obtained every 5 min , and 20% dextrose ( 1.11 mol / l ) infusion was adjusted to maintain a glucose level at 5 mmol / l . the rate of whole - body glucose uptake ( m value ) was calculated as the mean glucose infusion rate during the last 40 min of the clamp , corrected for the glucose space and normalized for fat - free mass ( ffm ) . then , participants were divided into subgroups of high insulin sensitivity ( high is ) ( above median from the clamp study , 8.86 mg / kg ffm / min , n = 8 ; m = 11.54 2.71 mg / kg ffm / min ) and low insulin sensitivity ( low is ) ( below median , n = 8 ; m = 7.19 1.87 mg / kg ffm / min ) . after the 4-h clamp , the second structural mri and h - mrs were performed . during the second brain structural mri and h - mrs the glucose infusion rate was continued as during the end of the 4-h period . to maintain euglycemia , the measurement of blood glucose was performed every 10 min , and 20% dextrose infusion was appropriately adjusted . steady - state insulin concentration did not differ during the clamp and the second brain structural mri and h - mrs . brain structural mri imaging and h - mrs examinations were performed on a 1.5 t scanner ( picker eclipse ; picker international inc . , highlands heights , oh ) with the use of a standard circularly polarized head coil . h - mrs examinations were performed with a single voxel point - resolved single voxel localized spectroscopy ( press ) sequence with repetition time ( tr ) = 1,500 ms , echo time ( te ) = 35 ms , number of excitations = 192 , and 2-khz bandwidth . voxels of 2 2 2 cm were positioned in the following regions of interest : left frontal lobe , left temporal lobe , and left thalamus . the signal over the voxel of interest was shimmed to within a line width of 37 hz , and then an unsuppressed signal of water was acquired with eight excitations . subsequently , the multiply optimized insensitive suppression train ( moist ) method was used for suppressing the signal from water . exponential to gaussian transformation was applied as a time - domain apodizing filter . then data were fourier transformed and phase corrected . after application of a legendre polynomial function to approximate the baseline , an automated curve fitting was performed using an iterative , nonlinear least squares fitting procedure by the means levenberg - marquardt algorithm . line shapes of the simulated peaks used in the fitting process were fixed with 85% gaussian and 15% lorentzian fractions . metabolites to cr ratios were analyzed , and the ratios of metabolites to nonsuppressed water signal were calculated according to the following formula : metabolite area 1,000/nonsuppressed water area . the following peaks were assessed : naa at 2.01 ppm and cr at 3.03 ppm , cho at 3.22 ppm , glx complex in the area from 2.11 to 2.45 ppm , and myo - inositol at 3.56 ppm ( fig . h - mrs spectrum obtained from left frontal lobe of the subject from the examined group . thick gray line represents the raw spectrum ; thin black line is the best - fitted spectrum . plasma glucose was measured immediately by the enzymatic method using a glucose analyzer ( ysi 2300 stat plus ; ysi life sciences , yellow springs , oh ) for the oral glucose tolerance test and clamp studies . serum total cholesterol , hdl cholesterol , and triglycerides were assessed by an enzymatic method using commercial kits produced by analco - gbg ( warsaw , poland ) . the concentration of ldl cholesterol was calculated from the friedewald formula : ldl cholesterol = total cholesterol triglycerides/5 hdl cholesterol ( mg / dl ) ( 31 ) . before estimation of the serum concentrations of insulin , the serum insulin was measured with immunoradiometric assay ( biosource europe , nivelles , belgium ) . the sensitivity of the assay was 1 iu / ml , and the intra - assay and interassay coefficients of variation were below 2.2 and 6.5% , respectively . the statistics were performed with the statistica 8.0 program ( statsoft , krakow , poland ) . the variables , which did not have a normal distribution , were log transformed before analyses . for the purpose of the data presentation , these variables were antilog transformed again to absolute values in the results . the differences between the groups were estimated with anova . repeated - measures anova with planned comparisons was used to assess the difference between the brain metabolites on h - mrs at baseline and in response to hyperinsulinemia . the relationship between the variables was studied with the pearson product - moment correlation analysis . the groups did not differ in bmi , waist circumference , % body fat , fasting plasma glucose , and insulin concentrations ( table 1 ) , although subjects with low is tended to have higher fasting serum insulin ( p = 0.052 ; ns ) . clinical characteristics of the studied groups baseline naa , cho , myo - inositol , and glx in the frontal and temporal regions and thalamus were not different between high - is and low - is groups after correction for both cr and h2o ( tables 2 and 3 ) . naa / cr and naa / h2o in the frontal region increased in response to hyperinsulinemia in high - is subjects ( p = 0.0018 and p = 0.007 , respectively ) , but not in the low - is group . in the high - is group , a decrease in frontal region cho / cr ( p = 0.044 ) and a decrease in temporal region cho / h2o ( p = 0.032 ) and myo - inositol / h2o ( p = 0.015 ) during insulin infusion were observed , which was not the case in the low - is group . frontal and temporal region glx / cr and glx / h2o increased in response to hyperinsulinemia in the high - is group ( frontal region , glx / cr , p = 0.0033 , glx / h2o , p = 0.0065 ; temporal region , glx / cr , p = 0.002 , glx / h2o , p = 0.054 , ns ) . there were no significant changes in the estimated parameters in the low - is group ( all p > 0.15 , except frontal glx / cr , p = 0.078 ) ( tables 2 and 3 ) . brain metabolite - to - cr ratios at baseline and during hyperinsulinemia in the studied groups brain metabolite - to - h2o ratios at baseline and during hyperinsulinemia in the studied groups in consequence of these changes , frontal naa / cr and naa / h2o ratios assessed during the second h - mrs were higher in the high - is group in comparison with the low - is group ( p = 0.0004 and p = 0.01 , respectively ) . temporal myo - inositol / cr and myo - inositol / h2o ratios assessed during insulin infusion were lower in the high - is group in comparison with the low - is group ( p = 0.032 and p = 0.015 , respectively ) . frontal and temporal glx / cr and glx / h2o ratios assessed during the second h - mrs were higher in the high - is group in comparison with the low - is group ( frontal region , glx / cr , p = 0.04 , glx / h2o , p = 0.029 ; temporal region , glx / cr , p = 0.057 , ns , glx / h2o , p = 0.032 ) ( tables 2 and 3 ) . when all 16 subjects were analyzed together , an increase in frontal glx / cr ( p = 0.017 ) and glx / h2o ( p = 0.008 ) and temporal glx / cr ( p = 0.015 ) and a decrease in temporal cho / h2o ( p = 0.005 ) were observed during insulin infusion . we observed significant correlations between insulin sensitivity and frontal naa / cr ( r = 0.78 , p < 0.001 ) ( fig . 2 ) and naa / h2o ( r = 0.65 , p = 0.006 ) and temporal myo - inositol / cr ( r = 0.67 , p = 0.012 ) , myo - inositol / h2o ( r = 0.81 , p < 0.001 ) ( fig . 2 ) , and glx / h2o ( r = 0.63 , p = 0.012 ) assessed during the second h - mrs . relationships between insulin sensitivity and frontal naa / cr ( a ) and temporal myo - inositol / h2o ( b ) during insulin infusion in the entire study population ( n = 16 ) . we also observed significant correlations between percent body fat and baseline temporal myo - inositol / h2o ( r = 0.55 , p = 0.027 ) . waist circumference was related to baseline temporal myo - inositol / h2o ( r = 0.57 , p = 0.02 ) . baseline temporal myo - inositol / cr and myo - inositol / h2o were related to total cholesterol ( r = 0.61 , p = 0.011 and r = 0.56 , p = 0.025 , respectively ) and ldl cholesterol ( r = 0.62 , p = 0.01 and r = 0.54 , p = 0.03 , respectively ) . baseline temporal myo - inositol / h2o and cho / h2o were also related to triglycerides ( r = 0.60 , p = 0.015 and r = 0.57 , p = 0.022 , respectively ) . the correlations and all significant differences ( before and during insulin infusion ) observed in the high - is group were still significant after excluding one subject with the highest insulin sensitivity . the main finding of our study is that insulin infusion resulted in significant changes of brain neurochemistry on h - mrs in young healthy men with high is but not with low is . in particular , we observed an increase in frontal naa / cr and naa / h2o and frontal and temporal glx / cr and glx / h2o and a decrease in frontal cho / cr and temporal cho / h2o and myo - inositol/ h2o in young healthy men with high insulin sensitivity . the metabolites in our study were calculated for cr , which is altered in hyperglycemic conditions ( 27 ) . however , we conducted our experiments under a euglycemic state , which should not affect cr , and we also corrected all metabolites for h2o signal . we found that naa after correction for both cr and h2o in the frontal cortex increased in insulin - stimulated conditions in high - is subjects but not in the low - is group . in consequence of these changes , frontal naa / cr and naa/ h2o ratios assessed during the second h - mrs were higher in the high - is group in comparison with the low - is group . the baseline naa / cr and naa / h2o ratios in the frontal lobe were not different between the high- and low - is groups . of note is the fact that insulin infusion revealed differences in young healthy men according to their insulin sensitivity our data suggest that insulin could improve neuronal metabolism in healthy subjects with better insulin action . ( 28 ) found decreased naa / cr ratios in the frontal cortex of type 2 diabetic patients with higher hemoglobin a1c levels ( > 10% ) . reduced naa has been also reported in patients with atherosclerotic cerebral disease ( 32 ) . chronic exposure to hyperglycemia and cerebrovascular disease independently will turn out neuronal dysfunction or accelerate neuronal damage . however , our data indicate that the young healthy subjects with normal glucose metabolism and without vascular diseases but with low is could have impaired neuronal metabolism . this indicates that altered insulin signaling could be a primary risk factor for neuronal dysfunction . our data provide the first evidence that low naa might be associated with an impaired insulin action even without clinically evident pathology . this might provide a potential link for the previously observed associations between apparent pathological conditions and low naa and might also reflect reduced neuronal mass in these diseases . naa is synthesized mostly in mitochondria ( 17 ) , and our data indicate that insulin might affect mitochondrial processes in neurons . impaired insulin signaling could play a role in alzheimer disease pathogenesis . a decrease in the naa / cr ratios , in both cortical and/or limbic structures , was observed in patients with alzheimer disease ( 21 ) . importantly , we observed significant positive correlations between insulin sensitivity and frontal lobe naa / cr and naa / h2o ratios in insulin - stimulated conditions . it was shown that homeostasis model assessment of insulin resistance levels were inversely correlated with parietal white matter naa / cr ratios in type 2 diabetic patients ( 28 ) . interestingly , in another study , an altered insulin effect on cerebrocortical activity as assessed by magneto - encephalography was shown in conditions associated with peripheral insulin resistance ( 33 ) . frontal and temporal lobe glx / cr and glx / h2o increased during the clamp in the high - is group . the balance of glutamatergic and gabaergic transmission is required to maintain proper brain function ( 10 ) . emerging evidence has suggested that insulin signaling plays a role in synaptic plasticity by modulating the activities of excitatory and inhibitory receptors , such as glutamate and gaba receptors , and by triggering signal transduction cascades , leading to an alternation of gene expression that is required for memory consolidation ( 10 ) . our data indicate that insulin could influence glutamatergic and gabaergic transmission in frontal and temporal lobes , and the insulin influence of this neurotransmission could be altered in healthy humans with low is . the limitation of our study is that we can not discriminate between glutamate , glutamine , and gaba within the glx spectrum . however , previous data ( 17 ) indicate that glutamate is the main metabolite in the glx spectrum . as glutamate mainly reflects neuronal metabolism , we hypothesize that the observed increase in glx after insulin indicates the hormone influence on neuronal metabolism and transmission . because glutamate metabolism is connected with mitochondria ( 23 ) , insulin could influence glutamatergic activity by affecting mitochondrial processes in neurons . in future studies , it would be advisable to also assess the glutamine / glutamate ratio , as it is a better marker of glutamatergic activity than each concentration separated ( 17 ) . importantly , we observed significant correlations between insulin sensitivity and the insulin - stimulated temporal glx / h2o ratio . it has been discovered by functional mri that insulin , when given to healthy human adults under euglycemic clamp to maintain normal blood glucose levels , results in activation of the medial temporal lobe , including the hippocampus , a structure critically required for processing declarative memory ( 35 ) . we observed a decrease in the temporal lobe myo - inositol / h2o ratio in the high - is group , which was not the case in the low - is group . myo - inositol is involved in the metabolism of cell membrane inositol phospholipids , which is fundamental for cellular signal transduction . the activation of the insulin receptor stimulates the routes for inositol lipid metabolism associated with phosphatidylinositol 3-kinase and production of the phosphorylated derivatives of phosphoinositide , which are second messengers in cellular signal transduction ( 10 ) . the decrease in myo - inositol during insulin infusion in the high - is group could be connected with an increase in the concentrations of phosphorylated derivatives of phosphoinositide . impaired insulin signaling in insulin - resistant subjects may result in decreased concentrations of the inositol metabolites in these pathways and could lead to an increase in brain myo - inositol levels in insulin resistance . in our study , insulin sensitivity inversely correlated with temporal myo - inositol / cr and myo - inositol / h2o ratios assessed during the second h - mrs . in the study of sahin et al . ( 28 ) , there was an increase in the myo - inositol / cr ratio in the frontal cortex in type 2 diabetic subjects in comparison with controls . in the same study , the change in the myo - inositol / cr ratio obtained from the frontal cortex , thalamus , and parietal white matter was similar for patients with impaired glucose tolerance and type 2 diabetes . these findings may suggest a role for increased insulin resistance in causing the observed changes ( 28 ) . some data indicate that the accumulation of myo - inositol within the brain of diabetic subjects is associated with chronic hyperglycemia . myo - inositol as a major organic osmolyte could regulate brain osmotic adaptation in hyperglycemic conditions ( 17 ) , which is not the case in our study . interestingly , reduced phosphoinositide concentrations were found in the anterior temporal cortex of a brain with alzheimer disease ( 36 ) . furthermore , it has been shown that the activity of phosphatidylinositol kinase was reduced in the postmortem brains of patients with alzheimer disease ( 36 ) . these data may suggest that alteration in phosphoinositide pathways could promote the enhancement of myo - inositol levels in the brain . indeed , an increase in myo - inositol levels has been frequently reported in the brain with alzheimer disease . it has been suggested that myo - inositol was a marker of glial proliferation in this disease ( 37 ) . in several neurological disorders , myo - inositol was found to be increased , and this is generally assumed to be a marker of gliosis , based on the fact that higher myo - inositol levels are found in cultured astrocytes as compared with neurons in vitro ( 17 ) . ( 27 ) brain myo - inositol levels did not correlate with the molecular marker of gliosis . in our study , in the high - is group , but not in the low - is group , a decrease in frontal region cho / cr and temporal region cho / h2o in the insulin - stimulated condition was observed . the decrease in cho during the clamp could be the result of using cho - containing compounds in the synthesis of neurotransmitters . some data showed an increase of cho in both the white matter and the gray matter of type 2 diabetic patients when compared with patients with type 1 diabetes ( 38 ) . ( 28 ) showed an increase in frontal cortical cho / cr in patients with impaired glucose tolerance . our results indicate that insulin could influence membrane turnover , and in insulin - resistant subjects , these processes could be altered . we can not exclude the possibility that the low - is group may have shown changes if studied longer . on the other hand , a 4-h period was sufficient to induce changes in high - is subjects with no significant changes in the low - is group . in our study , the changes in the brain neurochemistry under hyperinsulinemic conditions were observed only in the frontal and temporal region and not in the thalamus . lack of the changes in neurometabolites in the thalamus might indicate that the alterations observed in other areas of interest are not related to a different amount of glucose infused . it might be related to the higher density of insulin receptors in these regions in comparison with the thalamus ( 39 ) . the potential limitation of our study is the clinical relevance of low is / insulin resistance derived from clamp , given the lack of differences in other studied parameters , for instance , bmi . however , this approach allowed us to analyze the results only with respect to insulin action , without any confounding factors . clamp - derived insulin sensitivity was reduced by 37.7% in the low - is group in comparison with the high - is group . insulin resistance is an important risk factor for type 2 diabetes , cardiovascular disease , and alzheimer disease ( 1 ) . indeed , in a recently published study , an impaired insulin action in the postmortem brains of patients with alzheimer disease was demonstrated ( 40 ) . our data show that insulin might influence cerebral metabolites , and this action is impaired in subjects with low whole - body insulin sensitivity . thus , our results provide a potential link between insulin resistance and altered metabolism of the central nervous system .

objectiveinsulin may play important roles in brain metabolism . proton magnetic resonance spectroscopy ( 1h - mrs ) of the central nervous system gives information on neuronal viability , cellular energy , and membrane status . to elucidate the specific role of insulin action in the brain , we estimated neurometabolites with 1h - mrs and assessed their regulation by insulin infusion and their relationship with insulin sensitivity.research design and methodswe studied 16 healthy young men . 1h - mrs was performed at baseline and after 240 min of euglycemic - hyperinsulinemic clamp . voxels were positioned in the left frontal lobe , left temporal lobe , and left thalamus . the ratios of n - acetylaspartate ( naa ) , choline - containing compounds ( cho ) , myo - inositol , and glutamate / glutamine/-aminobutyric acid complex ( glx ) to creatine ( cr ) and nonsuppressed water signal were determined . the participants were divided into subgroups of high ( high is ) and low ( low is ) insulin sensitivity.resultsbaseline neurometabolic substrates were not different between the groups . insulin infusion resulted in an increase in frontal naa / cr and naa / h2o and frontal and temporal glx / cr and glx / h2o and a decrease in frontal cho / cr and temporal cho / h2o and myo - inositol / h2o ( all p < 0.05 , except temporal glx / h2o , p = 0.054 , ns ) in the high - is , but not in the low - is , group . insulin sensitivity correlated positively with frontal naa / cr and naa / h2o and temporal glx / h2o and negatively with temporal myo - inositol / cr and myo - inositol / h2o assessed during the second 1h - mrs ( all p < 0.05).conclusionsinsulin might influence cerebral metabolites , and this action is impaired in subjects with low whole - body insulin sensitivity . thus , our results provide a potential link between insulin resistance and altered metabolism of the central nervous system .

RESEARCH DESIGN AND METHODS Study group Study protocol Anthropometry Insulin sensitivity MRI and Biochemical analysis Statistical analysis RESULTS CONCLUSIONS

before entering the study , all participants underwent a 1-day study protocol that included a brain structural mri and h - mrs at basal state and then the 240-min euglycemic - hyperinsulinemic clamp study , combined with the second brain structural mri and h - mrs after the clamp , in the conditions of euglycemic hyperinsulinemia . the study protocol was approved by the local ethics committee of the medical university of biaystok , and all subjects gave their informed written consent before participation . , insulin sensitivity was evaluated by the euglycemic - hyperinsulinemic clamp technique according to defronzo et al . two venous catheters were inserted into antecubital veins , one for the infusion of the insulin and glucose and the other in the contralateral hand for the blood sampling , with that hand heated to about 60c ( 30 ) . the rate of whole - body glucose uptake ( m value ) was calculated as the mean glucose infusion rate during the last 40 min of the clamp , corrected for the glucose space and normalized for fat - free mass ( ffm ) . then , participants were divided into subgroups of high insulin sensitivity ( high is ) ( above median from the clamp study , 8.86 mg / kg ffm / min , n = 8 ; m = 11.54 2.71 mg / kg ffm / min ) and low insulin sensitivity ( low is ) ( below median , n = 8 ; m = 7.19 1.87 mg / kg ffm / min ) . after the 4-h clamp , the second structural mri and h - mrs were performed . during the second brain structural mri and h - mrs the glucose infusion rate was continued as during the end of the 4-h period . to maintain euglycemia , the measurement of blood glucose was performed every 10 min , and 20% dextrose infusion was appropriately adjusted . steady - state insulin concentration did not differ during the clamp and the second brain structural mri and h - mrs . h - mrs examinations were performed with a single voxel point - resolved single voxel localized spectroscopy ( press ) sequence with repetition time ( tr ) = 1,500 ms , echo time ( te ) = 35 ms , number of excitations = 192 , and 2-khz bandwidth . voxels of 2 2 2 cm were positioned in the following regions of interest : left frontal lobe , left temporal lobe , and left thalamus . line shapes of the simulated peaks used in the fitting process were fixed with 85% gaussian and 15% lorentzian fractions . metabolites to cr ratios were analyzed , and the ratios of metabolites to nonsuppressed water signal were calculated according to the following formula : metabolite area 1,000/nonsuppressed water area . the following peaks were assessed : naa at 2.01 ppm and cr at 3.03 ppm , cho at 3.22 ppm , glx complex in the area from 2.11 to 2.45 ppm , and myo - inositol at 3.56 ppm ( fig . h - mrs spectrum obtained from left frontal lobe of the subject from the examined group . the sensitivity of the assay was 1 iu / ml , and the intra - assay and interassay coefficients of variation were below 2.2 and 6.5% , respectively . the differences between the groups were estimated with anova . repeated - measures anova with planned comparisons was used to assess the difference between the brain metabolites on h - mrs at baseline and in response to hyperinsulinemia . before entering the study , all participants underwent a 1-day study protocol that included a brain structural mri and h - mrs at basal state and then the 240-min euglycemic - hyperinsulinemic clamp study , combined with the second brain structural mri and h - mrs after the clamp , in the conditions of euglycemic hyperinsulinemia . , insulin sensitivity was evaluated by the euglycemic - hyperinsulinemic clamp technique according to defronzo et al . two venous catheters were inserted into antecubital veins , one for the infusion of the insulin and glucose and the other in the contralateral hand for the blood sampling , with that hand heated to about 60c ( 30 ) . the rate of whole - body glucose uptake ( m value ) was calculated as the mean glucose infusion rate during the last 40 min of the clamp , corrected for the glucose space and normalized for fat - free mass ( ffm ) . then , participants were divided into subgroups of high insulin sensitivity ( high is ) ( above median from the clamp study , 8.86 mg / kg ffm / min , n = 8 ; m = 11.54 2.71 mg / kg ffm / min ) and low insulin sensitivity ( low is ) ( below median , n = 8 ; m = 7.19 1.87 mg / kg ffm / min ) . during the second brain structural mri and h - mrs the glucose infusion rate was continued as during the end of the 4-h period . to maintain euglycemia , the measurement of blood glucose was performed every 10 min , and 20% dextrose infusion was appropriately adjusted . steady - state insulin concentration did not differ during the clamp and the second brain structural mri and h - mrs . h - mrs examinations were performed with a single voxel point - resolved single voxel localized spectroscopy ( press ) sequence with repetition time ( tr ) = 1,500 ms , echo time ( te ) = 35 ms , number of excitations = 192 , and 2-khz bandwidth . voxels of 2 2 2 cm were positioned in the following regions of interest : left frontal lobe , left temporal lobe , and left thalamus . metabolites to cr ratios were analyzed , and the ratios of metabolites to nonsuppressed water signal were calculated according to the following formula : metabolite area 1,000/nonsuppressed water area . the following peaks were assessed : naa at 2.01 ppm and cr at 3.03 ppm , cho at 3.22 ppm , glx complex in the area from 2.11 to 2.45 ppm , and myo - inositol at 3.56 ppm ( fig . h - mrs spectrum obtained from left frontal lobe of the subject from the examined group . before estimation of the serum concentrations of insulin , the serum insulin was measured with immunoradiometric assay ( biosource europe , nivelles , belgium ) . the sensitivity of the assay was 1 iu / ml , and the intra - assay and interassay coefficients of variation were below 2.2 and 6.5% , respectively . for the purpose of the data presentation , these variables were antilog transformed again to absolute values in the results . repeated - measures anova with planned comparisons was used to assess the difference between the brain metabolites on h - mrs at baseline and in response to hyperinsulinemia . the groups did not differ in bmi , waist circumference , % body fat , fasting plasma glucose , and insulin concentrations ( table 1 ) , although subjects with low is tended to have higher fasting serum insulin ( p = 0.052 ; ns ) . clinical characteristics of the studied groups baseline naa , cho , myo - inositol , and glx in the frontal and temporal regions and thalamus were not different between high - is and low - is groups after correction for both cr and h2o ( tables 2 and 3 ) . naa / cr and naa / h2o in the frontal region increased in response to hyperinsulinemia in high - is subjects ( p = 0.0018 and p = 0.007 , respectively ) , but not in the low - is group . in the high - is group , a decrease in frontal region cho / cr ( p = 0.044 ) and a decrease in temporal region cho / h2o ( p = 0.032 ) and myo - inositol / h2o ( p = 0.015 ) during insulin infusion were observed , which was not the case in the low - is group . frontal and temporal region glx / cr and glx / h2o increased in response to hyperinsulinemia in the high - is group ( frontal region , glx / cr , p = 0.0033 , glx / h2o , p = 0.0065 ; temporal region , glx / cr , p = 0.002 , glx / h2o , p = 0.054 , ns ) . there were no significant changes in the estimated parameters in the low - is group ( all p > 0.15 , except frontal glx / cr , p = 0.078 ) ( tables 2 and 3 ) . brain metabolite - to - cr ratios at baseline and during hyperinsulinemia in the studied groups brain metabolite - to - h2o ratios at baseline and during hyperinsulinemia in the studied groups in consequence of these changes , frontal naa / cr and naa / h2o ratios assessed during the second h - mrs were higher in the high - is group in comparison with the low - is group ( p = 0.0004 and p = 0.01 , respectively ) . temporal myo - inositol / cr and myo - inositol / h2o ratios assessed during insulin infusion were lower in the high - is group in comparison with the low - is group ( p = 0.032 and p = 0.015 , respectively ) . frontal and temporal glx / cr and glx / h2o ratios assessed during the second h - mrs were higher in the high - is group in comparison with the low - is group ( frontal region , glx / cr , p = 0.04 , glx / h2o , p = 0.029 ; temporal region , glx / cr , p = 0.057 , ns , glx / h2o , p = 0.032 ) ( tables 2 and 3 ) . when all 16 subjects were analyzed together , an increase in frontal glx / cr ( p = 0.017 ) and glx / h2o ( p = 0.008 ) and temporal glx / cr ( p = 0.015 ) and a decrease in temporal cho / h2o ( p = 0.005 ) were observed during insulin infusion . we observed significant correlations between insulin sensitivity and frontal naa / cr ( r = 0.78 , p < 0.001 ) ( fig . 2 ) and naa / h2o ( r = 0.65 , p = 0.006 ) and temporal myo - inositol / cr ( r = 0.67 , p = 0.012 ) , myo - inositol / h2o ( r = 0.81 , p < 0.001 ) ( fig . 2 ) , and glx / h2o ( r = 0.63 , p = 0.012 ) assessed during the second h - mrs . relationships between insulin sensitivity and frontal naa / cr ( a ) and temporal myo - inositol / h2o ( b ) during insulin infusion in the entire study population ( n = 16 ) . we also observed significant correlations between percent body fat and baseline temporal myo - inositol / h2o ( r = 0.55 , p = 0.027 ) . waist circumference was related to baseline temporal myo - inositol / h2o ( r = 0.57 , p = 0.02 ) . baseline temporal myo - inositol / cr and myo - inositol / h2o were related to total cholesterol ( r = 0.61 , p = 0.011 and r = 0.56 , p = 0.025 , respectively ) and ldl cholesterol ( r = 0.62 , p = 0.01 and r = 0.54 , p = 0.03 , respectively ) . baseline temporal myo - inositol / h2o and cho / h2o were also related to triglycerides ( r = 0.60 , p = 0.015 and r = 0.57 , p = 0.022 , respectively ) . the correlations and all significant differences ( before and during insulin infusion ) observed in the high - is group were still significant after excluding one subject with the highest insulin sensitivity . the main finding of our study is that insulin infusion resulted in significant changes of brain neurochemistry on h - mrs in young healthy men with high is but not with low is . in particular , we observed an increase in frontal naa / cr and naa / h2o and frontal and temporal glx / cr and glx / h2o and a decrease in frontal cho / cr and temporal cho / h2o and myo - inositol/ h2o in young healthy men with high insulin sensitivity . however , we conducted our experiments under a euglycemic state , which should not affect cr , and we also corrected all metabolites for h2o signal . we found that naa after correction for both cr and h2o in the frontal cortex increased in insulin - stimulated conditions in high - is subjects but not in the low - is group . in consequence of these changes , frontal naa / cr and naa/ h2o ratios assessed during the second h - mrs were higher in the high - is group in comparison with the low - is group . the baseline naa / cr and naa / h2o ratios in the frontal lobe were not different between the high- and low - is groups . of note is the fact that insulin infusion revealed differences in young healthy men according to their insulin sensitivity our data suggest that insulin could improve neuronal metabolism in healthy subjects with better insulin action . ( 28 ) found decreased naa / cr ratios in the frontal cortex of type 2 diabetic patients with higher hemoglobin a1c levels ( > 10% ) . however , our data indicate that the young healthy subjects with normal glucose metabolism and without vascular diseases but with low is could have impaired neuronal metabolism . this might provide a potential link for the previously observed associations between apparent pathological conditions and low naa and might also reflect reduced neuronal mass in these diseases . naa is synthesized mostly in mitochondria ( 17 ) , and our data indicate that insulin might affect mitochondrial processes in neurons . a decrease in the naa / cr ratios , in both cortical and/or limbic structures , was observed in patients with alzheimer disease ( 21 ) . importantly , we observed significant positive correlations between insulin sensitivity and frontal lobe naa / cr and naa / h2o ratios in insulin - stimulated conditions . it was shown that homeostasis model assessment of insulin resistance levels were inversely correlated with parietal white matter naa / cr ratios in type 2 diabetic patients ( 28 ) . frontal and temporal lobe glx / cr and glx / h2o increased during the clamp in the high - is group . our data indicate that insulin could influence glutamatergic and gabaergic transmission in frontal and temporal lobes , and the insulin influence of this neurotransmission could be altered in healthy humans with low is . as glutamate mainly reflects neuronal metabolism , we hypothesize that the observed increase in glx after insulin indicates the hormone influence on neuronal metabolism and transmission . importantly , we observed significant correlations between insulin sensitivity and the insulin - stimulated temporal glx / h2o ratio . it has been discovered by functional mri that insulin , when given to healthy human adults under euglycemic clamp to maintain normal blood glucose levels , results in activation of the medial temporal lobe , including the hippocampus , a structure critically required for processing declarative memory ( 35 ) . we observed a decrease in the temporal lobe myo - inositol / h2o ratio in the high - is group , which was not the case in the low - is group . myo - inositol is involved in the metabolism of cell membrane inositol phospholipids , which is fundamental for cellular signal transduction . the decrease in myo - inositol during insulin infusion in the high - is group could be connected with an increase in the concentrations of phosphorylated derivatives of phosphoinositide . impaired insulin signaling in insulin - resistant subjects may result in decreased concentrations of the inositol metabolites in these pathways and could lead to an increase in brain myo - inositol levels in insulin resistance . in our study , insulin sensitivity inversely correlated with temporal myo - inositol / cr and myo - inositol / h2o ratios assessed during the second h - mrs . ( 28 ) , there was an increase in the myo - inositol / cr ratio in the frontal cortex in type 2 diabetic subjects in comparison with controls . in the same study , the change in the myo - inositol / cr ratio obtained from the frontal cortex , thalamus , and parietal white matter was similar for patients with impaired glucose tolerance and type 2 diabetes . some data indicate that the accumulation of myo - inositol within the brain of diabetic subjects is associated with chronic hyperglycemia . myo - inositol as a major organic osmolyte could regulate brain osmotic adaptation in hyperglycemic conditions ( 17 ) , which is not the case in our study . these data may suggest that alteration in phosphoinositide pathways could promote the enhancement of myo - inositol levels in the brain . indeed , an increase in myo - inositol levels has been frequently reported in the brain with alzheimer disease . in several neurological disorders , myo - inositol was found to be increased , and this is generally assumed to be a marker of gliosis , based on the fact that higher myo - inositol levels are found in cultured astrocytes as compared with neurons in vitro ( 17 ) . in our study , in the high - is group , but not in the low - is group , a decrease in frontal region cho / cr and temporal region cho / h2o in the insulin - stimulated condition was observed . the decrease in cho during the clamp could be the result of using cho - containing compounds in the synthesis of neurotransmitters . ( 28 ) showed an increase in frontal cortical cho / cr in patients with impaired glucose tolerance . we can not exclude the possibility that the low - is group may have shown changes if studied longer . on the other hand , a 4-h period was sufficient to induce changes in high - is subjects with no significant changes in the low - is group . in our study , the changes in the brain neurochemistry under hyperinsulinemic conditions were observed only in the frontal and temporal region and not in the thalamus . the potential limitation of our study is the clinical relevance of low is / insulin resistance derived from clamp , given the lack of differences in other studied parameters , for instance , bmi . clamp - derived insulin sensitivity was reduced by 37.7% in the low - is group in comparison with the high - is group . insulin resistance is an important risk factor for type 2 diabetes , cardiovascular disease , and alzheimer disease ( 1 ) . indeed , in a recently published study , an impaired insulin action in the postmortem brains of patients with alzheimer disease was demonstrated ( 40 ) . our data show that insulin might influence cerebral metabolites , and this action is impaired in subjects with low whole - body insulin sensitivity . thus , our results provide a potential link between insulin resistance and altered metabolism of the central nervous system .